Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae.

Bioconversion of xylose-the second most abundant sugar in nature-into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.

Deletion of JEN1 and ADY2 reduces lactic acid yield from an engineered Saccharomyces cerevisiae, in xylose medium, expressing a heterologous lactate dehydrogenase.

Microorganisms have evolved to produce specific end products for many reasons, including maintaining redox balance between NAD+ and NADH. The yeast Saccharomyces cerevisiae, for example, produces ethanol as a primary end product from glucose for the regeneration of NAD+. Engineered S. cerevisiae strains have been developed to ferment lignocellulosic sugars, such as xylose, to produce lactic acid by expression of a heterologous lactate dehydrogenase (ldhA from Rhizopus oryzae) without genetic perturbation to the native ethanol pathway. Surprisingly, the engineered yeast strains predominantly produce ethanol from glucose, but produce lactic acid as the major product from xylose. Here, we provide initial evidence that the shift in product formation from ethanol to lactic acid during xylose fermentation is at least partially dependent on the presence of functioning monocarboxylate transporter genes/proteins, including JEN1 and ADY2, which are downregulated and unstable in the presence of glucose, but upregulated/stable on xylose. Future yeast metabolic engineering studies may find the feedstock/carbon selection, such as xylose, an important step toward improving the yield of target end products.

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii.

The probiotic yeast Saccharomyces boulardii has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. S. boulardii displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate S. boulardii from Saccharomyces cerevisiae The majority of prior studies stated that S. boulardii exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used S. boulardii strain, ATCC MYA-796, was examined under various culture conditions, the S. boulardii strain could consume galactose, but at a much lower rate than that of S. cerevisiae While all GAL genes were present in the S. boulardii genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in PGM2, which codes for phosphoglucomutase, was identified in the genome of the S. boulardii strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of S. cerevisiaePGM2 in S. boulardii restored galactose utilization. After reverting the point mutation to a full-length PGM2 in S. boulardii by Cas9-based genome editing, the growth rates of wild-type (with a truncated PGM2 gene) and mutant (with a full-length PGM2) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length PGM2) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated PGM2 might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in PGM2 might be involved in multiple phenotypes with different effects.IMPORTANCESaccharomyces boulardii is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of S. boulardii remain unknown. Our study reports that a point mutation in PGM2, which codes for phosphoglucomutase, is responsible for inferior galactose utilization by S. boulardii After correction of the mutated PGM2 via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the PGM2 mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other S. boulardii strains revealed that the PGM2 mutation is evolutionarily conserved. Interestingly, the PGM2 mutation was beneficial for growth at a higher temperature on glucose. We speculate that the PGM2 mutation was enriched due to selection of S. boulardii in the natural habitat (sugar-rich fruits in tropical areas).

Changes of Serum Procalcitonin (PCT), C-Reactive Protein (CRP), Interleukin-17 (IL-17), Interleukin-6 (IL-6), High Mobility Group Protein-B1 (HMGB1) and D-Dimer in Patients with Severe Acute Pancreatitis Treated with Continuous Renal Replacement Therapy (CRRT) and Its Clinical Significance.

BACKGROUND The aim of this study was to investigate the changes in serum levels of procalcitonin (PCT), C-reactive protein (CRP), interleukin-17 (IL-17), interleukin-6 (IL-6), high mobility group protein-B1 (HMGB1), and D-dimer in severe acute pancreatitis (SAP) patients during treatment with continuous renal replacement therapy (CRRT) and the clinical significance. MATERIAL AND METHODS A total of 92 SAP patients admitted to our hospital from January 2017 to December 2017 were selected and randomly divided into the observation group and the control group using a random number table method, with 46 cases in each group. The control group was given conventional therapy, and the observation group was given CRRT in addition to conventional therapy. RESULTS After 1 week, the total effective rate of treatment in the observation group was significantly higher than that in the control group (P<0.05). In the observation group, each index showed a continuous downward trend at 6, 12, and 24 hours after treatment, and at different time points after treatment, the indexes were significantly lower than those in the control group (P<0.05). CONCLUSIONS CRRT is more effective in the treatment of SAP, and its effects are more obvious in removing a variety of inflammatory factors and reducing the serum levels of PCT, HMGB1, and D-dimer, which is of great clinical significance.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A) exhibited a 14% higher ethanol yield and 46% lower byproduct yield than the IIK1 strain from anaerobic fermentation of the Miscanthus hydrolysate. Our results demonstrate that industrial yeast strains can be engineered via haploid isolation. The isolated haploid strain (4124-S60) can be used for metabolic engineering to produce fuels and chemicals.

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc.

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD+-linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Short communication: Conversion of lactose and whey into lactic acid by engineered yeast.

Lactose is often considered an unwanted and wasted byproduct, particularly lactose trapped in acid whey from yogurt production. But using specialized microbial fermentation, the surplus wasted acid whey could be converted into value-added chemicals. The baker's yeast Saccharomyces cerevisiae, which is commonly used for industrial fermentation, cannot natively ferment lactose. The present study describes how an engineered S. cerevisiae yeast was constructed to produce lactic acid from purified lactose, whey, or dairy milk. Lactic acid is an excellent proof-of-concept chemical to produce from lactose, because lactic acid has many food, pharmaceutical, and industrial uses, and over 250,000 t are produced for industrial use annually. To ferment the milk sugar lactose, a cellodextrin transporter (CDT-1, which also transports lactose) and a ß-glucosidase (GH1-1, which also acts as a ß-galactosidase) from Neurospora crassa were expressed in a S. cerevisiae strain. A heterologous lactate dehydrogenase (encoded by ldhA) from the fungus Rhizopus oryzae was integrated into the CDT-1/GH1-1-expressing strain of S. cerevisiae. As a result, the engineered strain was able to produce lactic acid from purified lactose, whey, and store-bought milk. A lactic acid yield of 0.358g/g of lactose was achieved from whey fermentation, providing an initial proof of concept for the production of value-added chemicals from excess industrial whey using engineered yeast.

Metabolic Engineering of Probiotic Saccharomyces boulardii.

Saccharomyces boulardiiis a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae Therefore, S. boulardiiis an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2,ura3,his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools forS. cerevisiae We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome ofS. boulardii We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii Our results suggest that more sophisticated genetic perturbations to improveS. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineeredS. boulardii.

Optimization of an acetate reduction pathway for producing cellulosic ethanol by engineered yeast.

Xylose fermentation by engineered Saccharomyces cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD+ -linked xylitol dehydrogenase (XDH) suffers from redox imbalance due to cofactor difference between XR and XDH, especially under anaerobic conditions. We have demonstrated that coupling of an NADH-dependent acetate reduction pathway with surplus NADH producing xylose metabolism enabled not only efficient xylose fermentation, but also in situ detoxification of acetate in cellulosic hydrolysate through simultaneous co-utilization of xylose and acetate. In this study, we report the highest ethanol yield from xylose (0.463 g ethanol/g xylose) by engineered yeast with XR and XDH through optimization of the acetate reduction pathway. Specifically, we constructed engineered yeast strains exhibiting various levels of the acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) activities. Engineered strains exhibiting higher activities of AADH and ACS consumed more acetate and produced more ethanol from a mixture of 20 g/L of glucose, 80 g/L of xylose, and 8 g/L of acetate. In addition, we performed environmental and genetic perturbations to further improve the acetate consumption. Glucose-pulse feeding to continuously provide ATPs under anaerobic conditions did not affect acetate consumption. Promoter truncation of GPD1 and gene deletion of GPD2 coding for glycerol-3-phosphate dehydrogenase to produce surplus NADH also did not lead to improved acetate consumption. When a cellulosic hydrolysate was used, the optimized yeast strain (SR8A6S3) produced 18.4% more ethanol and 41.3% less glycerol and xylitol with consumption of 4.1 g/L of acetate than a control strain without the acetate reduction pathway. These results suggest that the major limiting factor for enhanced acetate reduction during the xylose fermentation might be the low activities of AADH and ACS, and that the redox imbalance problem of XR/XDH pathway can be exploited for in situ detoxification of acetic acid in cellulosic hydrolysate and increasing ethanol productivity and yield. Biotechnol. Bioeng. 2016;113: 2587-2596. © 2016 Wiley Periodicals, Inc.

GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae.

Rapid advances in the capabilities of reading and writing DNA along with increasing understanding of microbial metabolism at the systems-level have paved an incredible path for metabolic engineering. Despite these advances, post-translational tools facilitating functional expression of heterologous enzymes in model hosts have not been developed well. Some bacterial enzymes, such as Escherichia coli xylose isomerase (XI) and arabinose isomerase (AI) which are essential for utilizing cellulosic sugars, cannot be functionally expressed in Saccharomyces cerevisiae. We hypothesized and demonstrated that the mismatching of the HSP60 chaperone systems between bacterial and eukaryotic cells might be the reason these bacterial enzymes cannot be functionally expressed in yeast. The results showed that the co-expression of E. coli GroE can facilitate the functional expression of E. coli XI and AI, as well as the Agrobacterium tumefaciens D-psicose epimerase in S. cerevisiae. The co-expression of bacterial chaperonins in S. cerevisiae is a promising post-translational strategy for the functional expression of bacterial enzymes in yeast. Biotechnol. Bioeng. 2016;113: 2149-2155. © 2016 Wiley Periodicals, Inc.

Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, ß-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Deletion of PHO13, encoding haloacid dehalogenase type IIA phosphatase, results in upregulation of the pentose phosphate pathway in Saccharomyces cerevisiae.

The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.

Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR.

Genomic integration of expression cassettes containing heterologous genes into yeast with traditional methods inevitably deposits undesirable genetic elements into yeast chromosomes, such as plasmid-borne multiple cloning sites, antibiotic resistance genes, Escherichia coli origins, and yeast auxotrophic markers. Specifically, drug resistance genes for selecting transformants could hamper further industrial usage of the resulting strains because of public health concerns. While we constructed an efficient and rapid xylose-fermenting Saccharomyces cerevisiae, the engineered strain (SR8) might not be readily used for a large-scale fermentation because the SR8 strain contained multiple copies of drug resistance genes. We utilized the Cas9/CRISPR-based technique to refactor an efficient xylose-fermenting yeast strain without depositing any undesirable genetic elements in resulting strains. In order to integrate genes (XYL1, XYL2, and XYL3) coding for xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis, and delete both PHO13 and ALD6, a double-strand break formation by Cas9 and its repair by homologous recombination were exploited. Specifically, plasmids containing guide RNAs targeting PHO13 and ALD6 were sequentially co-transformed with linearized DNA fragments containing XYL1, XYL2, and XYL3 into S. cerevisiae expressing Cas9. As a result, two copies of XYL1, XYL2, and XYL3 were integrated into the loci of PHO13 and ALD6 for achieving overexpression of heterologous genes and knockout of endogenous genes simultaneously. With further prototrophic complementation, we were able to construct an engineered strain exhibiting comparable xylose fermentation capabilities with SR8 within 3 weeks. We report a detailed procedure for refactoring xylose-fermenting yeast using any host strains. The refactored strains using our procedure could be readily used for large-scale fermentations since they have no antibiotic resistant markers.

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.

Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease.

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.

3' Truncation of the GPD1 promoter in Saccharomyces cerevisiae for improved ethanol yield and productivity.

Glycerol is a major by-product in bioethanol fermentation by the yeast Saccharomyces cerevisiae, and decreasing glycerol formation for increased ethanol yield has been a major research effort in the bioethanol field. A new strategy has been used in the present study for reduced glycerol formation and improved ethanol fermentation performance by finely modulating the expression of GPD1 in the KAM15 strain (fps1Δ pPGK1-GLT1 gpd2Δ). The GPD1 promoter was serially truncated from the 3' end by 20 bp to result in a different expression strength of GPD1. The two engineered promoters carrying 60- and 80-bp truncations exhibited reduced promoter strength but unaffected osmostress response. These two promoters were integrated into the KAM15 strain, generating strains LE34U and LE35U, respectively. The transcription levels of LE34U and LE35U were 37.77 to 45.12% and 21.34 to 24.15% of that of KAM15U, respectively, depending on osmotic stress imposed by various glucose concentrations. In very high gravity (VHG) fermentation, the levels of glycerol for LE34U and LE35U were reduced by 15.81% and 30.66%, respectively, compared to KAM15U. The yield and final concentration of ethanol for LE35U were 3.46% and 0.33% higher, respectively, than those of KAM15U. However, fermentation rate and ethanol productivity for LE35U were reduced. On the other hand, the ethanol yield and final concentration for LE34U were enhanced by 2.28% and 2.32%, respectively, compared to those of KAM15U. In addition, a 2.31% increase in ethanol productivity was observed for LE34U compared to KAM15U. These results verified the feasibility of our strategy for yeast strain development.

Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae.

We have investigated whether simultaneous modification of cofactor metabolism and glycerol in a strain of Saccharomyces cerevisiae can eliminate glycerol synthesis during ethanol production. Two strains, S812 (gpd1Δ gpd2Δ PGK1p-GLT1) and LE17 (gpd1Δ gpd2Δ PGK1p-GLT1 PGKp-STL1) were generated that showed a 8 and 8.2 % increase in the ethanol yield, respectively, compared to the wild type KAM-2 strain. The ethanol titer was improved from 90.4 g/l for KAM-2 to 97.6 g/l for S812 and 97.8 g/l for LE17, respectively. These results provide a new insight into rationalization of metabolic engineering strategies for improvement of ethanol yield through elimination of glycerol production.

Decreased xylitol formation during xylose fermentation in Saccharomyces cerevisiae due to overexpression of water-forming NADH oxidase.

The recombinant xylose-fermenting Saccharomyces cerevisiae strain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis requires NADPH and NAD(+), creates cofactor imbalance, and causes xylitol accumulation during growth on d-xylose. To solve this problem, noxE, encoding a water-forming NADH oxidase from Lactococcus lactis driven by the PGK1 promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD(+) by XDH and the formation of NAD(+) by water-forming NADH oxidase. Overexpression of noxE significantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initial d-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing to noxE overexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due to noxE overexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression of noxE.

Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination.

Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain's performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches.