Towards industrial pentose-fermenting yeast strains

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.     

工业酵母菌株转化体系的建立

本文通过敏感度实验测定了工业酵母菌株SK—1、SPSC—1、NAN—27在不同的培养基上对G418及Cu^2 的敏感浓度,确定了3株工业菌株转化子的筛选方法。针对它们各自的生理生化、遗传特性,用改进的醋酸锂完整细胞转化和原生质体转化两种方法,分别建立了3株菌的转化系统,初步解决了工业酵母菌株转化的问题,并为下一步工业酵母菌株的代谢工程改造奠定了基础。     

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.     

FLO gene-dependent phenotypes in industrial wine yeast strains

Most commercial yeast strains are nonflocculent. However, controlled flocculation phenotypes could provide significant benefits to many fermentation-based industries. In nonflocculent laboratory strains, it has been demonstrated that it is possible to adjust flocculation and adhesion phenotypes to desired specifications by altering expression of the otherwise silent but dominant flocculation (FLO) genes. However, FLO genes are characterized by high allele heterogeneity and are subjected to epigenetic regulation. Extrapolation of data obtained in laboratory strains to industrial strains may therefore not always be applicable. Here, we assess the adhesion phenotypes that are associated with the expression of a chromosomal copy of the FLO1, FLO5, or FLO11 open reading frame in two nonflocculent commercial wine yeast strains, BM45 and VIN13. The chromosomal promoters of these genes were replaced with stationary phase-inducible promoters of the HSP30 and ADH2 genes. Under standard laboratory and wine making conditions, the strategy resulted in expected and stable expression patterns of these genes in both strains. However, the specific impact of the expression of individual FLO genes showed significant differences between the two wine strains and with corresponding phenotypes in laboratory strains. The data suggest that optimization of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach.     

Differentiation of industrial wine yeast strains using microsatellite markers

AIMS: To differentiate nine industrial wine strains of Saccharomyces cerevisiae using microsatellite (simple sequence repeats, SSR) markers. METHODS AND RESULTS: Six of the strains were indigenous yeasts currently used as high-density starter monocultures by the Uruguayan wine industry. Unequivocal differentiation of these six native strains and three commercial S. cerevisiae wine strains was achieved by PCR amplification and polymorphism analysis of loci containing microsatellite markers. CONCLUSION: We recommend the use of this reproducible and simple molecular method to routinely discriminate wine yeast strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Microsatellites are superior to other methods for typing yeasts because the results can be exchanged as quantitative data. Knowledge of the frequencies of the alleles for different SSR markers will eventually lead to an accurate typing method to identify industrial wine yeast strains.     

A novel strategy to construct yeast Saccharomyces cerevisiae strains for very high gravity fermentation

Very high gravity (VHG) fermentation is aimed to considerably increase both the fermentation rate and the ethanol concentration, thereby reducing capital costs and the risk of bacterial contamination. This process results in critical issues, such as adverse stress factors (ie., osmotic pressure and ethanol inhibition) and high concentrations of metabolic byproducts which are difficult to overcome by a single breeding method. In the present paper, a novel strategy that combines metabolic engineering and genome shuffling to circumvent these limitations and improve the bioethanol production performance of Saccharomyces cerevisiae strains under VHG conditions was developed. First, in strain Z5, which performed better than other widely used industrial strains, the gene GPD2 encoding glycerol 3-phosphate dehydrogenase was deleted, resulting in a mutant (Z5ΔGPD2) with a lower glycerol yield and poor ethanol productivity. Second, strain Z5ΔGPD2 was subjected to three rounds of genome shuffling to improve its VHG fermentation performance, and the best performing strain SZ3-1 was obtained. Results showed that strain SZ3-1 not only produced less glycerol, but also increased the ethanol yield by up to 8% compared with the parent strain Z5. Further analysis suggested that the improved ethanol yield in strain SZ3-1 was mainly contributed by the enhanced ethanol tolerance of the strain. The differences in ethanol tolerance between strains Z5 and SZ3-1 were closely associated with the cell membrane fatty acid compositions and intracellular trehalose concentrations. Finally, genome rearrangements in the optimized strain were confirmed by karyotype analysis. Hence, a combination of genome shuffling and metabolic engineering is an efficient approach for the rapid improvement of yeast strains for desirable industrial phenotypes.     

Breeding of a distiller's yeast by hybridization with a wine yeast

Summary Hybrids were constructed between auxotrophic mutants of a heterothallic distiller's strain and a homothallic wine yeast. The hybridization resulted in a significant increase in both ethanol production and tolerance against exogenous ethanol. The hybrids were heterogeneous in ploidy, probably due to segregation of aneuploids during culturing. Sporulation of the hybrids broke down the high productivity, producing spore clones that were mostly of various intermediate levels of performance. However, a meiotic product superior to both crossing partners was also found. The results demonstrate that fermentation capacity can be improved by crossing with a low performance strain. Offprint requests to: M. Sipiczki     

A comparison of stress tolerance in YPD and industrial lignocellulose-based medium among industrial and laboratory yeast strains

In general, it is believed that fermentation by yeast under harsh industrial conditions, especially if substrates such as wood hydrolysate or lignocellulosic substrates are used, requires the use of so-called industrial strains. In order to check whether this is always true, a comparison of performance was made using two industrial strains and four commonly used laboratory strains, the haploid and diploid versions of CEN-PK and X2180, under industrially relevant stress conditions. The industrial strains were a Swedish commercial baker’s yeast strain and a strain previously isolated from an industrial bioethanol production plant using lignocellulosic substrate. Stress conditions included, apart from growth in the lignocellulosic substrate itself, elevated concentrations of glucose, NaCl, ethanol, and lactate as well as low pH. Results showed that, indeed, the strain adapted to lignocellulosic substrate also possessed the highest growth rate as well as shortest duration of the lag phase in this type of medium. However, the higher the additional stress level, the lower the difference compared to other strains, and X2180 in particular displayed a high resistance to these additional stress conditions. Furthermore, no difference in performance could be detected between the haploid or diploid versions of the laboratory strains. It might be that, at least under some circumstances, a laboratory strain such as X2180 could be an industrially attractive production organism with the advantage of facilitating the possibilities for making controlled genetic manipulations.     

Protoplast fusion as a means of producing new industrial yeast strains

The ability of the yeast Saccharomyces cerevisiae to produce ethanol and carbon dioxide from carbohydrates has been exploited by man for thousands of years. During its brief existence protoplast fusion has already become an invaluable tool for investigating the molecular genetics of yeast, as well as an important part of the arsenal of genetic manipulations available to develop new strains. In the case of industrial strains, a mating reaction is usually lacking. Protoplast fusion overcomes this barrier and allows for the genetic analysis of commercially valuable traits. A major block toward broader applicability of fusion is that hybrids becomes more unstable as the genetic backgrounds of the parents diverge. As greater progress in overcoming this problem is made, fusion, by itself and in conjunction with classical hybridization, will become increasingly important in the development of new strains. The incorporation of cytoplasmic elements into yeast protoplasts has the potential to vastly expand the array of biochemical reactions performed by yeasts, thereby increasing the importance of this microbe to mankind.     

Fermentation of biomass sugars to ethanol using native industrial yeast strains

In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn-ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production.     

Efficient use of DNA molecular markers to construct industrial yeast strains

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.     

A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteins

An epitope tag introduced to a gene of interest (GOI) greatly increases the ease of studying cellular proteins. Rapid PCR-based strategies for epitope tagging a protein's C-terminus at its native gene locus are widely used in yeast. C-terminal epitope tagging is not suitable for all proteins, however. Epitope tags fused to the C-terminus can interfere with function of some proteins or can even be removed by C-terminal protein processing. To overcome such problems, proteins can be tagged with epitopes at their amino-termini, but generating yeast strains expressing N-terminal epitope tagged genes under control of the endogenous promoter at the native locus is comparatively more difficult. Strategies to introduce N-terminal epitope tags have been reported previously but often introduce additional sequences other than the epitope tag into the genome. Furthermore, N-terminal tagging of essential genes by current methods requires formation of diploid strains followed by tetrad dissection or expression of an additional copy of the GOI from a plasmid. The strategies described here provide a quick, facile means of epitope tagging the N-terminus of both essential and nonessential genes in a two-step PCR-based procedure. The procedure has the significant advantage of leaving tagged genes under the control of their endogenous promoters, and no additional sequences other than the epitope tag encoding nucleotides are inserted into the genome.     

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24 h and then shifted to 5.0 after 24 h. Using this strategy, the highest β-carotene production (50.39 mg/l) and the formation rate (1.40 mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H₂O₂ enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.     

Stably inherited killer activity in industrial yeast strains obtained by electrotransformation

Killer-sensitive strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were transformed by electroinjection using double-stranded RNA isolated from a superkiller strain. Various recipient strains were used: both thermo-resistant and thermo-sensitive as well as mutants of industrial strains. Conversion of respiratory competent (rho+) into respiratory deficient (rho-) strains (mutants) resulted in a significant increase of the yield of electrotransformants and/or of longterm killer stability. Electrotransformation of rho- mutants of distillery and brewery strains resulted in more than 100 clones, which exhibited weak or strong killer activity over some or all of the experimental period of 10 months.     

Identification of industrial yeast strains of Saccharomyces cerevisiae by fatty acid profiles

Summary The fatty acid composition of 20 Saccharomyces cerevisiae strains (laboratory and industrial strains, top and bottom brewery yeasts, distillery and wine yeasts) was determined. Fatty acids with chain lengths of 12 to 28 carbons were separated. It has been found that individual strain groups differ in their fatty acid profiles.     

Role of cultivation media in the development of yeast strains for large scale industrial use

The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.