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Starch utilization by wild and genetically engineered yeasts grown in batch and continuous culture

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Title:
Starch utilization by wild and genetically engineered yeasts grown in batch and continuous culture
Creator:
Altman, Hanna
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
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xi, 83 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Saccharomyces cerevisiae ( lcsh )
Saccharomyces cerevisiae ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (M.A.)--University of Colorado at Denver, 1994. Biology
Bibliography:
Includes bibliographical references (leaves 79-83).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Hanna Altman.

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University of Colorado Denver
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|Auraria Library
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All applicable rights reserved by the source institution and holding location.
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32480741 ( OCLC )
ocm32480741

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STARCH UTILIZATION BY WILD AND GENETICALLY ENGINEERED YEASTS GROWN IN BATCH AND CONTINUOUS CULTURE by Hanna Altman M.S., Warsaw Agricultural University, 1984 A thesis submitted to the Faculty of the Graduate School of the University of Colorado at Denver in partial fulfillment of the requirement for the degree of Master of Arts Biology 1994

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This thesis for the Master of Arts degree by Hanna Altman has been approved for the Graduate School by James R. Mattoon Gerald J. Audesirk Linda K. Dixon

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Altman, Hanna (M.A., Biology) Starch Utilization by Wild and Genetically Engineered Yeasts Grown in Batch and Continuous Culture Thesis directed by Professors James R. Mattoon and Gerald J. Audesirk ABSTRACT Naturally-occurring strains of Saccharomyces capable of utilizing starch as a primary carbon source [Saccharomyces diastaticus] secrete glucoamylase encoded by one or more of three unlinked STA genes. Common baking and brewing strains of Saccharomyces cerevisiae or Saccharomyces carlsbergensis lack STA genes, and do not exhibit significant utilization of st"arch. Conditions were opti mized for aerobic continuous culture of S. diastaticus, and production of biomass and extracellular glucoamylase were followed. Two different plasmids (yeast cen tromere and episomal-multicopy), containing the same STA2K allele, and a third plasmid containing a-amylase eDNA were used to transform various laboratory strains of Saccharomyces cerevisiae. The S. cerevisiae strains transformed with these plasmids were grown in batch and continuous culture on starch media. lll

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In all cases, the yeast strains transformed with the yeast centromeric plasmid produced substantial quantities of biomass while secreting significant amounts of glucoamylase and maintaining relatively high plasmid stability. On the other hand, the yeast strains transformed with the multi copy plasmid showed a low pro duction of biomass, no detectable glucoamylase secretion, and progressive loss of the plasmid, in both batch and continuous experiments. Also, a S. cerevisiae strain carrying both a-amylase and multicopy glucoamylase plasmids was pre pared. In batch experiments, the a-amylase plasmid, when carried together with the glucoarnylase plasmid, significantly improved the ability of S. cerevisiae to grow on starch. However, this improvement was not achieved in a continuous culture in which very poor growth of yeast cells and lack of the expression of the amylolytic genes was observed. Progressive loss of the multicopy plasmid from the cells occurred even though starch was the primary carbon source. Starch alone was shown to be insufficient as a selective pressure for the maintenance of the a-amylase and glucoamylase multicopy plasmids. Finally, two mutants with enhanced glucoamylase secretion and/or multicopy plasmid retention were isolated. IV

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This abstract accurately represents the content of the candidate's thesis. I recommend its publication. r James R. Mattoon Signed, Gerald J. Audesirk v

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CONTENTS Chapter 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Background and Survey . . . . . . . . . . . . . . . . . . . . . 4 1.1.1 Expression of Amylolytic Genes in S. cerevisiae ................... 5 1.1.2 S. diastaticus Amylolytic Yeasts ................................. 5 1.1.3 Origin of the STA Genes in S. diastaticus ....................... 6 1.1.4 Manipulating Yeast Genome Using Plasmid Vectors .............. 7 1.1.5 Selection Systems for Improved Plasmid Stability ................ 9 1.2 Objectives. of This Study ........................................ 12 2. Materials and Methods .............................................. 19 2.1-Yeast Strains and Plasmids ................... : .................. 19 2.2 -Growth Media .................................................... 24 2.3 Growth Conditions and Measurements . . . . . . . . . . . . . 24 2.4 Plasmid DNA Extraction from E. coli ............................ 26 2.5 Transformation of Yeast Cells .................................... 28 2.6 Glucoamlyase Assay ............................................. 31 2. 7 Plasmid Stability Measurements . . .. .. . .. . . .. . . .. . . .. 32 3. Experimental Results and Discussion . . . . . . . . . . . . . . . . 33 3.1 Shake Flask Culture Experiments ................................ 33 3.1.1 Growth of Yeast Strains on Starch ..... ........................ 33 3.1.2 Effects of STA21< Gene on Yeast Growth ....................... 37 3.1.3 Effects of a-amylase Gene on Yeast Growth .................... 49 3.2 Continuous Culture of Yeast on. Starch Medium .................. 51 3.2.1 Optimization of Conditions for Continuous-chemostat Culture .. 51 3.2.2 Behavior of Transformants in Continuous Culture . . . . . . . 64 Vl

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4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 References ................................................................ 79 Vll

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FIGURES Figure 1.1 The starch molecule and action patterns of amylolytic enzymes ......... 2 2.1 Construction of yeast multicopy plasmid pSTA325 .................... 21 2.2 Construction of yeast centromeric plasmid pSTA316 .................. 22 2.3 Restriction map of a-amylase plasmid pMS12 ........................ 23 3.1 Relative growth of S. cerevisiae and S. diastaticus strains on complete starch medium in shake flask experiments ............................ 36 3.2 Effects of the pSTA325 multicopy plasmid on the growth of S. cerevisiae strains on starch medium ............................................ 38 3.3 Expression of the STA2K gene by S. cerevisiae strains 1403-7A and AP1-5C transformed by the multicopy plasmid pSTA325 ...................... 40 3.4 Comparison of the levels of secreted glucoamylase production by S. cere-. t .. ST'A2K 46 vzszae s rams carrymg .11 gene ................................. 3.5 Effect of the STA2K gene carried on centromeric and multicopy plasmids on the growth of various S. cerevisiae strains ......................... 48 3.6 Time course of growth of transformed and untransformed strain of S. cerevisiae SHU32a in sha_ke flask culture ................................. 50 3.7 Effects of the dilution rate (D) on growth and glucoamylase secretion by S. diastaticus SD2-A8/C5 in continuous culture ...................... 53 3.8 Effects of the agitation speed on growth and glucoamylase secretion by S. diastaticus SD2-A8/C5 in continuous culture ...................... 55 3.9 Effects of the aeration rate on growth by S. diastaticus SD2-A8/C5 in continuous culture ................................................... 58 Vlll

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3.10 Effects of starch concentration on growth and glucoamylase secretion by S. diastaticus SD2-A8/C5 in continuous culture ...................... 60 3.11 A continuous culture of S. diastaticus SD2-A8/C5 under optimized conditions ................................................................ 62 3.12 Time course of cell growth and plasmid stability in S. cerevisiae strain MMY2 transformed by the multicopy plasmid pSTA325 in continuous culture ................................................................. 65 3.13 Cell growth, plasmid stability, and glucoamylase secretion by S. cerevisiae 1403-7A transformed by the centromeric plasmid pSTA316 in continuous culture ............................................................... 67 3.14 The stability of plasmids pSTA325 and pMS12 in S. cerevisiae SHU32o: grown in continuous culture .......................................... 71 3.15 Screening of transformed cells for amylolytic activity .................. 73 3.16 Screening of transformed cells for mutations showing increased glucoamylase expression .... ................................................... 75 lX

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TABLES Table 2.1 List of yeast strains, transformants and their relevant genotypes ....... 20 2.2 Chemostat optimal operating conditions .............................. 25 3.1 Relative growth of S. cerevisiae and S. diastaticus .................... 34 3.2 Cell growth and plasmid stability of selected S. cerevisiae strains transformed by multicopy plasmid pSTA325 ............................... 42 3.3 Cell growth, plasmid stability, and glucoamylase production of S. cerevisiae transformed by centromeric plasmid pSTA316 ........................ 43 X

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ACKNOWLEDGMENTS I would like to express my appreciation to my thesis co-advisors Professors James R. Mattoon and Gerald J. Audesirk for their guidance and enthusiastic support throughout this work. I am also very grateful to Professor Linda K. Dixon for her assistance and willingness to serve on my committee. Finally, a special thanks to Professor George Bajszar for his assistance and encouragement. Xl

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1 Introduction Starch is an important, renewable biological resource, produced by the process of photosynthesis in higher plants. This polysaccharide consists of the simple sugar glucose, molecules of which may be linked in either of two patterns: amylose or amylopectin. Amylose has a linear chain-like structure formed by a-1,4-linked a-D-glucose residues and a few a-1,6 branching points [42]. Amylopectin has a highly branched pattern i:riwhich straight chains of a-1,4-linked a-D-glucose units are interlinked by branching via a-1 ,6-glucosidic bonds. The relative amounts of amylose and amylopectin in starch depend upon the source, but the average starch preparation contains about 27% amylose and 73% amylopectin [42]. that are capable of catalyzing the hydrolysis of starch are widely produced by bacteria, fungi, plants, animals and yeasts other than brewing and baking strains. There are seven classes of amylolytic enzymes of microbial ori gin, as listed below. Their action patterns on the starch molecule are shown schematically in Figure 1.1, where the hexagons represent glucose units and the hydrolyzed linkages are indicated by disconnected, white hexagons [42]. MICROBIAL CLASSES OF AMYLOLYTIC ENZYMES 1. a-amylase. An extracellular endoenzyme that catalyzes the hydrolysis of the a-1,4-glucosidic linkages and is capable of by-passing a-1,6-linkages, producing polyand oligosaccharide chains of varying length. This enzyme

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6' l 0 8 .... t Oooo. o .. .0 p-amylase / O O O 0 u-amytase glucoarnylase 0 0 -._ 0 ... 7 putlulanase -.-a _p / \ /soamylase .._.-O f Cyclodxlrin \ .. l 0 .:;oyl"anslro: ... "'e(j (f Fig. 1.1. The starch molecule and action patterns of amylolytic enzymes 2

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produces very limited amounts of free glucose. 2. ,8-amylase. An extracellular exoenzyme that hydrolyzes alternate a-1,4glucosidic linkages starting from the non-reducing end to produce the ,Banomeric form of maltose and a high molecular-weight limit dextrin. It is incapable of bypassing a-1,6-glucosidic linkages to attack a-D-1,4 linkages on the other (reducing) side of the branch point. 3. 1-amylase I glucoamylase I amyloglucosidase. An extracellular exoact ing enzyme that splits a-1,4-, and in some cases, a-1,6-glucosidic linkages as well as some 1,3-glucosidic linkages starting from the non:..reducing ends of a-glucans to yield ,8-D-glucose. 4. Pullulanase. An extracellular debranching enzyme that hydrolyzes a-1,6linkages of pullulan and other branched oligosaccharides to form maltotriose and dextrins, respectively. 5. Isoamylase. An extracellular debranching enzyme that hydrolyzes a-1,6glucosidic linkages of amylopectin, glycogen, various branched dextrins and oligosaccharides. It has no activity on pullulan. 6. Cyclodextrin glycosyltransferase. An extracellular enzyme that pro duces a series of non-reducing cyclodextrins (rings of 6, 7, and 8 glucose units) from starch and other polysaccharides. 3

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7. a-glucosidase. An extracellular or intracellular enzyme that appears to hydrolyze short-chain a-1,4 or a-1,6-linked saccharides arising from the action of other enzymes on starch to produce glucose. The conversion of starch biomass to glucose involves two phases: (i) liquefac tion by aand ,8-amylases and (ii) saccharification of the liquefied starch by glucoamylases and debranching enzymes. 1.1 Background and Survey The growing demand for the production of ethanol (as a fuel extender) and single cell protein (as food and feed supplements) has led to an increased utilization of grain starch for their production. The use of commercial enzymes for starch degradation represents a significant cost for the fermentation industry and has led to intensive research on one-step utilization of starch by yeasts. Out of more than 500 yeast species that have been recognized, there are approximately 150 yeast strains that are capable of using starch as a carbon and energy source [7, 23, 42). However, amylolytic yeast of genera other than Saccharomyces are not suitable for the production of ethanol because they have low tolerance for alcohol and exhibit slow fermentation rates [7). S. cerevisiae (baker's or brewer's yeast) has had a long history of use in the food and beverage industry. It has a high ethanol tolerance, a fast growth rate, and is an efficient ethanol producer [6). Brewing strains have the capability of fermenting glucose, fructose, maltose, and 4

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maltotriose, while leaving larger oligosaccharides unfermented [11]. In general, the wild strains of S. cerevisiae are unable to utilize starch because they do not express amylolytic activity. The introduction of the genes encoding amylolytic enzymes into industrial strains of the S. cerevisiae would result in the synthesis and secretion of the amylolytic enzymes as well as in a direct, one-step, conversion of starch to ethanol or single-cell proteins. 1.1.1 Expression of Amylolytic Genes in S. cerevisiae Heterologous amylase genes derived from different organisms have been cloned and expressed inS. cerevisiae. These include the a-amylase genes from mice [41] and human [37] salivary glands, wheat [35, 36], Bacillus amyloliquefaciens [21], and Schwanniomyces castellii [8]; and the glucoamylase genes from Rhizopus oryzae [2], Saccharomycopsis fibuligera [46], and Saccharomyces diastaticus [9, 20, 25, 28, 43, 47]. In addition, both the a-amylase gene (from Bacillus amyloliq uefaciens) and the glucoamylase-encoding gene of S. diastaticus were previously co-expressed in S. cerevisiae [20, 40]. 1.1.2 S. diastaticus Amylolytic Yeasts The first strain of S. cerevzszae var diastaticus, in their paper referred to as S. diastaticus, was discovered by Andrews and Gilliland in 1952 [1]. According to them, S. diastaticus was able to ferment starch and dextrins. Later, other 5

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S. diastaticus strains were discovered. Most of them were originally isolated from superattenuated beer. Studies following the S. diastaticus isolation showed that its power to grow on starch was due to its ability to produce extracellular glucoamylase [11]. Superattenuation, or lowering of specific gravity, was found to be caused by dextrin hydrolysis resulting from the action of glucoamylase. 1.1.3 Origin of the STA Genes in S. diastaticus Even though S. cerevisiae lacks extracellular glucoamylase activity, a single glu coamylase gene (SGAJ) is expressed exclusively during the sporulation phase of the life cycle in both S. cerevisiae and S. diastaticus [33]. Its presence has been found in all examined S. cerevisiae strains [9, 43, 47]. InS. cerevisiae, this intracellular glucoamylase degrades internal glycogen at the time of spore formation. However, this degradation was found not to be essential for the sporulation [47]. Three additional glucoamylase genes exist in various S. diastaticus strains [33], containing one, two, or even three STA (glucoamylase) genes each. These unlinked STAl, STA2 or STA3 genes give the cells a capacity to produce and secrete glucoamylase during vegetative growth [9, 11]. All three of them have been cloned [40, 47]. Also, the nucleotide sequences of the STAl and STA2 genes have been determined [9, 25, 28]. Currently, it is accepted that starch and dextrin utilization in S. diastaticus can result from the presence of any one of the polymorphic family STA genes. Enzymatic and 6

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nucleic acid studies have shown that the secreted enzymes of S. diastaticus are closely related to the product of the SGA1 gene [28]. Thus, it has been proposed that the STA genes of S. diastaticus originated from the ancestral SGA1 gene by some genetic rearrangement [10, 45, 47]. Many S. cerevisiae strains also carry the STA10 gene whose protein product inhibits the expression of the STA1, STA2 and STA3 genes [30]. Additional study on the expression of the STA2 and SGA1 genes in presence of the STA10 gene confirmed STA2 and SGA1 repression inS. cerevisiae [28]. Genetic studies have shown that STA10 also represses the expression of the STA1, STA2 and STA3 genes in haploid hybrid strains that result from crossingS. diastaticus and S. cerevisiae [30). The molecular mechanism of STA10 inhibition of STA gene expression has not been clearly established. 1.1.4 Manipulating Yeast Genome Using Plasmid Vectors In general, there are four types of vectors used in yeast genetic engineering [13]. They are classified on the basis of the manner in which they are maintained in yeast cells. 1. The Yip (yeast integrating plasmid) vectors lack a yeast replication origin, so they must be propagated as integrated elements in a yeast chromosome, usually in a single copy per genome. 7

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2. The YRp (yeast replicating plasmid) vectors have a chromosomally derived autonomously replicating sequence ( ARS) representing a yeast chromosomal origin of replication, and are propagated as medium-copy-number plasmids. The YRp plasmids exhibit high frequencies of transformation, but transformants are very unstable (both mitotically and meiotically). During mitosis this instability is due to a strong bias of plasmids to segregate to the mother cell [13]. Incorporation of DNA segments from yeast centromeres ( CEN elements) into YRp plasmids (to generate the YCp vectors) greatly increases plasmid stability during both mitosis and meiosis. 3. The YCp (yeast centromere plasmid) vectors, as noted above, have both a replication origin ( ARS) and a centromere sequence from a yeast chromo. some. They are propagated as low-copy-number, autonomously replicating, stably segregated plasmids. 4. The YEp (yeast episomal plasmid) vectors contain a fragment of the yeast 2-pm plasmid. They are propagated as high-copy-number, autonomously replicating, irregularly segregated plasmids [13]. The behavior of the YCp and YEp vectors in transformed yeast is of special interest in this study because the pSTA316 plasmid is a YCp (centromere) type vector, while pSTA325 is oftype YEp (2-pm, multicopy). 8

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The YCp vectors have a yeast centromere sequence ( CEN4 in pSTA316) that allows them to be fairly regularly segregated during mitosis and meiosis. Possessing yeast origin of replication (ARSJ in case of pSTA316), they behave in a yeast cell as a circular miD:ichromosome [13]. Plasmids bearing a centromere sequence, however, are not as stable as natural linear yeast chromosomes. Whereas, for example, chromosome V is lost approximately once in 105 divisions, YCp vectors are typically lost once in 102 divisions [13]. The YCp plasmids are present in one to few copies per cell. The YEp vectors contain sequences from naturally occurring yeast plas mid known as the 2-J..Lm ci,rcle. These 2-J..Lm sequences allow extrachromosomal replication of the plasmid and its presence in high-copy number in yeast cells. In general, the YEp vectors are used for high-level gene expression in yeast due to their high-copy number. However, they are known to be irregularly segregated during mitosis and meiosis [13] and are gradually lost after many generations in the absence of selective pressure. 1.1.5 Selection Systems for Improved Plasmid Stability When present in the host yeast cells for the purpose of production of heterologous gene-products, recombinant plasmids have to be reasonably stable. Stability can be defined as the ability of plasmid-bearing cells to maintain the plasmid un changed during their growth, while manifesting the expected phenotypic charac-9

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teristics [4r The stability of a plasmid can be affected by the genetic characteristics of the host cells, characteristics of the plasmid itself (e.g., plasmid type, copy number, and type of promoter), physiological consequences of the expression of the foreign gene carried on the plasmid, and culture conditions such as selective medium and growth rate [4]. Plasmid instability can be caused by segregational or structural instability. The segregational instability may be caused by the loss of the entire plasmid from the cell, whereas rearrangements and losses of some of the plasmid sequences (via deletions) are responsible for the structural instability. Plasmids need to be regularly and efficiently partitioned to each daughter cell during the cell division if they are to be stably inherited in a population [4]. In general, yeast host cells harboring plasmids based on YEp high-copy vectors need to be grown in the presence of selective pressure in order to maintain the plasmids. Otherwise, these plasmids are usually very unstable and are quickly lost from the cells [34]. The most commonly used yeast selection system is based on the complementation of an auxotrophic mutation in the host by a plasmid borne wild-type gene. Any other autoselection system that would allow the transforming plas mids to be stably maintained, regardless of the medium composition, is of great interest in biotechnology. One method for increasing the stability of plasmids 10

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is the isolation of yeast host mutants showing an increased ability to maintain plasmids. One study [16] has shown that the smp1 mutation that confers a respiration-deficient phenotype, caused by lack of mitochondrial DNA, improved the stabilities of YRp plasmids, but had little or no effect on the stabilities of YEp and YCp based vectors. Further studies [17] of S. cerevisiae respiration deficient mutants (carrying smp2 and smp3 mutations) also showed increased plasmid maintenance. Another study [34] used an autoselection system based on fragile srb1-1 mutants for stable plasmid maintenance. S. cerevisiae srb1-1 mutants grow nor mally only in the presence of osmotic stabilizers in the medium. In the absence of the stabilizers, cells lyse spontaneously upon osmotic shock. Only when trans formed with the cloned SRB 1 gene (carried on the plasmid vectors), do these mutants lose their growth dependence on the presence of the osmotic stabiliz ers and their susceptibility to lysis upon osmotic shock. In this autoselection system, any low osmolarity medium becomes selective for transformants which harbor plasmids containing the SRB1 gene. In this study, srb1-1 fragile S. cere visiae mutants, transformed with plasmids based on a YEp vector, were shown to be stably maintained in both batch and continuous cultures for at least 80 generations. Moreover, this system provided conditions for the survival of only the plasmid-containing cells, since the fragile cells eventually lysed upon loss of the SRB1-containing transforming plasmid. 11

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Another approach to improve plasmid stability is to study the influence of the ploidy of the host cell on plasmid behavior. A study of effects of the host ploidy on plasmid stability and copy number [39] has shown that the stability and copy number of chimaeric plasmids (i.e., the YEp plasmids) can be increased by increasing the ploidy of the host yeast. However, the expression of the recombi-nant proteins and the productivity of the high ploidy host were not investigated in that study [39]. 1.2 Objectives of This Study In a batch fermentation mode, the yeast is exposed to a constantly changing environment. Continuous culture at steady state, on the other hand, provides relatively stable growth conditions. It eliminates the dependence of physiological state on time and is a useful tool in studying the growth kinetics of yeasts. A . major objective of this project was to determine optimal conditions for continuous stable culture of yeast capable of utilizingstarch as a carbon source while secreting amylases into the culture medium. The first goal of this study was to develop conditions for carrying out continuous culture in a small laboratory fermentor using S. diastaticus. Because this yeast carries one or more STA genes within its chromosomes, glucoamylase production was expected to be stable. The second goal was to prepare transformants of S. cerevisiae containing 12

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the STA2K gene on a multicopy plasmid and on a centromeric plasmid, and to determine their relative growth behavior in both batch and continuous cultures. The ability to maintain plasmids with starch as the only selection employed was also of particular interest of this study. Because S. cerevisiae cannot utilize starch directly, it was expected that a STA gene on a plasmid would act as an autos election system when starch was the primary carbon source. It was also hoped that continuous culture of S. cerevisiae transformed with STA plasmids would permit selection of strains bearing mutations that enhanced plasmid retention. The last goal of this project was to prepare a transformed S. cerevisiae strain which would. carry both the glucoamylase and the a-amylase genes on plasmids and to study its growth in starch medium in both batch and continuous cultures. It was considered possible that the ability of such a double transformant to hydrolyze starch almost completely would provide sufficient selective pressure to maintain plasmid-containing cells in starch medium indefinitely. Mattoon, Kim, and Laluce collected a number of S. diastaticus strains [24, 26] and compared thein for efficiency of direct starch fermentation. Strain SD2A8 exhibited the best ability to produce high levels of glucoamylase while at the same time producing high levels of alcohoL In the same study [26], the Lintner starch preparation was shown to be the most suitable substrate for routine testing of amylolytic yeasts. Also, an optimal pH of 4.2 was established, which represents an optimum balance of pH effects on glucoamylase action and cell growth. 13

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On the basis of the above studies, S. diastaticus strain SD2-A8/C5, an adenine auxotroph of SD2-A8, was selected for growth in a continuous culture to determine the optimal conditions for aerobic growth of yeast capable of secreting amylases. The optimum pH and the medium composition, including Lintner starch and succinic acid as a buffering agent were also chosen as in [26]. To meet the first objective, effects of feed rate, aeration and stirring speed on cell growth and glucoamylase secretion were studied. Conditions were optimized for aerobic continuous culture of SD2-AB/C5. As before, various amylolytic enzymes of different origins have been cloned and expressed in yeast S. cerevisiae [2, 8, 21, 41, 46]. To meet the second goal, a STA gene of Saccharomyces diastaticus, instead of a glucoamylase gene from some other fungal or bacterial source, was chosen to modify S. cerevisiae strains. Plasmids containing the STA2K gene encoding a glucoamylase of S. diastaticus, that was previously cloned by Kim et al. [19], were used to transform various laboratory strains of S. cerevisiae. The STA2I< gene was isolated from a strain of S. diastaticus that ex. hibited extremely efficient glucoamylase production [19]. The physical map of the DNA sequence of the STA2K gene exhibited differences both in its structural gene and in thepromoter region when compared to the STA genes cloned in other laboratories [9, 15, 25]. Self-replicating yeast plasmids containing sequences and the STA2K gene were constructed by Kim et al. [19]. A laboratory strain 14

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and various brewing strains transformed with these plasmids secreted glucoamy lase and produced superattenuated beers. Interestingly, these studies [19] also revealed that the glucoamylase activity by the investigated transformants was only 14-21% of that produced by S. diastaticus from which the STA2K gene had been derived. The best mitotic stability of these multicopy plasmids determined after 96 and 164 hours was 69.3 and 55.2%, respectively. In order to improve the plasmid stability and glucoamylase secretion, Bajszar et al. [3] further investigated the cloned STA2K gene. The focus of their study was on the properties and engineering of the promoter region of the STA2K gene. They demonstrated a regulated glucoamylase secretion by placing the STA2K gene under the control of either the PH05 (acid phosphatase gene) or CYCl ( iso-1-cytochrome c gene) upstream regulatory sequences. The expres sion of the secreted-STA2K glucoamylase, controlled by the engineered promoter variants, was monitored in S. cerevisiae hosts using self-replicating single-copy and multicopy yeast vectors. On high-copy-number vectors, induction of the VASPHo5-STA2K chimeric promoter by phosphate depletion resulted in a de structive overexpression of the secreted glucoamylase, which completely halted cell growth, and promoted tell decay. In contrast, U AScyc1 was shown to mediate a fine-tuned regulation both by glucose concentration and, indirectly, by starch, the substrate of the glucoamylase to produce glucose. [3] Plasmids containing the STA2K gene (placed under the CYC1 upstream 15

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regulatory sequences) appear. to be very promising. For the further investiga tion of the STA2K gene expression, a new G-STA2K expression cassette was constructed. In that construction, the original STA2K promoter was replaced by the strong constitutive GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene) promoter. Self-replicating centromeric and multicopy yeast plasmids con taining the G-STA2K cassette were constructed. They were tested, in the real ization of the second goal of this project, for their productivity and stability in various S. cerevisiae strains in both batch and continuous cultures. The transforming plasmids have two functions. The first function is to complement the uracil auxotrophy of the mutant host cells (the host cells con tained the selectable ura3 marker). However, this function was mainly used for the selection of the transformed yeast colonies, because, in general, the testing of the transformant strains was carried out in complete medium which contains uracil. This permitted evaluation of the utiljty of STA2K as a selective marker. The second function of the transforming plasmids is to provide the STA2K gene-product (glucoamylase) necessary for starch hydrolysis required to release the glucose needed for the yeast growth (when starch served as primary carbon source). A preliminary screening on the minimal starch-agar medium was used to select those strains that produced the largest halos for further investigation. A secondary testing of the strains showing the best expression of the STA2K gene (large halo producers) was performed in shake flask experiments 16

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in complete starch medium. In this test, relative growth, glucoamylase secre tion, and retention of the plasmids were investigated. Those strains exhibiting the most favorable combination of these properties were selected for studies in continuous culture. A single, fast-growing colony exhibiting enhanced glucoamylase produc tion and mitotic stability was found among the STA2K brewing yeast transformants by Kim et al. [19]. This mutant, called BY6-A6, grew much more rapidly and produced almost times as much glucoamylase as the parental trans formant. In order to determine whether spontaneous mutations which enhance plasmid stability can be selected, the ability to maintain STA was the only se lection employed in continuous culture experiments. To meet that objective, the stability of the plasmids was monitored throughout the culture period. Plasmid maintenance was assessed by determining the loss of URA3 and by the loss of halo formation. Pilot experiments using sequential shake :flask cultures instead of the ferinentor were pe_rformed as required. Kim et al. [20] described the construction of a hybrid yeast strain se creting both glucoamylaseand a-amylase into the culture medium. This strain was obtained by transforming an S. diastaticus derivative with an episomal plasmidcontaining a mouse salivary a-amylase gene. However, the disadvantage of using this transformant for industrial applications was the difficulty in further manipulation of the glucoamylase gene located on the chromosome of the recip-17

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ient S. diastaticus hybrid strain. Moreover, a relatively high instability of the a-amylase plasmid was observed. The above study [20] was extended in this work to satisfy the last objective of this project. A S. cerevisiae strain was successfully transformed with two different plasmids. Strain SHU32a, already transformed with the multicopy plasmid carrying the STA2K gene was subsequently transformed with. the pMS12 plasmid containing a mouse salivary a-amylase eDNA. The cell growth and the stability of both plasmids was monitored throughout the continuous culture period. Also, three different transformants of SHU32a (SHU32a/STA325, SHU32ajpMS12, and SHU32a/STA325 and jpMS12) were investigated in shake flask cultures, and their growth was compared. 18

PAGE 30

2 Materials and Methods 2.1 Yeast Strains and Plasmids The S. cerevisiae strains and their genotypes used in this study are presented in Table 2.1. The E. coli strains DH5a.F' and ED8654 were used to maintain and amplify the plasmids. All strains were obtained from the UCCS stock culture collection. The multicopy plasmid pSTA325 and a yeast centromeric plasmid pSTA316 containing the G-STA2K gene, which is an allelic variant of the STA2 gene ( S. diastaticus glucoamylase) equipped with a GAPDH promoter, both have a URA3 selectable marker. They were constructed by Dr. George Bajszar, and the meth ods used in their construction and their restriction maps are presented in Figures 2.1 and 2.2, respectively. A plasmid, pMS12, containing a mouse salivary a.-amylase eDNA and TRPJ selectable marker [20, 41] is shown in Figure 2.3. This plasmid was supplied by Dr. Karl Thomsen [41]. All plasmids used to transform the studied yeasts are yeast-E. coli shuttle vectors that allow propagation in E. coli for convenient manipulation and large scale preparation of their DNA. 19

PAGE 31

Yeast Strains and Relevant Plasmid Relevant Transformants Genotype Markers S. cerevisiae MMY2 MATa ura3 SHU32a MATa leu2 trpl ura3 6gal2 SHU32a MATa leu2 trpl ura3 6gal2 1403-7A MATa MAL4 mel MGL3 gal3 gal4 ura3 lys AP1-5C MATa ura3 trpl MAL4C S. diastaticus SD2-A8 MATa STA SD2-A8/C5 MATa ade STA KK1-R1 trpl lys7 STA3 Trans formants MMY2/pSTA325 MATa ura3 URA3 STA2K. MMY2/pSTA316 MATa ura3 URA3 STA2K SHU32a/pSTA325 MATa leu2 trpl ura3 6.gal2 URA3 STA2K SHU32ajpSTA316 MATa leu2 trpl ura3 6gal2 URA3 STA2K SHU32ajpMS12 MATa leu2 trpl ura3 6gal2 TRP1 AMY SHU32ajpSTA325 MATa leu2 trpl ura3 6gal2 URA3 STA2K and pMS12 TRP1 AMY SHU32ajpSTA316 MATa leu2 trpl ura3 6gal2 URA3 STA2K AP1-5CjpSTA325 MATa ura3 trpl MAL4C URA3 STA2K AP1-5CjpSTA316 MATa ura3 trpl MAL4C URA3 STA2K 1403-7AjpSTA325 MATa MAL4 mel MGL3 URA3 STA2K gal3 gal4 ura3 lys 1403-7A/pSTA316 MATa MAL4 mel MGL3 URA3 STA2K gal3 gal4 ura3 lys Table 2.1. List of yeast strains, transformants and their relevant genotypes 20

PAGE 32

7691,Scal 10365, Stu!, lislE II Nhol N

    PAGE 33

    T779;Kpnl_l 7n3,Apal Smal,1296 Sac1,2005 Xbal,2029 l lf 8amHI,2041 Smal 2047 i\f EcoRi,2059 l\" Hindlll,2071 rsan,2086 l Apal,2101 Kpnl,2107 ... :! ...... :r P(GAPDH) Ncol,622 Smal,1296 f Sacl,2005 : i Xbal,2029 /act> pST A316P(GAPDH) 10500 bps f. BamHI,2041 Smal,2047 EcoRI,2059 ': Hindll1,2071 BamH1,2853 H i ndlll,2918 \ EcoRI,4304 Sph1,4342 Fig. 2.2. Construction of yeast centromeric plasmid pSTA316 22

    PAGE 34

    6450,8amHI 8250, Sphl. .._ 8200, San.:.EcoRI,1 ,Apal,100 pMS12 "TOOOO bps 2uORI Ap Xbal,4300 Pstl,4900 .. Xbal,2350 Fig. 2.3. Restriction map of a-amylase plasmid pMS12 23

    PAGE 35

    2.2 Growth Media The yeast strains were grown in starch media containing Lintner soluble potato starch as a carbon source. Complete yeast growth media contained 1% yeast extract, 2% peptone, and either 1.5% or 2% starch. The medium for plates and for preculture also contained 0.5% glucose. Minimal media contained 0.67% yeast nitrogen base (without amino acids), nutritional supplements as required, 2% starch and glucose: 2% for plates and 0.1% for liquid medium, respectively. The starch liquid media were buffered with 0.1M of succinic acid at pH 4.2. Stock cultures were grown on complete 2% glucose medium (YPD), except for transformants that were grown on minimal media without uracil, tryptophan, or lacking both supplements for double transformants. For solid media, 2.0% agar was added. When required, the additions were as follows: uracil20 mg/1, adenine sulfate 60 mg/1, L-leucine 200 mg/1, L-lysine HCl 40 mg/1, and 1-tryptophan 10 mg/1. 2.3 Growth Conditions and Measurements Growth (dry weight) was estimated as absorbance at 570 nmx0.5312. All shake flask cultures were grown at 30C on a rotary shaker operated at 300 revolutions per minute (RPM). Each 500-ml Erlenmeyer flask containing 100 ml of liquid medium was inoculated with 1 mg (dry weight) of yeast cells per 100 ml of medium. All continuous culture fermentations were carried out in a BioFlow 24

    PAGE 36

    chemostat (New Brunswick Scientific C-30) equipped with a 750-rnl vessel which possessed a working volume of 350 ml. For each run the fermentor was inoculated with 7 mg (dry weight) of yeast cells / 350 ml of medium. The cells for inoculation of flask or chemostat cultures were previously grown in a preculture in minimal or complete medium for 24 hours. All continuous cultures of transformed yeast were carried out under the optimal operating conditions in a complete medium containing 1.5% starch. Medium for continuous culture was supplemented with of polyethyleneglycol which served as an antifoam reagent. Dilution rate (D) is defined as the ratio between the flow rate (F) of the medium (ml/hr) and the working volume of the fermentor. The optimal <:hemostat operating conditions are displayed in Table 2.2. Temperature Agitation speed pH 30C 400 RPM (revolutions per minute) -4.2 Aeration 0.4 LPM (liters per minute) Dilution Rate '(D) 0.12h-1 Table 2.2. Chemostat optimal operating conditions 25

    PAGE 37

    2.4 Plasmid DNA Extraction from E. coli For E. coli nucleic acid extraction, a 125-ml culture flask containing 25 ml of LB (Luria broth, 0.5% yeast extract, 1% tryptone, 0.5% NaCl in doubly deion ized water) containing 50 J.Lg/ml Ampicillin was used to grow the plasmid DNA containing E. coli cells. The cultures were incubated overnight in a 37C shaker operated at 300 RPM. The overnight culture was transferred into a sterile 250-ml plastic bottle and harvested in a Sorvall centrifuge with a GSA rotor operated at 5,000 RPM for 5 minutes. The supernatant fluid was discarded and the pellet was resuspended in 3 ml of STET solution (8% sucrose, 8% Triton X-100, 50 mM EDTA, 50mM TRIS-HCI, pH 8.0). The resuspended cells were then enzymat ically treated with 80 J.Ll of lysozyme solution (20 mg/ml in STET) mixed and boiled for exactly 3 minutes. Then the mixture was again centrifuged, this time for 15 minutes at 10,000 RPM in the Sorvall centrifuge (rotor GSA). The supernatant fluid was transferred into a 25-ml Corex tube (the pellet was discarded) and after 4 ml of isopropanol had been added, the mixture was allowed to stand in a -20C freezer for 20 minutes. The precipitated nucleic acids were collected in a Sorvall centrifuge (rotor SS-34) operated at 10,000 RPM for 15 minutes. The supernatant fluid was discarded and the pellet was in 3 ml of 1 x TE buffer (10 mM Trizma base, 1 mM EDTA, pH 7.5). To eliminate the RNA, the nucleic acids were treated with 5 J.Ll of RNase A solution (5 mg/ml, 26

    PAGE 38

    preboiled) and incubated for 30 minutes at 37C. Next, 3 ml of 4 M lithium chloride were added and the mixture was incubated for 30 minutes at 45C. After the mixture had been centrifuged ( Sorvall rotor SS-34), the supernatant fluid was collected and transferred to a fresh sterile Corex tube. The plasmid DNA solution was extracted with an equal volume of phenol : chloro form : isoamyl alcohol (24:24:1). The mixture was then mixed with a vortex mixer and centrifuged again in a Sorvall centrifuge (Sorvall rotor SS-34) for 5 minutes at 10,000 RPM. After the phenol layer had been discarded, the phe nol remaining in the water phase was extracted with one volume of chloroform : isoamyl alcohol (24:1 ). Again, the mixture was centrifuged (Sorvall rotor SS-34), and the water layer collected. The water layer containing the purified plasmid DNA was then mixed with 2.5 volumes of absolute ethanol and 0.1 volume of 3 M Na-acetate. The tube was then allowed to stand for 24 hours at -20C. The precipitate of plasmid DNA was collected in a centrifuge (Sorvall rotor SS-34) for 20 minutes at 10,000 RPM. The supernatant fluid was discarded, and the pellet was washed twice with 2 ml of 80% ethanol, centrifuging for 10 minutes at 10,000 RPM in a Sorvall centrifuge (rotor SS-34) after each wash. The harvested pellet of plasmid DNA was then dried under vacuum. Finally, the dry pellet was resuspended in a 200 J.Ll of 1 x TE buffer, and the plasmid DNA concentration was determined spectrophotometrically at 260 nm. 27

    PAGE 39

    2.5 Transformation of Yeast Cells The yeast cells were transformed to uracil, tryptophan, or uracil and tryptophan prototrophy using a method recently developed by Dr. Carlos Stella. In this procedure, a 500-ml Erlenmeyer flask containing 100 ml of YPD medium was in oculated with 4 mg (dry weight) of yeast cells. Minimal medium with nutritional supplements as required was used when the previously transformed cells were grown for transformation with the second plasmid. The culture was incubated at 30C and 300 RPM for 18 hours, until an optical density (A570) of 3.30 was reached. At that time, 1 ml of culture (containing 1.65 mg /dry weight/ of cells) was aseptically removed from the flask, put in a sterile 1.5-ml Eppendorf tube, and centrifuged for 5 minutes at 10,000 RPM. The supernatant fluid was removed, and the collected cells were resus pended in 250 J.Ll of the One-Step Buffer Solution, which is detailed below. Next, 18 J.Ll of single-stranded salmon sperm DNA (10 mg/ml) was added to the cell suspension and the tube contents were gently mixed. A description of the denaturation process of the salmon sperm DNA follows shortly. Aliquots of 100 J.Ll of cell suspension were placed in each of 3 sterile 1.5-ml Eppendorf tubes. Two of the tubes, containing the cells to be transformed, each received 5 J.Ll (0.2 J.Lg) of the plasmid DNA. The third tube, which did not receive the plasmid DNA, was used as a control. The tubes were incubated at 30C for 30 minutes without shaking. 28

    PAGE 40

    Next, 5 Ill of dimethyl sulfoxide (DMSO) were' added to each tube and the cell suspensions were mixed with a vortex mixer. The mixtures were again incubated, but this time for 10 minutes, at 45C without shaking. The contents of each tube were then plated on an appropriate selection medium. The number of transformant colonies was recorded after 2 to 3 days of incubation at 30C. The transformants carrying plasmids were selected on minimal medium lacking uracil for the pSTA325 and pSTA316 transformants, lacking tryptophan for the pMS12 transformants, and lacking both nutrients for the double transfor mants. All transformed strains produced clear zones of starch hydrolysis (halos) around individual colonies when plated on starch-agar medium. The halo assay involved plating of transformants producing glucoamylase or a-amylase on fresh (previously not refrigerated) plates of minimal medium containing 0.5% glucose and 2% starch, and incubation at 30Gfor 4 days followed by refrigeration at 4C for 3 days [24]. In order to photograph the results, the agar plates were exposed to iodine vapors. Immediately before each photograph was taken, the plates were inverted onto beakers containing the iodine solution (0.33%h, 0.67% KI) sitting in a 60C water bath. 29

    PAGE 41

    CONTENTS OF THE ONE-STEP BUFFER SOLUTION 0.2 M lithium acetate 40% polyethylene glycol (PEG 3350) 100 mM dithiothreitol (DTT) The ingredients were dissolved in 1 x TE buffer and the pH was adjusted to 7.5. Finally, the solution was filter-sterilized under vacuum. DENATURATION OF SALMON SPERM DNA The DNA-sodium salt from salmon testes, catalog #D-1626, was supplied by Sigma. First, the DNA was dissolved in doubly deionized water at a concentration of 10 mg/ml. Intensive mixing on a magnetic stirrer (at room temperature) was required in order to completely dissolve the DNA. Next, the DNA was sheared by passing it through an 18-gauge hypodermic needle. After that, the DNA solution was boiled for 10 minutes. Then 50-pl samples were dispensed into sterile 1.5-ml Eppendorf tubes and stored at -20C. Just before use a tube with the DNA was boiled again for 5 minutes and placed on ice until used. 30

    PAGE 42

    2.6 Glucoamylase Assay The reaction mixture for the enzyme assay contained 0.2 ml of 1.6% solution of Lintner starch, 0.3 ml of 2 M sodium acetate buffer, pH 5.0, and 0.5 ml of centrifuged culture fluid as crude enzyme solution that was diluted as required. A tube containing the reaction mixture was incubated for 20 minutes at 55C; then the starch hydrolysis was stopped by boiling the contents of the tube for 10 minutes. The tube was then cooled, and the amount of released glucose was measured with the glucose oxidase-peroxidase method using the PGO enzymes supplied by Sigma (catalog #510-6). Absorbance was measured at 450 nm with a Beckman spectrophotometer (DU. 50). A calibration curve and glucose standards (a 20-fold dilution of the Glucose Standard Solution, catalog #635-100) were used to relate the absorbance at 450 nm to the actual amount of released glucose. A standard curve was constructed using glucose concentrations ranging from 0 to 556 nmoles/ml. A reaction mixture containing the inactive culture fluid, which had previously been treated for 10 minutes in a boiling water bath, was used as a blank. One unit of enzyme is defined as the amount that liberates 1-p mol of glucose per min per 1 ml of enzyme sample under the assay conditions. 31

    PAGE 43

    2. 7 Plasmid Stability Measurements To check the stability of strains transformed with pSTA325 or pSTA316, diluted samples of cell suspensions of transformed yeast strains harboring plasmids con taining both the STA2K and URA3 genes were plated onto plates of minimal medium with or without added uracil. The plasmid stability was calculated as lOOx (the ratio of the number of colonies formed on medium without uracil to the number of colonies on medium containing uracil). Similarly, to check the stability of transformants containing plasmid pMS12, diluted cell suspensions of transformed yeast strains carrying a plasmid contain ing the TRPJ and AMY gene were plated onto plates of minimal medium with or without added tryptophan. The plasmid stability was calculated as 100 x (the ratio of the number of colonies formed on the medium without tryptophan to the number of colonies formed on medium containing tryptophan. Also, a second independent assay for plasmid-containing colonies was performed and the average of the two values was recorded. For all transformants, diluted cell suspensions were plated onto complete starch media and 100 x (the ratio of the number of halo-forming colonies to the total number of colonies) was calculated. The two assays always led to the same results, i.e., the plasmid stability percentages computed by the two assays were almost always identical. The cell cultures were diluted as needed to obtain between 200 and 300 colonies per plate. 32

    PAGE 44

    3 Experimental Results and Discussion 3.1 Shake Flask Culture Experiments 3.1.1 Growth of Yeast Strains on Starch The ability of S. cerevisiae strains SHU32o:, SHU32a, AP1-5C, MMY2, and 1403-7A to grow on soluble starch was compared to that of S. diastaticus strains SD2-A8, SD2-AB/C5, and J(J(l-RJ that are known to produce an extracellular glucoamylase. Relative cell growth and glucoarnylase production in liquid cul tures and ability to produce clear zones of starch hydrolysis (halos) on starch agar plates of investigated yeasts are shown in Table 3.1. None of the tested strains of S. cerevisiae exhibited significant utilization of starch. However, they were able to grow in the medium containing starch as a primary carbon source, increasing their biomass from 0.01 mg dry wt/ml to between 1 and 2 mg dry wt/ml. Strain AP1-5C gave the least growth (1.12 mg/ml), whereas strain MMY2 gave the most (2.24 mg/ml), which was the best growth among the tested S. cerevisiae strains. Although no glucose was added to the complete growth medium, it was found to contain 47 mg/1 glucose (0.0047%), which was derived from either the Lintner starch or from the yeast extract and peptone, or both. This small amount of contaminating glucose, together with the aminoacids and peptides in the yeast extract and peptone, provided the carbon source which supported the limited growth observed in the S. cerevisiae strains. 33

    PAGE 45

    However, these strains did not secrete any detectable glucoamylase, nor did they produce any halos on a starch-agar plates, as shown in Table 3.1. On the other hand, the S. diastaticus strains produced 5 to 10 times as much biomass, secreted significant amounts of glucoamylase, and all produced large halos. Cell Growth Glucoamylase Halo Yeast Strain mg dry wt/ml activity production U/(mg dry wtx10-2 ) ability MMY2 2.24. NJD No SHU32a 1.75 N/D No SHU32a 1.75 NfD No 1403-7A 1.74 NJD No AP1-5C 1.12 NfD No SD2-A8 8.30 11.86 Yes SD2-AB/C5 9.23 9.09 Yes KK1-R1 10.07 10.50 Yes Table 3.1. Relative growth of S. cerevzszae and S. diastaticus on complete medium containing 2% measured after 4 days of aerobic incubation in shake flask culture. (N/D =not detected). When grown in liquid complete medium containing 2% starch, S. cerevisiae strains stopped growing after approximately 48 hours, while S. diastaticus strains continued to increase biomass progressively until about 70 hours. As shown in Figure 3.1, S. cerevisiae 1403-7A was a better growing strain (with a doubling time of 180 minutes) and completed 75% of its growth during the first 24 hours. It reached the final cell concentration of 1.74 mg dry wt/ml much 34

    PAGE 46

    faster than S. cerevisiae SHU32a which grew much slower (doubling time of 210 minutes) and reached the comparable growth of 1.75 mg dry wt/ml only after about 55 hours. A brewing strain shown for comparison exhibited extremely slow growth under the investigated conditions. As displayed in Figure 3.1, all tested S. diastaticus strains showed similar growth patterns up to about 30 hours. They all had a doubling time during exponential growth of about 180 minutes, and only later did their growth patterns differentiate. Strain /(/(1-Rl was the best biomass producer, whereas strain SD2A8 gave the least cell growth during the first 96 hours. However, strain SD2-AB produced the highest levels of secreted glucoamylase among the S. diastaticus strains, as shown in Table 3.1. As previously reported by Mattoon et al. [26], SD2-AB showed not only the best ability to produce high levels of enzyme, but also produced the highest levels of alcohol among the investigated S. diastaticus strains. Although alcohol production was not measured in this study, the growth and amylolytic activity data obtained are in complete agreement with the results previously reported in [24, 26]. 35

    PAGE 47

    0") E 0') E -.c. 0') "iii "C 1/J ca .c. j 0 .... (9 12 10 8 6 4 2 0 ----SHU32o<} s. cerevislae -&-1403-?A Brewing strain --oSD2-A8 } ..........-SD2-A8/C5 S. diastaticus --
    PAGE 48

    3.1.2 Effects of STA2K Gene on Yeast Growth STA2K carried on the multicopy plasmid pSTA325 S. cerevisiae strains SHU32a, MMY2, AP1-5C, and 1403-7A were transformed to uracil prototrophy with the multicopy plasmid pSTA325. All transformant colonies produced clear halos on plates of starch medium. The transformed S. cerevisiae strains were grown in liquid, minimal or complete medium contain ing 2% starch. The minimal medium containing starch as the sole carbon source was not sufficient to support any cell growth whatsoever. Therefore, minimal medium was supplemented with 0.1% glucose, in order to initiate cell growth. Host strains were grown as controls under the same conditions except for the addition of uracil to the minimal medium. The effects of the STA2K gene on the growth of different S. cerevisiae strains are shown in Figure 3.2. When compared to other S. cerevisiae strains, MMY2 transformed by pSTA325 showed the best expression of the STA2K gene under the selective conditions. When grown in minimal medium, MMY2/STA325 produced 130% more cell. biomass than untransformed MMY2. However, only 11.6% more growth was recorded for the transformed MMY2 in complete medium. The strain 1403-7A displays a good expression of the pSTA325 plasmid under both selective and nonselective conditions. As shown in Figure 3.2, trans formed strain 1403-7AjpSTA325 grown in minimal medium gave a 73% increase in cell growth. This strain exhibited the best expression of the STA2K gene 37

    PAGE 49

    "'-=' 00 3....--------------, = 2.5 .e Cl .s 2 E Cl 'ii) 1.5 "0 Ill Ill i 1 (5 0.5 0 MMY2 1403-. SHU AP1-7A 320: 5C Minimal medium o Untransformed 13 Transformed by the pSTA325 plasmid 3,...--------------......., = 2.5 .s 2 E Cl 'ii) 1.5 "0 Ill Ill 1 (.!) 0.5 0 I I 1""" 1 I I MMY2 1403-?A SHU 320G' Complete medium AP15C Fig. 3.2. Effects of the pSTA325 multicopy plasmid on the growth of S. cerevisiae strains on starch medium (measured after 4 days of aerobic incubation in shake flask culture)

    PAGE 50

    among all multicopy plasmid transformants when grown in complete medium, giving 47% more growth than untransformed 1403-1A. Either the genetic back ground of strain 1403-7A may enhance a good expression of the STA2K gene from the multicopy plasmid, or strain 1403-7A may be less sensitive to the inhibitory effects of the overexpression of the multicopy plasmid than the other strains studied. In addition, Figure 3.2 demonstrates that the presence of STA2K gene carried on the multicopy plasmid pSTA325 did not benefit to any significant extent strains SHU32a or AP1-5C in either complete or minimal medium. The degree to which STA2K is expressed in solid medium of glucoamy lase screening plates is shown in Figure 3.3. Colonies derived from individual transformed cells of the strains 1403-1A and APJ-5C were transferred with ster ile toothpicks to minimal, selective medium containing starch. Agar plates were incubated for 6 days at 30C, then refrigerated for 3 days at 30C. To better visu alize the clear zones of starch hydrolysis around glucoamylase secreting colonies, starch containing agar plates were stained with hKI (iodine dissolved in KI). Strains 1403-1A and SHU32a were selected to relate the difference in the halo size and its intensity in starch solid medium to level of STA2K expression, measured by cell growth in flask shake culture shown in Figure 3.2. As stated before, strain 1403-7A displayed a relatively good expression of the multicopy plasmid pSTA325, whereas strain SHU32a did not. This result corresponds well to the photographs in Figure 3.3. The transfotmant of 1403-1A formed larger 39

    PAGE 51

    1403-7A/pSTA325 AP1-5C!pSTA325 Fig. 3.3 Expression of the STA2K gene by S. cerevisiae strains 1403-?A and AP1-5C, transformed by the multicopy vector pSTA325

    PAGE 52

    halos of stronger intensity than the transformant of SHU32a:. It should be pointed out that negligible glucoamylase activity was detected in supernatant fluids of any liquid culture of the S. cerevisiae transformed with the multicopy vector (including 1403-7A and SHU32a: transformants), grown in minimal as well as complete media. However, the starch must have been hy drolyzed by the extracellular glucoamylase and the glucose taken up by the cells, since the transformed cells exhibited slightly more growth than the untransformed control. Apparently, the concentrations of secreted glucoamylase were below the level of detection of the applied assay. On the other hand, Pretorius et al. (32) reported that batch cultures of S. diastaticus grown in synthetic media did not produce detectable amounts of STA mRNA, or extracellular glucoamylase. In addition, they observed that halo reflecting extracellular glucoamylase by STA-containing strains, on synthetic agar-starch plates was similar to that formed on complex agar-starch plates. Cell growth and plasmid stability of selected S. cerevisiae strains trans formed by multicopy plasmid pSTA325 are shown in Table 3.2. The yeast were grown in complete medium containing 2% starch. 41

    PAGE 53

    Yeast Cell Growth Plasmid Strain mg dry wt/ml Stability (%) M MY2 (control) 2.24 N/A MMY2/pSTA325 2.50 36.7 SHU32a (control) 1.75 N/A SHU32ajpSTA325 1.80 li.5 1403-7A (control) 1.75 N/A 1403-7AjpSTA325 2.57 29.5 Table 3.2. Cell growth and plasmid stability of selected S cerevzszae strains transformed by multi copy plasmid pSTA 325. The cell growth and plasmid sta bility were determined after 3 days of aerobic incubation. (N /A= not applicable). As shown in Table 3.2, only 17.5% of the SHU32a cells still retained the plasmid after 3 days of aerobic growth, and the presence of plasmid caused only a minor increase in growth. The MMY2 and 1403-7A strains that show some expression of the pSTA325 plasmid (measured as enhanced growth) had retention levels of 36.7% and 29.5%, respectively. Poor expression of the STA2K gene in yeast harboring the multicopy plasmid is apparently due to the high segregational instability of that plasmid. STA2K carried on the Yeast Centromeric Plasmid pSTA316 Various S. cerevisiae strains were transformed to uracil prototrophy with the centromeric plasmid pSTA316 in the procedure identical to the transformation by the multi copy plasmid pSTA325. All transformed strains produced clear zones of starch hydrolysis on starch plates. The relative halo sizes were determined 42

    PAGE 54

    on minimal agar plates containing 2% starch and 0.5% glucose after 6 days of incubation at 30C followed by 3 days of refrigeration at 4C. The results of the experiments monitoring cell growth, glucoamylase production, and mitotic stability of various S. cerevisiae strains transformed with the yeast centromeric pSTA316 plasmid are shown in Table 3.3. As shown in Table 3.3, strain MMY2 exhibited exceptionally good expression of the STA2K gene. This is reflected by its growth as well as by the production of the highest levels of secreted glucoamylase. Strain Cell Growth Plasmid Glucoamylase Relative transformed mg dry wtjml Stability activity halo with pSTA316 (%) U /(mg dry wt x 10-2 ) SIZe MMY2 11.58 89.0 17.27 large SHU32a 9.40 96.0 9.53 medium SHU32a 9.88 97.8 10.97 medium AP1-5C 4.17 86.3 2.00 small 1403-7A 10.25 89.8 13.90 large Table 3.3. Cell growth, plasmid stability, and glucoamylase production of var ious S. cerevisiae strains transformed by yeast centromeric plasmid pSTA316. Cells were cultured in shake flasks in complete medium containing 2% starch, and assayed after 4 days of aerobic incubation. 43

    PAGE 55

    Table 3.3 shows that strain 1403-7A was also very good in expressing the STA2K gene as indicated both by growth and by glucoamylase secretion. The best expres sion of the pSTA316 plasmid by MMY2 and 1403-7A strains in liquid medium correlates well with the large size of the halos produced by both strains in the starch-agar-plate assay. Both strains showed high plasmid stability of 89% after 4 days of aerobic incubation. Similarly, transformants of SHU32a and SHU32a strains both showed good cell growth, correlated well with their glucoamylase secretion, and medium sized halos on starch plates. It should be noted that strains SHU32a and SHU32 a both exhibited exceptionally good plasmid stability of 96.0 and 97.8%, respec tively. As shown in Table 3.3, strain AP1-5C exhibited the lowest level of ex pression of the pSTA316 plasmid. while showing only moderate growth in liquid culture. Although plasmid retention was also quite good (86%) in strain AP1-5C, cell growth was only 35% that of the best growing strain. This transformant generated very small halos on starch-agar plates. and secreted only a small amount of glucoamylase ( 12% of that produced by the MMY2 strain). The low levels of cell growth and glucoamylase activity of the AP1-5C transformant carrying the pSTA316 plasmid may reflect the presence of an in hibitory gene, such as the STA 10 gene described by Polaina and Wiggs [30] or INHJ found by Yamashita and Fukui [45]. The molecular mechanisms of their 44

    PAGE 56

    action and possible target sites of these inhibitory genes have not yet been defined (see (31] for review). The presumed presence of the inhibitory gene(s) in APJ-SC could also explain the absence of STA2K expression from the multicopy plasmid in this strain. A plate assay was employed, in addition to the tests in shake flask cultures, to estimate the levels of glucoamylase secretion of S. cerevisiae strains trans formed with the centromeric and multicopy plasmids. Yeast cells were grown on complete starch agar plates to allow growth of the nontransformed host colonies. Incubation of plates at 30C for 3 days was followed by a 3-day refrigeration at 4C. In order to photograph the results, the agar plates were exposed to iodine vapor. Figure 3.4 displays the pattern of expression of the STA2K gene carried on centromeric and multicopy plasmids. As shown in the figure, the glucoamylase producing cells that were transformed with the STA2K gene formed halos around their colonies, while the nontransformed parental colonies did not. It can also be seen in Figure 3.4 and Table 3.3 that halo formation, which gives a simple estimation of the level of expression of the STA2K gene on a plate, reflected the cell growth and glucoamylase activity of the yeast in liquid cultures. 45

    PAGE 57

    .;:,. O'l A controls B MMY2 c 1403-lA 0 AP1-5C A. Colonies of the non-transformed host strains; upper row MMY2, middle row 1403-lA and bottom row AP1-5C. B, C, D. upper row: colonies of the strains transformed by the centromeric plasmid pSTA316. bottom row: colonies of the strains transformed by the multicopy plasmid pSTA325. Fig. 3.4. Comparison of the levels of secreted glucoamylase production by S. cerevisiae strains carrying STA2K gene on centromeric and multicopy vectors.

    PAGE 58

    In order to assess the extent to which the type of the vector affected the expression of the 5TA2K gene, cell growth of various 5. cerevisiae strains transformed by centromeric plasmid p5TA316 was compared to that of the same strains transformed with multicopy plasmid p5TA325. Figure 3.5 displays the effects of that comparison and confirms the visual results presented in Figure 3.4. As shown in Figure 3.5, the cell growth of p5TA316 transformants (with the exception of AP 1-5C) was close to the growth obtained for the 5. diastaticus strain KK1-R1, the best biomass producer studied. Strain MMY2 showing the best p5TA 316 expression produced even more cell biomass than the 5. diastaticus strain. The above results did not hold for the p5TA325 transformants. They showed a very poor expression of the multicopy plasmid when compared to that of the centromeric vector. This difference in cell growth can be directly attributed to the levels of glucoamylase expression presented in Figure 3.4 with which they are in complete agreement. 47

    PAGE 59

    ..,.. 00 E -Cl E --.s:::. Cl a; !\t "C Vl 111 .s:::. j e (!) 12.----------------------------------------------------, 10 I 8 s f ..... -1' ,_,;1! .ill'-tf ; ,:; 4 r j'" I J .. ....... (1 !il 2 Q I I 1 1 I I I ... I '"'"I I I I I II' l 'lk'lil"f1 I MMY2 SHU32oc. AP1-5C Yeast strain 14037A I I KK1-R1 Control II 0 Untrans f o rmed 0 T r ansformed by mult i copy p l asm i d pSTA325 l:J Transformed by centromeric plasm i d pSTA316 Fig 3.5 Effects of the STA2K gene carried on centromeric and multicopy plasmids on the growth of various S. cerevisiae strains grown in complete medium containing 2% starch (measured after 4 days of aerobic incubation in shake flask culture)

    PAGE 60

    3.1.3 Effects of a-amylase Gene on Yeast Growth Strain SHU32o:, already transformed with the pSTA325 was transformed to tryptophan prototrophy with the plasmid pMS12. The time courses of cell growth for S. cerevisiae SHU32o: (untransformed), harboring both plasmids, and each of them alone are shown in Figure 3.6. The results are for a 5-day growth period in a complete medium containing 2% starch. Untransformed strain SHU32o: exhibited only a trace of growth. Limited growth was observed for the SHU32o: harboring either of the plasmids alone (SHU32o:jpSTA325 or SHU32o:jpMS12). Neither of the plasmids alone im proved the ability of SHU32o: to grow on starch. In contrast, the transformant harboring both plasmids pSTA325 and pMS12 grew at a moderate rate, and after 6 days of aerobic growth reached high cell concentration of 12.7 mg/ml, which is 121% of the growth of SHU32o:jpSTA316 grown under the same conditions. In addition, supernatant fluids from a 48-hour culture exhibited amylolytic activity of 5.9 U/(mgxl0-2 ) as measured by the standard glucoamylase assay. 49

    PAGE 61

    CJ1 0 8 7 -E 6 0 E --5 .c. .Ql Q) !: 4 u C/) cu 3 .c. 0 (5 2 1 0 0 20 40 60 Time (hours) 80 100 120 --o-Strain SHU32 ( untransformed) --.-Strain SHU32oc transformed by a plasmid pSTA325 -+-Strain SHU32t:r transformed by plasmids pST A325 and pMS 12 -o-Strain SHU32 transformed by a plasmid pMS 12 _j Fig. 3.6. Time course of growth of transformed and untransformed strain of S. cerevisiae SHU320c in a shake flask culture

    PAGE 62

    3.2 Continuous Culture of Yeast on Starch Medium In order to use continuous culture as a tool in the investigation of the starch utilization by wild and genetically engineered yeast, it was of key importance to determine the optimal conditions for yeast cultivation in the available fermentor. Because very limited research (of this type) was previously conducted using this fermentor, no data regarding, e.g., the kinetics of yeast growth, were available. 3.2.1 Optimization of Conditions for Continuous-chemostat Culture S. diastaticus strain SD2-A8/C5 was selected to determine optimal conditions for aerobic growth of yeast capable of secreting amylases in continuous culture. The following parameters were varied: dilution rate (as a direct result of the feed rate) stirring speed -aeration rate -starch concentration in feed. Yeasts were grown in complete medium containing starch as pnmary carbon source. Cell growth, glucoamylase secretion, and medium glucose concentration were determined during continuous culture. 51

    PAGE 63

    Effects of the dilution rate on growth and glucoamylase activities Figure 3. 7 shows the time courses of cell growth and glucoamylase activity of runs 1 and 2 with the dilution rates of 0.12h-1 and 0.06h-i, respectively. Cells were grown in complete medium containing 2% starch. The chemostat operating conditions for the above runs were: agitation speed 300 RPM and air flow rate 0.3 LPM. Dilution rates of 0.07h-1 and 0.09h-1 were also used in the fermentor and observations similar to the results presented were made. The data from these runs is not displayed here because other chemostat operating conditions were also varied. As shown in Figure 3. 7, at the lower dilution rate, slightly higher lev els of the steady state biomass concentrations and glucoamylase activities were recorded. However, neither .growth, nor glucoamylase production were very sen sitive to the dilution rates. in the range 0.06 to 0.12h-1 No dilution rates above 0.12h-1 were investigated. In addition, irregular behavior of the peristaltic pump (supplying the culture with the fresh medium) was observed at rates below 0.12h-1 Because of the small effects of the dilution rate on the studied properties of the yeast culture and the technical limitations of the fermentor setup, after 3 runs with low dilution rate, the 0.12h-1 rate was selected for most of the later runs in this study. 52

    PAGE 64

    CJ1 3 2.5 E o, 2 E .E Ol a; 1.5 c=-"C 1/) nl .s:::. C) 0.5 50 40 -...... >a 30 u ci nl )( E"C nl Ol 20 8 .s ::J--(5=> 10 -+-Run 1 Cell growth -Run 2 Cell growth --6Run 1 Glucoamylase -oRun 2 Glucoamylase Run 1: D = 0.12 h l Run 2: D = 0.06 h.1 -------------------I I 0 I 0 I 200 0 50 100 150 Elapsed time (hours) Fig. 3.7. Effects of the dilution rate (D) on growth and glucoamylase secretion by S. diastaticus SD2-ABIC5 in continuous culture

    PAGE 65

    Effects of stirring speed on growth and glucoamylase activities Run number 4 was selected to illustrate the effects of agitation speed on the continuous culture of S. diastaticus. A stirring speed of 300 RPM, air flow of 0.3 LPM, and an average dilution rate of 0.07h-1 was applied for 167 hours. As shown in Figure 3.8, the aeration levels resulting form the combination of these paramenters resulted in a maximal biomass concentration of 2.08 mg/ml which was attained after about 100 hours. After that time, a slow but progressive decrease in cell concentration was observed over the next 67 hours. However, no corresponding negative effect on glucoamylase activity was noted. After the 167 hour period, the agitation speed was increased to 400 RPM. With this increase, a progressive (rapid) increase in the cell concentration and a decreased production of glucoamylase were recorded. Since the stirring speed also directly affected the aeration levels of the cell culture, it is very difficult to conclude if the better growth was a direct result of the stirring speed itself or if enhanced aeration stimulated the yeast growth. As shown in Figure 3.8(B), during the period when cell concentration was decreasing in value (from 100 to 167 hours) glucoamylase productivity of the cells was continually increasing until the 167th hour. When agitation speed was increased to 400 RPM, cells started to decrease their secretion of glucoamylase progressively. Figure 3.8(B) shows that maximum level of glucoamylase of 46.2 U/(mg dry wtxl0-2 ) was obtained when cell density reached its lowest concen-54

    PAGE 66

    C;1 C;1 4 T I"\ 6 T 400 B 4T T 6o 350 :J -E -3 300 01 E -E 3 .. -.. c: 40, 2 5 250 g 2.5 01 , 01 .. 01 2 200 c: --..-Cll E 2 30 :::l 0 u 5lci .s: c: .s: .. 1.5 150 0 .. ca u 1 5 20 1-0 Cll 0 ... 01 C/1 ... 0 01 ca Qj 1 100 u 1 0 :J Qj u u 8 u 10 .a 0.5 50 0.5 C) 0 C)"""' --+ ----\-0 0 --La 0 100 200 300 0 100 200 300 Time (hours) Time (hours) [=0--Cell growth ---o-Glucose concentration ] [=o-Cell growth GlucoamylasBJ Chemostat operating conditions : dilution rate 0 07 h '1 aeration 0 3 LPM, initial agitation speed 300 RPM, agitation speed after 167 hours 400 RPM Complete growth medium contained 2% starch. A Time course of growth and glucose concentration in the culture medium. B. Time course of growth and glucoamylase secretion. Fig. 3 .8. Effects of the agitation speed on growth and glucoamylase secretion by S. diastaticus SD2-ABIC5 in continuous culture. (run 4)

    PAGE 67

    tration level of 1. 7 mg/ml. After yeast started their second phase of intensive growth ( stimulated by the agitation increase) glucoamylase secretion from yeast cells started to recede progressively. It reached a low value of 22.1 U /(mg dry wt x 10-2 ) (50 % of the maximum level) by the end of the experiment. It should be pointed out that the lowest level of glucoamylase production was accompanied by the highest cell density obtained in that run. One possible explanation of this phenomenon could be that yeast confronted with the need to use more of their energy and metabolites for the intensified growth had to lower their glucoamylase productivity. However, some glucoamylase production must occur even at the higher growth rate in order to supply glucose to the culture. Figure 3.8(A) shows that glucose available in the medium at the time of inoculation was almost completely utilized by the cells during first 24 hours. After that time glucose concentration was fluctuating until glucose content stabilized to a near steady-state. During the second phase of intensive cell growth, glucose concentration decreased sharply. Effects of aeration rate on growth and glucoamylase secretion Runs 6, 7, and 8 were investigated to illustrate the effects of aeration on the cell growth and glucoamylase production by S. diastaticus in a continuous culture. In all runs, yeast were grown in complete medium containing 1.5% starch. Also, 0.2% glucose was added to the fermentor at the time of inoculation to decrease the time required to reach a large cell population. 56

    PAGE 68

    In runs 6 and 7 stirring speed of 400 RPM and an aeration rate of 0.3 LPM were applied for 143 hours. As shown in Figure 3.9(A), the aeration levels resulting from these parameters achieved steady-state cell concentration levels of about 2.5 mg/ml in both runs. After 143 hours, the air rate was increased to 0.4 LPM in run 7. As a direct result of this change, a progressive increase in the cell concentrations was observed over the next 48 hours. With the new aeration rate, a new steady-state cell growth level of 3.7 mg/ml was obtained after 190 hours. In run 6, coincidentally, an equipment failure after 143 hours caused a decrease in the aeration level. As the air intake dropped and fluctuated around 0.1 to 0.2 LPM, decreasing cell concentrations were observed until the end of the run. As shown in Figure 3.9(B), during the period of the limited air supply glu coamylase concentrations increased significantly and at the end of the experiment reached 36 U /(mg dry wt x 10-2 ) equal to 140% of the prior steady-state activity levels. At that point cell density had reached a low value of 1. 73 mg/ml (63% of the steady-state cell concentration level). Run number 8, displayed in Figure 3.9(A), illustrates the time course of cell growth resulting from the continuous application of 400 RPM and 0.4 LPM throughout the run. The cell growth in run 8 was excellent. Steady-state growth was obtained after 96 hours and was stably maintained at 3.4 mg/ml throughout the run. Based on the high level of cell concentrations obtained in run 8 during the steady-state growth, the chemostat operating conditions applied in this run 57

    PAGE 69

    C11 00 ... .J::. Cl a; :: 't:'J. en Cl ca E 0 4 3.5 3 2.5 2 1.5 A 0.5 Ou::-----+-----t--------i 0 100 200 300 Time (hours) 4 3.5 ... .J::. Cl 3 a; :: 't:'J 2 en Cl ca E .J::. -1.5 ... :: 1 0 ... (!) 0.5 0 B 40 35 >-... ..-30 C! 25 t)O ca >< Q) ... 20 :g :: 15 E 't:'l Cl 10 g E. --5 (!) ::l 0 0 100 200 300 Time (hours) Run #6 -oRun #7 --o-Run #Bj Cell growth Glucoamylase I Run 6: initial aeration rate 0.3 LPM, aeration rate after 143 hours 0.2-0.1 LPM. Run 7: initial aeration rate 0.3 LPM, aeration rate after 143 hours 0.4 LPM Run B: aeration rate was constant 0.4 LPM for the duration of this run. A Time course of growth for runs 6, 7 and B. B. Time course of growth and glucoamylase activities for run 6. In runs 6, 7 and B yeasts were grown in complete medium containing 1.5% starch; 0.2% dextrose was added to the fermentor at the time of inoculation. Chemostat operating condition : agitation speed 400 RPM, dilution rate 0.12 h"1 Fig. 3.9. Effects of the aeration rate on growth by S. diastaticus SD2-AB/C5 in continuous culture.

    PAGE 70

    (i.e., 400 RPM and 0.4 LPM) were selected as optimal for the aerobic cultivation of genetically engineered yeasts. Effects of starch concentration on growth and glucoamylase secretion Runs 1, 3, and 5 were selected to illustrate the effects of the starch content in the medium on growth and glucoamylase secretion from 5. diastaticus. The production of glucoamylase was examined in culture fluids of S. diastaticus grown in media containing 1, 1.5, and 2% starch. As shown in Figure 3.10, the concentration of starch in the culture medium had a significant effect on the amounts of glucoamylase secreted by the yeast cells. The largest amount of glucoamylase was produced in the medium containing 2% starch. However, the starch content in the medium did not affect the steadystate levels of cell concentrations obtained in these runs. Since no limitation of the cell growth was observed at the 1.5% starch level in the medium, and at the same time relatively high levels of secreted glucoamylase were recorded, the 1.5% starch content was chosen for the studies of the transformed yeast in continuous culture. It should be noted that technical difficulties were encountered with the 2% starch medium due to physical limitations of the available equipment. Specif ically, the medium containing 2% starch caused a ring formation of solidified starch on the heating element of the chemostat. The sizes of the rings were in versely proportional to the agitation speed. Near the end of experiments (and, 59

    PAGE 71

    O"l 0 E ..._ C) E ...; > .... "C .t:. .... e (!) 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 A 0 100 Elapsed time (hours) 200 -trRun 1 2% starch content --oRun 3 1.5% starch content - > .... E -c co C) 8 E :J:::::: -:::> "B 45 40 35 30. 25 20 15 10 5 0 0 100 Elapsed time (hours) Chemostat operating conditions: agitation speed 300 RPM, aeration rate 0.3 LPM, dilution rate 0.12 h"1 Fig. 3.1 0. Effects of starch concentration in the culture medium on growth and glucoamylase secretion by S. diastaticus SD2-A8/C5 in continuous culture. 200

    PAGE 72

    especially, at high agitation speeds) pieces of the ring would detach and plug the outlet valve of the chemostat. Optimized conditions for continuous growth of amylolytic yeast The chemos tat operating parameters (used in run 8) of agitation speed: 400 RPM, air rate: 0.4 LPM, and dilution rate: 0.12h-1 proved to be an optimal combination for efficient yeast biomass production. Steady-state continuous culture data for cell concentrations, glucoamylase activities, and glucose concentra tions of S. diastaticus grown under these conditions are displayed in Figure 3.11. The above conditions allowed for a good cell growth that was accompanied by a satisfactory level of stable glucoamylase secretion of 30 U /(mg dry wt x 10-2). In all runs, from 6 to 13 (with the exception of run 9), 0.2% glucose was added directly to the fermentor at the time of inoculation. The addition of that small amount of glucose at the beginning of a run allowed for a rapid initial biomass accumulation. This, in turn, permitted the secretion of significant amounts of glucoamylase early in the fermentation process. As shown in Figure 3.11 (A), gl ucoamy lase accumulation in the medi urn rose proportionally to the increasing cell concentrations until its activity reached a stable level of 30 U /(mg dry wt x 10-2 ) during the steady-state growth phase. These observations are consistent with those described by Pretorius et al. [32]. They reported that the synthesis of extracellular glucoamylase in hap loid yeast strains is continuous throughout the growth phase, and follows a typical 61

    PAGE 73

    Ol A B 3.5 T 0.. 140 3.5 _J 2500 3 -t-I r5 3 1 500 30 c: 2.5 ..... 0 0 2.5 ;:; t 25 400 -.._ -.._ -3: 23: 2 c: 0 3: >-0 3: Q)E 3 u _J 300 0, ='0 1.5 ='0 1.5 u.. Q) Cl 15 g '0 Q) Cl u E u E Q) Cl 200 :g (!) E 1 u 10 --:::1 ::::> 100 B 0.5 u 15 0.50 ---------1------+-----l 0 oc:r-t-o 0 100 200 300 0 100 200 300 Time (hours) Time (hours) growth -1:r-Glucoamylase] Ecell growth -oGlucose concentrationj Chemostat operating conditions: agitation speed 400 RPM, aeration rate 0.4 LPM, dilution rate 0.12 h"1 A complete growth medium contained 1.5% starch, 0.2% dextrose was added to the fermentor at the time of inoculation. Fig. 3.11. A continuous culture of S. diastaticus SD2-ABIC5 under optimized conditions. (run 8)

    PAGE 74

    growth-associated production pattern. They also found that the ratio of secreted to total glucoamylase increases during the exponential growth phase, reaching a maximum of 50% of the total glucoamylase synthesized during the early station ary phase in batch culture. As shown in Figure 3.11(B), during the first 24 hours of the 8th run, the yeast utilized 90% of glucose that was present in the medium at the time of inoculation. Moreover, rapidly increasing glucoamylase activity, starting after 24 hours, resulted in an accumulation of glucose in the medium that was recorded after 48 hours. Finally, after 72 hours of the continuous process (at the beginning of the steady-state growth) the glucose content reached a low level of about 30 mg/1. Starting from that point and until the end of the experiment, the glucose content in the medium oscillated around 50 mg/1. Steady-state cell growth of 3.5 mg/ml, a level that was stably maintained for about 100 hours, was presumably very close to the maximum level of biomass production that could be supported by the growth medium containing 1.5% starch at the applied dilution rate. 63

    PAGE 75

    3.2.2 Behavior of Transformants in Continuous Culture Study of the stability of the multicopy plasmid The S. cerevisiae strain MMY2 transformed by pSTA325 was grown in contin uous culture in complete medium containing 1.5% starch. The cell growth and plasmid stability of S. cerevisiae strain MMY2 transformed by the multicopy plasmid pSTA325 in a continuous culture of run 9 are displayed in Figure 3.12. Starch was the only selection employed in this experiment. Under the applied conditions, this strain showed a weak ability to maintain the plasmid. In an agreement with the shake flask cultures, the chemostat culture experiment showed that the multicopy plasmid pSTA325 was quickly lost from the cells, and after 117 hours, only about 7% of cells still contained the plasmid. However, the rate of plasmid loss decreased after about 65 hours suggesting that some selection might be occurring. Glucose present in the medium at the time of inoculation ( 4 7 mg/1) was no longer detectable after 48 hours, i.e., it was less than 10 mg/l. However, very low concentrations of glucose ( 13 mg/l) were detected in the culture fluids after 68 hours. Although no glucoamylase activity was detected in the cell-free extracts throughout the whole run 9, the starch must have been hydrolyzed by some small amounts of the extracellular glucoamylase. Its presence apparently was not detected because it was below the sensitivity threshold of the glucoamylase assay. Glucose resulting from starch hydrolysis by a trace amount of glucoamy-64

    PAGE 76

    Fig 3.12 Time course of cell growth and plasmid stability in S. cerevisiae strain MMY2 transformed by the multicopy plasmid pSTA325 in continuous culture

    PAGE 77

    lase was taken up by the cells and contributed to a limited cell growth. These observations are consistent with the ones made during shake flask experiments in which the transformed MMY2 strain grew more than the untransformed host. Expression of the centromeric plasmid in continuous culture The strain studied was 5. cerevisiae 14 03-7 A harboring the centromeric plasmid p5TA316. Continuous studies were performed using complete growth medium containing 1.5% starch as the primary carbon source. The culture was inocu lated with 35 mg (dry weight) of yeast cells/350 ml medium, and 0.2% glucose was added directly to the fermentor at the time of inoculation. The amount of preculture used for inoculation was 5 times greater than that used in other exper iments. This was done in order to speed up the process of reaching steady-state growth. The cell growth, glucoamylase secretion, and plasmid stability of 5. cere visiae strain 1403-7A transformed by yeast centromeric plasmid p5TA316 in a continuous culture (run 13) are presented in Figure 3.13. In this case, plasmid loss is very limited, and both growth and glucoamylase production are substan tial. A transformant of the investigated strain was shown to produce significant quantities of biomass, reaching the steady-state cell concentration of 2 mg/ml, which is 170% of that obtained for the multicopy transformant, grown under similar conditions. Again starch was the only selection employed in the experiment and proved 66

    PAGE 78

    0) --1 2 A 1.8 'E 1.6 --Cl E 1.4 1.2. 2'." 1 0.8 0 0.6 0.4 0.2 0 -t-------j--0 50 100 Elapsed time (hours) -trGrowth --oPlasmid stability '----------'-___J 100 90 80 --c: 70 ..... Q) 60 .e: 50 ::6 ro 40 iii "'0 30 .E Ill ro 20 a: 10 0 150 2.5 >2 .... .-s; 0 'fl ci IV ><1.5 Q) .... :a ;: ">2'." E -c IV Cl 8 E ::I ---::::> (!)-0.5 B 0 50 100 Elapsed time (hours) -o--amylase ......,._ J 2500 / 700 600 500 400 300 200 100 0 150 Fig. 3.13 Cell growth, plasmid stability, and glucoamylase secretion by S. cerevisiae 1403-7A transformed by the centromeric plasmid pSTA316 in continuous culture. ::J' --Ol g c: 0 .. -c: Q) 0 c: 8 Q) Ill 0 0 ::I {5

    PAGE 79

    to be sufficient to maintain reasonable stability of the centromeric plasmid. After 150 hours of the continuous culture, about 75% of the cells were still maintaining the plasmid. Figure 3.13(B) presents the time course of glucose concentration changes in the culture fluids of run 13. It was observed that the glucose available at the time of inoculation (2.5 g/1) decreased rapidly and reached 70 mg/1 after 24 hours. Higher glucose uptake by yeast during the first 24 hours in run 13, as compared to glucose utilized during the same time period in run 8, is a direct result of the much larger inoculum used in run 13. As displayed in Figure 3.13(B), even less glucose (21 mg/1) was detected after 48 hours, probably because it was utilized to support a rapid increase in biomass. Finally, after 72 hours the glucose concentration in the culture medium began to stabilize. Starting from that time, for the next 70 hours of run 13 (i.e., during the steady-state growth) the glucose content oscillated at a fairly stable level of 70 mg/l. The relatively low concentration of glucose in the culture medium indicates a rapid starch hydrolysis and utilization. The glucose level at steady-state growth in run 13 is fairly close to the concentration of glucose obtained in run 8 (50 mg/1) during the steady-state growth of S. diastaticus under the same growth conditions. As shown in Figure 3.13, the increase in glucoamylase activity was associated with cell growth and reached a maximum level of 2.2 U/(mg dry wtx10-2 ) 68

    PAGE 80

    at 120 hours. The glucoarriylase activities exhibited at the steady-state levels by the 1403-7A transformant, grown in continuous culture, were approximately 14 times less than that of the S. diastaticus SD2-A8/C5 cultivated under the same growth conditions. Apparently S. diastaticus produced far more enzyme than was needed. However, that difference in glucoamylase production was not observed in the flask culture experiments; where supernatant fluids from 1403-7AjpSTA316 cultures exhibited even higher glucoamylase activities than the SD2-AB/C5. Previous studies have shown that whereas glucoamylase production and STA2 transcription of S. diastaticus strains were found to be repressed by glu cose to a relatively small extent, the degree of repression was dependent upon the strain [32]. S. diastaticus strains can synthesize and secrete significant amounts of glucoamylase in complex media even with glucose as the sole carbon source. However, glucose brings about carbon catabolite repression of many other yeast enzymes involved in sugar catabolism, (see e.g., [31), for a review). Also, enzymes involved in the metabolism of starch were found to be subject to catabolite re pression in other yeast genera [8]. Possibly the low activities of glucoamylase obtained in culture supernatant fluids from the 1403-7AjpSTA316 transformant, grown in continuous culture, were due to STAf!< being a subject to carbon catabolite repression. As for the high activities of secreted glucoamylase present in the culture fluids from shake flask cultures of that transformant, one explanation could be that glucoamylase 69

    PAGE 81

    activities were measured at the end of the experiments, when glucose could no longer be detected in the culture medium. The glucoamylase activities in the culture fluids from shake flask cultures at earlier stages were not investigated. Studies of the double transformant Plasmid pMS12, carried together with the plasmid pSTA325, significantly im proved the ability of S. cerevisiae SHU32a to grow on starch when investigated in shake flask cultures. The behavior of that double transformant was further studied in continuous culture. Again starch was the only selection employed. Similar to run 13, the culture was inoculated with 35 mg of yeast cells/350 ml medium, and 0.2% glucose was directly added to the fermentor at the time of inoculation. Two continuous experiments were performed (runs 11 and 12) and data including cell growth, plasmid stability, amylolytic activities, and glucose content in the culture fluids were collected. Results that represent the averages of these two experiments are displayed in Figure 3.14. Strain SHU32a transformed by both pSTA325 and pMS12 plasmids ex hibited very poor growth under the investigated conditions. Initial growth was supported by 0.2% glucose added directly to the reaction vessel and allowed to obtain a cell density of 0.36 mg/ml during the first 24 hours. Yeast cell density continued to increase for the next 24 hours and reached the maximum level of 0.57 mg/ml after 48 hours. That level was no longer maintained during the rest of the experiment. Because this additional growth (from 0.36 to 0.57 mg/ml) 70

    PAGE 82

    -J ,....... 90 80 70 60 :ceo-1i) 50 "C -Q) ,!;40 co a.. 30 20 10 0 T I I I I =-=---+ 2 1.8 1.6 1 .4 I ......,.... percentage of cells containing pSTA325 1.2 E I plasmid 1 _._ percentage of cells "C containing pMS12 0 8 plasmid 0 6 percentage of cells containing both 0 4 1 plasmids (pSTA325 and pMS12) -o-growth 0.2 0 0 20 40 60 80 100 120 Elapsed time (hours) Fig. 3.14. The stability of plasmids pSTA325 and pMS12 inS. cerevisiae SHU32 grown in continuous culture.

    PAGE 83

    could no longer be attributed to the externally added glucose, there had to be some other reason. No amylolytic activity was detected in any of the samples taken through out the duration of the two runs. There could have been some trace amounts of amylolytic enzymes secreted into the culture medium that were not detectable (during the second 24 hours). However, because of the rapid loss of the plasmids by the cells, amylolytic activity never reached detectable levels. As displayed in Figure 3.14, SHU32a: cells carrying both plasmids (pSTA325 and pMS12) presented with uracil and tryptophan in the complete growth medium lost the plasmids quickly. Although both plasmids were segregationally unsta ble, plasmid pMS12 was still carried by about 40% of the cells after 120 hours, whereas only about 5% of the cells carried the pSTA325 plasmid. To determine the ability of the transformed yeast to produce amylase and glucoamylase, diluted samples of the culture medium (collected between 48 and 72 hours) were plated onto complete and selective starch-agar plates. The plates, when exposed to iodine vapors, showed clear zones of starch hydrolysis, as displayed in Figure 3.15. Although expressing amylolytic activity on solid medium, yeast bearing both plasmids pSTA325 and pMS12 did not produce any detectable levels of arnylolytic enzymes while in the chemostat. 72

    PAGE 84

    > 0 ro 0 >-0 >E ro 0 '+-(/) Q) 0 TI Q) E L--0 '+(/) c ro '...... '+-0 0) c c Q) Q) L-0 (/) (") 0) LL

    PAGE 85

    Screening of transformed yeasts for spontaneous mutations Ability to maintain STA was the only selection employed in all experiments car ried out in the chemostat and in most of the shake flask cultures. Diluted samples from the cultures of the transformed yeast were periodically plated onto starch agar selective media for the assessment of plasmid stability. To determine whether spontaneous mutations enhancing plasmid stability or increased glucoamylase secretion occurred, these plates were carefully screened for any remarkable features, such as an exceptionally large size of a yeast colony or the halo surrounding it. Figure 3.16 shows one such a colony among pSTA325 transformants of S. cerevisiae strain SHU32a. This colony exhibits a significantly larger zone of starch hydrolysis than the rest of the transformed colonies. It is a potential mutant having enhanced expression or secretion of amylolytic enzymes. A second distinct colony was found in a plated sample of the chemostat culture of a 1403-7A/pSTA316 transformant. This colony differed from the rest by its unusually large size. Apparently, mutations of this type are relatively rare. On more than 200 plates screened (each having between 200 to 300 colonies) only two colonies manifesting clear size differences were detected. Although such spontaneous mutations may be helpful for the expression and production of glu coamylase, it remains to be seen whether these mutants can yield higher levels of secreted enzyme. 74

    PAGE 86

    -1 .:.;1 Fig 3.16 Screening of transformed cells for mutations showing increased glucoamylase expression

    PAGE 87

    4 Conclusions Shake flask experiments The cell growth, mitotic stability of the plasmids, and glucoamylase secre tion parameters were significantly higher in transformants harboring the yeast centromeric plasmid pSTA316 than in transformants carrying the multicopy pSTA325 vector. Plasmid pMS12, when carried together with the plasmid pSTA325, signif icantly improved the ability of S. cerevisiae SHU32a to grow on starch, whereas plasmids pSTA325 and pMS12 alone, did not. Starch alone is not sufficient as a selective pressure for the maintenance of multicopy plasmids, encoding amylolytic enzymes, (pSTA325 and pMS12). Continuous culture experiments S. diastaticus Parameters were optimized for maximum cell growth of S. diastaticus under aerobic conditions in a continuous culture. Changing dilution rate did not show any significant effects on glucoamy lase secretion; however, lower dilution rates resulted in slightly higher cell densities obtained at steady-state growth. 76

    PAGE 88

    Aeration levels directly affected both cell growth and glucoamylase secretion by the cells. Whereas low aeration levels were limiting for cell growth, they also stimulated the cells to secrete increased amounts of glucoamylase. Glucoamylase secretion by S. diastaticus increased with increase in starch concentration even though growth was not affected significantly. The chemostat operating parameters of agitation rate: 400 RPM, aera tion rate: 0.4 LPM, and dilution rate: 0.12h-1 proved to be an optimal combination for efficient yeast biomass production by S. diastaticus. Transformants While in the chemostat, the strain carrying the STA2K gene on the mul ticopy plasmid pSTA32S exhibited relatively poor growth, no detectable secreted glucoamylase activity, and lost plasmid at a high rate. In contrast, the strain carrying the centromeric plasmid pSTA316 exhibited good expression of the STA2K gene and maintenance of the plasmid, which allowed for moderate secretion of glucoamylase, permitting relatively good growth of the transformant. Due to the high instability of the plasmids which were quickly lost from the host cells, the continuous culture of the SHU32o: transformed by two plasmids pSTA325 and pMS12 did not produce the expected results. 77

    PAGE 89

    Two mutants with enhanced glucoamylase secretion and/or multicopy plas mid retention were isolated. 78

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