Chapter 24

Chapter 24 Genetic Engineering of Industrial Strains of Saccharomyces cerevisiae Sylvie Le Borgne Abstract Genetic engineering has been successfully applied to Saccharomyces cerevisiae laboratory strains for different purposes: extension of substrate range, improvement of productivity and yield, elimination of by-products, improvement of process performance and cellular properties, and extension of product range. The potential of genetically engineered yeasts for the massive production of biofuels as bioethanol and other nonfuel products from renewable resources as lignocellulosic biomass hydrolysates has been recognized. For such applications, robust industrial strains of S. cerevisiae have to be used. Here, some relevant genetic and genomic characteristics of industrial strains are discussed in relation to the problematic of the genetic engineering of such strains. General molecular tools applicable to the manipulation of S. cerevisiae industrial strains are presented and examples of genetically engineered industrial strains developed for the production of bioethanol from lignocellulosic biomass are given. Key words: Saccharomyces cerevisiae, Industrial strain, Adaptation, Ethanol production, Pentose fermentation, Chromosomal integration, Lignocellulosic biomass

1. Introduction The yeast Saccharomyces cerevisiae has long been used in the food industry in baking, brewing, and winemaking as well as in industrial fermentations for the production of bioethanol from sugar and starch feedstocks (1, 2). Branduardi et al. (3) have reviewed the potential of metabolically engineered yeasts for industrial applications, including the production of biofuels and nonfuel petroleum-derived products from renewable resources as an alternative to the use of oil (Fig. 1). Nonfuel products include glycerol, pyruvate, and organic acids that are substances directly utilized in the pharmaceutical or chemical industry or used as building blocks

Argelia Lorence (ed.), Recombinant Gene Expression: Reviews and Protocols, Third Edition, Methods in Molecular Biology, vol. 824, DOI 10.1007/978-1-61779-433-9_24, © Springer Science+Business Media, LLC 2012

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Fig. 1. Traditional, modern, and emerging industrial applications of the yeast S. cerevisiae. Adapted from ref. 1.

for further chemical or enzymatic syntheses (4, 5). An example of biofuel is the production of bioethanol from sugars derived from biomass hydrolysates (6). S. cerevisiae is highly tolerant to low pH values and high sugar and ethanol concentrations lowering the risk of contamination in industrial fermentations; it is also able to grow anaerobically and is quite resistant to high osmotic pressure, oxidative stress, and inhibitors present in biomass hydrolysates (7, 8). There is a vast knowledge concerning its genetics, biochemistry, physiology, and fermentation technologies. This yeast has been safely used since ancient times in winemaking, brewing, and baking. It is classified as generally regarded as safe (GRAS) by the US Food and Drug Administration (FDA) and does not produce toxins or oncogenic and viral DNA. These features confer to S. cerevisiae a general robustness and potential applicability for industrial applications. In contrast to classical methods of genetic strain improvement, such as selection, mutagenesis, and mating, genetic engineering consists in the targeted manipulation of the cell’s genetic information to improve the production of a metabolite naturally formed by an organism in a low concentration or provide the organism with the ability to utilize atypical substrates allowing to extend the spectrum of usable industrial media, in particular renewable substrates as lignocellulosic biomass for example, or form metabolites not naturally produced. The genetic engineering of S. cerevisiae has mainly been performed with well-known laboratory strains. For industrial applications, the use of S. cerevisiae strains with an industrial background is necessary since industrial strains have adapted

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to stress conditions found in industrial processes. However, several interesting technical challenges arise concerning the genetic engineering of industrial strains in order to obtain yeast strains useful for commercial applications. This chapter addresses some general issues and problematic about the genetic engineering of industrial strains of S. cerevisiae for the production of bulk chemicals as biofuel and nonfuel products.

2. Importance of Using Industrial Strains of S. cerevisiae

Hahn-Hägerdal et al. (6) have pointed out the importance of using industrial yeast strains for the production of bioethanol from biomass residues. In general, practically all industrial biotechnological processes expose cells to simultaneous or sequential combinations of stressful conditions. So, desirable phenotype for industrial organisms is resistance to multiple stresses or, more generally, environmental robustness in addition to adequate yield and productivity. Industrial S. cerevisiae strains are highly specialized organisms, which have adapted to different stress conditions as those found in industrial fermentations. This process known as “domestication” involves genetic, molecular, and physiological mechanisms of adaptation and has been extensively reviewed for wine and brewery yeasts (9, 10). The ability of such strains to adapt to varying environmental conditions and physiological challenges governs their usefulness and applicability. In general, it has been shown that commercial yeasts are more tolerant to stress conditions than laboratory strains. Concerning distillers’ yeasts used for the production of fuel ethanol, Silva-Filho et al. (11) have demonstrated, by PCR fingerprinting of yeast samples from several Brazilian distilleries, that indigenous strains are more adapted to the industrial process than commercial yeasts. In fact, these indigenous yeasts tend to become dominant in the fermentative process even if commercial baker’s yeasts were used as starter cultures in a proportion of 500 g of indigenous yeast in 1 ton of commercial yeast (12). During industrial biomass production, conditioning, propagation, and fermentation, yeast cells are subject to a number of stresses as illustrated in Fig. 2. Minimizing fresh water usage and recirculation of process water streams leads to more concentrated solutions with increased ionic strength. The high sugar content in fermentation media also contributes to hyperosmotic stress. Yeast cell recycling and washing in acid solutions also impose a selective pressure on cells. It has been shown that strains isolated from an industrial winery showed higher values for characters typically subjected to selective pressure, such as maximum capability to produce ethanol, fermentation rate, and SO 2 resistance (13). So, continual selective

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Fig. 2. Environmental stress and transcriptional response of yeast during industrial bioethanol fermentation. Adapted from ref. 14. The black arrows indicate upregulation, i.e., transcriptional activation (up arrow ) or downregulation, i.e., transcriptional repression (down arrow ).

pressure imposed on cells in industrial environments has led to the adaptation and selection of strains with higher stress tolerance and improved fermentative behavior (12). The molecular and physiological response of an organism to changes in the environment is referred to as “stress response.” The two major stress response pathways in S. cerevisiae are the heatshock response and the general stress response activated by a number of environmental stresses, including oxidative, pH, heat, osmotic stresses, and nitrogen starvation (10). The general stress response is believed to be an evolutionary adaptation allowing yeast to respond to adverse environmental conditions in a nonspecific manner. Among the genes activated by stress in S. cerevisiae are the HSP genes, encoding heat-shock proteins most of which act as chaperones (9). The molecular basis of the technological properties of industrial yeast strains is still largely unknown. One possibility is that the adaptation of these strains is dependent on specific expression profiles of their genomes. The comparison of gene expression in industrial and nonindustrial strains could lead to the identification of genes involved in the adaptation to industrial environments. The transcriptional responses of S. cerevisiae during bioethanol industrial fermentation was evaluated by DNA microarray analysis showing that strong transcriptional changes occurred during both continuous and fed-batch fermentation processes (Fig. 2) (14). Repression of glycosylation was observed and upregulation of genes involved in the unfolded protein response, reserve metabolism, and glucose-repressible genes was

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thought to be a consequence of protein glycosylation deficiency. On the other hand, many of the overexpressed genes in a sherry flor yeast strain, compared with a laboratory strain, were found to be located within amplified regions in the genome, suggesting that changes in gene expression were due to an increase in DNA copy number and not due to differences in regulation (9).

3. Genomic Features of Industrial Strains of S. cerevisiae

The genetics of industrial Saccharomyces strains are more complex than that of laboratory reference or academic strains (1). Industrial strains are mainly diploid, aneuploid, or even polyploid. Most of the studies on the characteristics of the genomes of industrial strains have been performed with strains related to the production of alcoholic beverages. This knowledge has been recently reviewed (15). Codon et al. (16) measured the DNA content and ploidy of 17 industrial Saccharomyces strains, including bakers’ (11 strains), wine (2 strains), brewers’ (2 strains), and distillers’ (2 strains) yeasts and compared them to two well-known laboratory strains. All industrial strains had variable DNA contents ranging from 1.3 to 3n (n being the DNA content of an haploid strain). The nonentire ploidy numbers indicated partial amplifications of certain chromosomal regions and the presence of extra sets of chromosomes. Electrophoretic chromosomal patterns indicated strong polymorphisms between strains. However, all industrial strains possessed a high degree of DNA homology with laboratory yeasts. Hybridization studies indicated that all tested genes were located on the same chromosomes both in laboratory and industrial strains indicating that intrachromosomal arrangements had taken place in industrial strains. The SUC gene family includes six loci SUC1, 2, 3, 4, 5, and 7. These genes encode the invertase enzyme that splits sucrose, the major carbon source in molasses and sugar cane juice, into glucose and fructose. All of these genes, except SUC2, have telomeric location (near chromosome ends), highly homologous sequences, and are dispersed on different chromosomes. The different industrial yeasts tested presented multiple copies of different SUC genes in their genomes while laboratory strains only carried the SUC2 gene. This suggests that the amplification of SUC genes could be an adaptive mechanism conferring better fitness for fermentation on sucrose juices and molasses. Infante et al. (17) have used the DNA microarray technology to compare the 16 chromosomes of two yeasts strains isolated from sherry wine aging fermentation that differ on their ethanol and acetaldehyde resistance. Gross chromosomal rearrangements were responsible for the amplification of 116 genomic regions that

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Fig. 3. Genomic rearrangements in the chromosomes of a sherry wine yeast. Solid bars indicate amplified regions. Roman numbers indicate the chromosome number. The location of genes conferring specific traits is indicated. Adapted from ref. 17.

comprise 38% of their genomes. For one strain, the amplified regions contained genes that were clearly related to specific characteristics as biofilm formation, sulfite and copper resistance, ergosterol biosynthesis, and assimilation of glycerol, a major carbon source in sherry wine (Fig. 3). For the other strain, the amplified regions were related to “unknown biological processes.”

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Concerning yeasts used to produce fuel ethanol, Lucena et al. (18) have shown that yeasts cells adapt for survival through chromosome rearrangements that can be produced in laboratory media under simulated industrial conditions. Such rearrangements are produced only after 435 generations and are specific to parental genetic backgrounds. The comparison between karyotypes of original and evolved strains showed that the observed chromosome variations originated from the parental strains and not from contaminant yeasts. Microarray karyotyping and pulse field gel electrophoresis studies have shown that industrial strains of S. cerevisiae used in the production of fuel ethanol have amplifications of telomeric genes for the biosynthesis of vitamins B6 and B1 (19). These amplifications allow them to grow better in high sugar environments, where the demand of these vitamins is high and their bioavailability low. Thiamine pyrophosphate is the cofactor of the enzyme pyruvate decarboxylase. Such strains of S. cerevisiae have been selected during the industrial production of fuel ethanol and rapidly replace commercial baker’s yeast used as “starter” strains in a matter of weeks. They ferment cane juice or diluted molasses containing up to 200 g/L of total sugar, and produce high ethanol concentrations up to 12% (v/v) with 90–92% of the maximum theoretical yield. The fermentation lasts 6–10 h, allowing two to three fermentations per day. At the end of each fermentation, the cells are centrifuged, washed in dilute sulfuric acid, and recycled back for the next fermentation during 6–9 months. The fuel strains appear to cluster together and be more similar to baker’s yeast than to brewing or wine strains. Interestingly, none of the fuel strains showed amplification of the SUC2 gene, indicating that invertase activity is not limiting sucrose fermentation during industrial ethanol production by these yeasts. These authors propose that such a genetic adaptation can also be useful in the development and engineering of yeast strains for the efficient utilization of biomass-derived sugars. The genome of a haploid derivative of the PE-2 wild isolate currently used to produce bioethanol in 30% of the Brazilian distilleries was sequenced and analyzed (20). This strain possessed a high degree of DNA homology with other sequenced laboratory yeasts, but presented extensive structural differences at the periphery of its chromosomes. These regions typically contain genes that participate in alternative carbon source and vitamin metabolisms, ion and amino acid transport, flocculation, and other processes not essential for viability but related to ecological niche adaptation. Here, again, genes involved in vitamin B6 metabolism were amplified. According to theses authors, the development of genetically engineered yeast strains for industrial applications should be based on strains naturally adapted to industrial conditions; any other strain would be rapidly washed out of the process.

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From these studies, it is clear that industrial strains contain traits that enable them to perform well under a particular set of conditions.

4. Genetically Engineered Industrial Yeasts for the Production of Chemicals and Biofuels 4.1. Molecular Tools for the Genetic Engineering of Industrial Strains

Classical genetic techniques have been successfully used for years to improve the performance of production organisms. Such techniques are not site directed. The ability to perform directed genetic changes by genetic engineering techniques allows to add or remove specific features from strains allowing to design new cell factories or improve the existing ones (21). Laboratory strains of S. cerevisiae usually exist as stable haploids, exhibit good mating ability, readily take up exogenous DNA, and contain convenient selectable (auxotrophic) markers. Industrial strains lack many of these properties (1, 22). Such strains are typically prototrophic, diploid, and even polyploid, often heterozygous and homothallic. These characteristics restrict the use of traditional selection systems based on the complementation of recessive auxotrophic markers generally used in yeast genetic engineering. The use of classical genetic techniques is also limited because these strains tend to sporulate poorly, switch their mating type (mediated by the HO gene), and produce few viable spores. In haploid laboratory strains, single genes can be disrupted and the resulting mutants easily selected and studied. This is difficult to achieve in diploid or polyploid and heterozygous strains since multiple copies of a same gene are present and all the copies present on different chromosomes have to be inactivated. There are several ways of amplifying genes introduced by transformation in order to increase the amount of gene product. One possibility is to insert the gene of interest in a multicopy plasmid vector. Most plasmids are unstable and lost under nonselective conditions. Chromosomal integration of genetic material is a method to avoid the inherent instability of most plasmid vectors. Genomic conserved regions as ribosomal DNA (rDNA) sequences can be used as target for chromosomal integration of genes in industrial yeast strains. A yeast integration plasmid, pIARL28, containing an rDNA sequence as homologous recombination site and the bacterial kanamycin gene (kan from Tn903) that confers resistance to geneticin for selection of the integrants has been described (23). The rDNA sequence of this plasmid exists at about 140 copies in a yeast cell; integration at the rDNA genes occurs, therefore, very efficiently. The undesirable geneticin resistance sequence can be excised from the transformants by repetitive culture under nonselective conditions. So this plasmid is a useful vector for gene transfer into industrial Saccharomyces strains.

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In laboratory strains, plasmids containing auxotrophic markers can be introduced and selected (24). To avoid the use of antibiotic resistance markers, the successful generation of auxotrophic mutants in industrial diploid strains of S. cerevisiae has been described (25). His−, Met−, Lys−, Trp−, Leu−, Arg−, and Ura− auxotrophic mutants of five sake strains were obtained after UV mutagenesis and screening by conventional replica plating on minimal and rich media. HIS3 was used as a selectable marker for the insertion of a yeast overexpression promoter upstream of the gene encoding the alcohol acetyltransferase by one-step gene replacement in a his3 mutant, allowing the production of a larger amount of isoamyl acetate, a banana-like flavor in Sake. Due to obstacles concerning the acceptance of antibiotic selection markers on the industrial scale, the ability to eliminate or recycle markers is very important. The Cre–loxP system is usually used for marker cassette excision purposes in a variety of organisms, enabling marker recycling and elimination during the construction of multiply deletant strains or for industrial applications. Carter y Delneri (26) have developed dominant antibiotic resistance marker cassettes flanked with mutated loxP sites (loxLE and lox2272). The loxP cassettes contain resistance for geneticin (kanMX gene), nourseothricin (natNT2), or hygromycin (hphNT1) as selectable markers. These cassettes can be present in the yeast genome together with the widely used loxP–marker gene–loxP cassettes. The probability of interaction with and between the mutated loxP sites and consequent recombination between the cassettes, or undesirable chromosomal rearrangements between lox sequences, is minimized. A phleomycin-resistant Cre-expressing vector to excise multiple markers simultaneously has also been developed. Such a toolkit should be particularly useful for the genetic engineering of industrial strain bypassing the need of auxotrophic markers and the presence of residual heterologous antibiotic-resistance genes. 4.2. Examples of Genetically Engineered Industrial Yeasts

Many examples of genetic engineering of S. cerevisiae have appeared in the two last decades (Table 1), most of them developed in laboratory strains (27, 28). The development of industrial pentose-fermenting strains of S. cerevisiae has been described in the literature and is currently a very active area of research due to the future needs to produce large quantities of biofuels and other chemicals from renewable sources. Pentoses are 5-carbon sugars (C5) which are not naturally fermented by S. cerevisiae. They represent up to 25% of the total carbohydrates present in lignocellulosic raw materials (29). Initially, the genetic engineering of industrial strains had been limited to the introduction of xylose utilization pathways; since S. cerevisiae is unable to ferment pentoses, further improvements were obtained by nondirected adaptation strategies as reviewed by Hahn-Hägerdahl et al. (30). Industrial strains supplemented with

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Table 1 Genetic engineering of S. cerevisiae Target

Example

Type of strain

Extension of substrate range

Transformation of S. cerevisiae with xylose reductase and xylitol deshydrogenase from Pichia stipitis or with bacterial xylose isomerase. This enables S. cerevisiae to use pentose, a sugar abundant in lignocellulosic biomass

Laboratory and industrial

A GPD2 (glycerol dehydrogenase) mutant grown under Improvements of anaerobic conditions, had a higher ethanol yield of 8% productivity and in addition to a 40% reduction of the glycerol yield. yield, and elimination This is an example of redirecting the carbon flux of by-products

Laboratory

Industrial Improvement of FLO1 encodes a cell surface protein that plays a direct role process performance in the flocculation process. The FLO1 gene has successfully been integrated into the genome of a nonflocculent brewer’s yeast strain and a stable constitutive flocculating strain was obtained Improvements of cellular properties

MIG1 encodes a zinc finger protein that mediates glucose repression by controlling the expression of SUC2 (encoding the invertase). Deletion of MIG1 has allowed a ninefold increase in SUC2 expression of cells grown on glucose

Laboratory

Extension of product range

Heterologous expression of LDH (lactate dehydrogenase) allowed the NADH-dependent reduction of pyruvate to lactate

Laboratory

Adapted from ref. 21 with input from refs. 27, 28

the “yeast pathway” for xylose utilization and overexpressing the endogenous XK successfully cofermented xylose and glucose. These genes were generally integrated into the yeast chromosome (in the HIS3 locus, for example) and selected by their ability to grow on xylose. In some cases, the strains were transformed with multicopy plasmids overexpressing these genes and selected by geneticin resistance. The “yeast pathway” comprises two enzymes from Pichia stipitis, the XR and the XDH (Fig. 4). It can be redox neutral if the XR is linked to NADH instead of NADPH. The “bacterial pathway” uses the enzyme xylose isomerase (XI) to directly convert D-xylose to D-xylulose (Fig. 4). Other strategies for the chromosomal integration of the “yeast pathway” genes included the use of the pAUR101 shuttle vector which is a chromosomal integrating vector for S. cerevisiae containing the aureobasidin A-resistance gene for integration at the AUR1-C allele (31, 32). The industrial strains tested included bakery yeast, shochu yeasts, wine yeasts, and industrial alcohol fermentation yeasts, including flocculent yeast with high xylulosefermenting ability. Both xylose consumption and ethanol production remarkably increased in the latter.

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Fig. 4. Pathways for the conversion of xylose to ethanol. XI xylose isomerase, XK xylulose kinase, XR xylose reductase, XDH xylitol deshydrogenase. Adapted from ref. 41.

Karhumaa et al. (33) demonstrated the importance of increasing the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae. For this, the genes encoding the XK (XKS1) and enzymes of the pentose phosphate pathway (TAL1 encoding the TAL transaldolase, TKL1 encoding the TKL transketolase, RKI1 encoding the ribose-5-phosphate ketol-isomerase RKI, and RPE1 encoding the RPE ribulose 5-phosphate epimerase) were placed under the control of stronger promoters for overexpression and chromosomally integrated. Chromosomal integration was performed sequentially using a zeocin-resistance marker for selection and the loxP–cre system for marker excision. However, this work was only performed in laboratory strains. Genome-wide transcription analysis revealed that the expression of an ORF of unknown function (YLR042c) was reduced 6.0fold in strains with improved xylose utilization compared with their respective parental strain; deletion of this ORF improved aerobic growth on xylose (34). Deletion of YLR042c improved ethanolic xylose fermentation in several recombinant strains of baker’s yeast. However, the magnitude of the effect was strain dependent (35).

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The obtained results suggested that YLR042c influences the assimilation of carbon sources other than glucose and has a role at the membrane level when cell damage occurs. The increased xylose flow into the cell resulted in increased xylitol formation, rather than increased ethanol formation. These authors concluded that the transfer of this property to industrial strains might not be so attractive. Bera et al. (36) have reported industrial strains of S. cerevisiae engineered for simultaneous xylose and arabinose utilization. The new strains were based on the previously developed S. cerevisiae yeast 424A(LNH-ST) with multiple copies of the XR, XDH, and XK stably integrated in the chromosome. The new strain was constructed by overexpressing fungal genes for L-arabinose utilization in plasmids with hygromycin resistance selection. An ethanol production, about 72.5% the theoretical yield, was achieved from sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose. An inconvenience of this system is that the fermentation medium contained hygromycin. Other industrial strains cofermenting xylose and arabinose were reported (37, 39). In this case, bacterial genes for arabinose utilization were chromosomally integrated in rDNA genes. Evolution of the genetically engineered strains in the presence of xylose and arabinose as sole carbon sources allowed to obtain strains with increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions and improved anaerobic ethanol production at the expense of xylitol and glycerol; however, arabinose was almost stoichiometrically converted to arabitol. These improvements were attributed to mutations in the arabinose genes, duplication of the xylose utilization genes, and increased transport capacity. Concerning the introduction of “bacterial pathways” for xylose utilization in industrial strains, Brat et al. (40) have described the functional expression of the Clostridium phytofermentans XI with high activity. The codon usage of the corresponding gene was adapted to that of highly expressed glycolytic genes of S. cerevisiae and chromosomally integrated into the FAA2 gene encoding the long-chain fatty acid CoA ligase 2 using kanMX as the selection marker and geneticin in the selection and fermentation media. This enzyme was less inhibited by xylitol, a side product during xylose fermentation. This study is encouraging for further improvement of xylose fermentation in industrial yeast strains. On the other hand, Karhumaa et al. (38) compared the performance of strains containing the bacterial and the yeast pathways, concluding that the yeast xylose utilization pathway resulted in faster ethanol production. However, the comparison was performed between two laboratory strains transformed with plasmids, one containing the Pyromyces XI gene and the other the P. stipitis XR y XDH genes; the performance of industrial strains containing these pathways was not compared and is difficult to predict.

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5. Final Remarks S. cerevisiae is extensively used in the industry in baking, brewing, winemaking, and bioethanol production from conventional carbohydrates (starch and sucrose). The interest for producing bioethanol and other interesting compounds from renewable resources has grown. The process robustness of S. cerevisiae makes this yeast a potential platform for the development of genetically engineered yeasts for these new applications. However, this requires the development of stable genetically engineered strains which is not an easy task due to the peculiar characteristics of industrial strains as aneuploidy, polyploidy, frequent chromosomal rearrangements, difficulty of using auxotrophic markers, and problematic of using antibiotic-resistance markers at a commercial scale. Examples of genetically engineered strains for the production of ethanol from lignocellulosic biomass have been described; however, contrary to baking, brewing, and wine applications, there is no knowledge concerning the genetic stability and performance of such strains under real process conditions. References 1. Walker, G.M. (1998) Yeast technology. In: Yeast physiology and biotechnology. p. 265–320. John Wiley and Sons Ltd, Chichester, England. 2. Bai, F.W., Anderson, W.A. and Moo-Young, M. (2008) Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol Adv. 26(1):89–105. 3. Branduardi, P., Smeraldi, C. and Porro, D. (2008) Metabolically engineered yeasts: “Potential” industrial applications. J Mol Microbiol Biotechnol. 15(1):31–40. 4. Werpy, T. and Petersen, G. (2004) Top Value added chemicals from biomass. Vol. 1 Results of screening for potential candidates from sugars and synthesis gas. 5. BREW (2006) Medium and long-term opportunities and risks of the biotechnological production of bulk chemicals from renewable resources – final report. Prepared under the European Commissions GROWTH program, Utrecht. 6. Hahn-Hägerdal, B., Galbe, M., GorwaGrauslund, M.F., Lidén, G. and Zacchi, G. (2006) Bio-ethanol-the fuel of tomorrow from the residues of today. Trends Biotechnol. 24(12): 549–556. 7. Gibson, B.R., Lawrence, S.J., Leclaire, J.P., Powell, C.D. and Smart, K.A. (2007) Yeast

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24. Mumberg, D., Müller, R. and Funk, M. (1995) Yeasts vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156(1):119–122. 25. Hashimoto, S., Ogura, M., Aritomi, K., Hoshida, H., Nishizawa, Y. and Akada, R. (2005) Isolation of auxotrophic mutants of diploid industrial yeast strains after UV mutagenesis. Appl Environ Microbiol. 71(1):312–319. 26. Carter, Z. and Delneri, D. (2010) New generation of loxP-mutated deletion cassettes for the genetic manipulation of yeast natural isolates. Yeast 27(9):765–775. 27. Ostergaard, S., Olsson, L. and Nielsen, J. (2000) Metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 64(1): 34–50. 28. Nevoigt, E. (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 72(3):379–412. 29. Lee, J. (1997) Biological conversion of lignocellulosic biomass to ethanol. J Biotechnol. 56:1–24. 30. Hahn-Hägerdal, B., Karhumaa, K., Fonseca, C., Spencer-Martins, I. and Gorwa-Grauslund, M.F. (2007) Towards industrial pentosefermenting yeast strains. Appl Microbiol Biotechnol. 74(5):937–953. 31. Matsushika, A., Inoue, H., Watanabe, S., Kodaki, T., Makino, K. and Sawayama, S. (2009a) Efficient bioethanol production by a recombinant flocculent Saccharomyces cerevisiae strain with a genome-integrated NADP+dependent xylitol dehydrogenase gene. Appl Environ Microbiol. 75(11):3818–3822. 32. Matsushika, A., Inoue, H., Murakami, K., Takimura, O. and Sawayama, S. (2009b) Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae. Bioresour Technol. 100(8):2392–2398. 33. Karhumaa, K., Hahn-Hägerdal, B. and GorwaGrauslund, M.F. (2005) Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 22(5):359–368. 34. Bengtsson, O., Jeppsson, M., Sonderegger, M., Parachin, N.S., Sauer, U., Hahn-Hägerdal, B. and Gorwa-Grauslund, M.F. (2008) Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering. Yeast 25(11): 835–847. 35. Parachin, N.S., Bengtsson, O., Hahn-Hägerdal, B. and Gorwa-Grauslund, M.F. (2010) The deletion of YLR042c improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae. Yeast 27(9):741–751.

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36. Bera, A.K., Sedlak, M., Khan, A. and Ho, N.W. (2010) Establishment of L-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering. Appl Microbiol Biotechnol. 87(5):1803–1811. 37. Karhumaa, K., Wiedemann, B., Hahn-Hägerdal, B., Boles, E. and Gorwa-Grauslund, M.F. (2006) Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains. Microb Cell Fact. 5:18. 38. Karhumaa, K., Garcia Sanchez, R., HahnHägerdal, B. and Gorwa-Grauslund, M.F. (2007) Comparison of the xylose reductasexylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microb Cell Fact. 6:5. 39. GGarcia Sanchez, R., Karhumaa, K., Fonseca, C., Sánchez Nogué, V., João RM Almeida,

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ARKANSAS

BIOSCIENCES INSTITUTE

L~SU

ARKANSAS STATE UNIVERSITYT" P.O. Box 639 State University. AR 72467-0639

Phone.

870-972-2025 Fax; 870-972-2026

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December 14th, 2010

Jonesboro. Arkansas

To whom it may concern:

Per request of Dr. Sylvie Le Borgne Le Gall, this letter has the purpose to communicate that the book chapter entitled: "Genetic engineering of industrial strains of Saccharomyces cerevisiae" by Dr. Le Borgne Le Gall has been accepted for publication in the third edition of "Methods in Molecular Biology", volume on "Recombinant Gene Expression, Reviews and Protocols", Humana/Springer, NY. The schedule of publication is as follows: book chapters arrived in July-August 2010, the editor of the entire series Prof. John Walker and me provided editorial comments on all contributions in August-September 2010, authors worked on making corrections and returned final manuscripts for print in September-October. The final product is expected to be in press by February 2011. I am very pleased with the excellent contribution of Dr. Le Borgne Le Gall to this project.

Dr. Arqelia Lorence Associate Professor in Metabolic Engineering Editor [email protected]

METHODS

IN

MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Recombinant Gene Expression Reviews and Protocols, Third Edition Edited by

Argelia Lorence Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA

Editor Argelia Lorence, Ph.D. Arkansas Biosciences Institute and Department of Chemistry & Physics Arkansas State University, Jonesboro University Loop E. 504 State University, AR 72401, USA [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-432-2 e-ISBN 978-1-61779-433-9 DOI 10.1007/978-1-61779-433-9 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011943340 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Dedication To David and Noah, my two big loves

v

Preface Formidable progress in the field of recombinant gene expression has taken place since 2004, the date of publication of the previous edition of this book. In particular, the emergence of the “omics” technologies has revolutionized all areas of biology. In the industrial arena, Escherichia coli, Saccharomyces cerevisiae, and insect cells continue to be the dominant production platforms of recombinant proteins. However, in the last few years plants and animals have grown in importance as viable sources of more complex proteins. This third edition of Recombinant Gene Expression is a lot more than just an update of the previous edition. Although some of the authors that contributed in 2004 were also invited to participate in this new project, this volume contains brand new protocols and topics not covered before. I am indebted to the experts in the field and their students and post-doctoral associates whose talent and experience is reflected in the outstanding quality of the chapters here included. I would like to thank Dr. Paulina Balbás, from whom I have learned a lot about managing a project like this, and two members of my laboratory, Austin Slaven and Gwendolyn Wilson who helped me edit the reference section of each chapter. While organizing a book of such an extensive topic as gene expression, it was indispensable to pick and choose from the multitude of strategies, vectors, promoters, and so on, so the coverage of topics is far from exhaustive. Some expression systems were omitted because of size limitations and even within areas presented unavoidably; some research approaches were unevenly treated. The information provided in Recombinant Gene Expression, is organized in sections by biological host: Bacteria, lower eukaryotes, fungi, plants and plant cells, and animals and animal cells, presenting one or two authoritative reviews and several protocol chapters in each section. Each chapter concludes with a section containing excellent notes where authors offer their valuable expertise of scientists and their personal views of strategy planning, as well as a variety of approaches, and alternatives that will surely be useful and inspiring to you, the reader. Jonesboro, AR, USA

Argelia Lorence

vii

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

GENERAL ASPECTS

1 Using Folding Promoting Agents in Recombinant Protein Production: A Review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beatrix Fahnert 2 Routine Identity Confirmation of Recombinant Proteins by MALDI-TOF Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brett J. Savary and Prasanna Vasu 3 A Matter of Packaging: Influence of Nucleosome Positioning on Heterologous Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . María de la Cruz Muñoz-Centeno, Gonzalo Millán-Zambrano, and Sebastián Chávez 4 Tools of the Trade: Developing Antibody-Based Detection Capabilities for Recombinant Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maureen C. Dolan, Giuliana Medrano, Anthony McMickle, and Carole L. Cramer

PART II

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PROKARYOTES

5 Heat-Shock Protein Fusion Vectors for Improved Expression of Soluble Recombinant Proteins in Escherichia coli. . . . . . . . . . . . . . . . . . . . . . . . . Christos A. Kyratsous and Christos A. Panagiotidis 6 The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ferenc Vonderviszt, Ráchel Sajó, József Dobó, and Péter Závodszky 7 Optimization of Purification Protocols Based on the Step-by-Step Monitoring of the Protein Aggregates in Soluble Fractions . . . . . . . . . . . . . . . . . . . Ario de Marco 8 Heterologous Protein Expression by Lactococcus lactis. . . . . . . . . . . . . . . . . . . . . . . Julio Villatoro-Hernández, Oscar P. Kuipers, Odila Saucedo-Cárdenas, and Roberto Montes-de-Oca-Luna 9 An Extended Suite of Genetic Tools for Use in Bacteria of the Halomonadaceae: An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Montserrat Argandoña, Carmen Vargas, Mercedes Reina-Bueno, Javier Rodríguez-Moya, Manuel Salvador, and Joaquín J. Nieto

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10 Regulated Recombinant Protein Production in the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valentina Rippa, Rosanna Papa, Maria Giuliani, Cinzia Pezzella, Ermenegilda Parrilli, Maria Luisa Tutino, Gennaro Marino, and Angela Duilio 11 A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125 . . . . . . . . . . . . Maria Giuliani, Ermenegilda Parrilli, Cinzia Pezzella, Valentina Rippa, Angela Duilio, Gennaro Marino, and Maria Luisa Tutino 12 A New Bacterial Co-expression System for Over-expressing Soluble Protein and Validating Protein–Protein Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jumei Zeng and Zheng-Guo He 13 Heterologous High-Level Gene Expression in the Photosynthetic Bacterium Rhodobacter capsulatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nadine Katzke, René Bergmann, Karl-Erich Jaeger, and Thomas Drepper 14 Plasmid DNA Production for Therapeutic Applications. . . . . . . . . . . . . . . . . . . . . . Alvaro R. Lara and Octavio T. Ramírez

PART III

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LOWER EUKARYOTES

15 Recombinant Protein Production in the Eukaryotic Protozoan Parasite Leishmania tarentolae: A Review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Tomoaki Niimi 16 Expression of Multisubunit Proteins in Leishmania tarentolae . . . . . . . . . . . . . . . . . 317 Marisa Sugino and Tomoaki Niimi

PART IV

FUNGI

17 Recombinant Protein Production in Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diethard Mattanovich, Paola Branduardi, Laura Dato, Brigitte Gasser, Michael Sauer, and Danilo Porro 18 Yeasts as a Tool for Heterologous Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . Raja Mokdad-Gargouri, Salma Abdelmoula-Soussi, Nadia Hadiji-Abbès, Ines Yacoubi-Hadj Amor, Istabrak Borchani-Chabchoub, and Ali Gargouri 19 The Cre/Lox System: A Practical Tool to Efficiently Eliminate Selectable Markers in Fungal Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simona Florea, Caroline Machado, Kalina Andreeva, and Christopher L. Schardl 20 Aptamer-Regulated Expression of Essential Genes in Yeast . . . . . . . . . . . . . . . . . . . Beatrix Suess, Karl-Dieter Entian, Peter Kötter, and Julia E. Weigand 21 Cloning and Expression of Hemicellulases from Aspergillus nidulans in Pichia pastoris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prasanna Vasu, Stefan Bauer, and Brett J. Savary 22 A Thiamine-Regulatable Epitope-Tagged Protein Expression System in Fission Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tiina Tamm

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23 Heterologous Gene Expression by Chromosomal Integration in Fission Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433 Akihisa Matsuyama and Minoru Yoshida 24 Genetic Engineering of Industrial Strains of Saccharomyces cerevisiae . . . . . . . . . . . . 451 Sylvie Le Borgne

PART V

PLANTS AND PLANT CELLS

25 Recombinant Protein Production in Plants: Challenges and Solutions . . . . . . . . . . . Elizabeth E. Hood and Deborah V. Vicuna Requesens 26 A Novel Plant Cell Bioproduction Platform for High-Yield Secretion of Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jianfeng Xu and Marcia J. Kieliszewski 27 Super-promoter:TEV, a Powerful Gene Expression System for Tobacco Hairy Roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Luis Ñopo, Bonnie J. Woffenden, Deborah G. Reed, Scott Buswell, Chenming Zhang, and Fabricio Medina-Bolivar 28 Bioseparation of Recombinant Proteins from Plant Extract with Hydrophobin Fusion Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jussi J. Joensuu, Andrew J. Conley, Markus B. Linder, and Rima Menassa 29 Quality Assessment of Recombinant Proteins Produced in Plants . . . . . . . . . . . . . . Giuliana Medrano, Maureen C. Dolan, Jose Condori, David N. Radin, and Carole L. Cramer 30 Cell-Free Protein Synthesis as a Promising Expression System for Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xumeng Ge and Jianfeng Xu

PART VI

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ANIMALS AND ANIMAL CELLS

31 The Use of Bacterial Artificial Chromosomes for Recombinant Protein Production in Mammalian Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leander Blaas, Monica Musteanu, Beatrice Grabner, Robert Eferl, Anton Bauer, and Emilio Casanova 32 Engineering the Chaperone Network of CHO Cells for Optimal Recombinant Protein Production and Authenticity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lyne Jossé, C. Mark Smales, and Mick F. Tuite 33 High-Throughput Baculovirus Expression in Insect Cells . . . . . . . . . . . . . . . . . . . . Richard B. Hitchman, Robert D. Possee, and Linda A. King 34 Recombinant Protein Expression in Milk of Livestock Species . . . . . . . . . . . . . . . . . Zsuzsanna Bösze and László Hiripi Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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