Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption

ABSTRACT Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113–7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

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Effective fermentation of sugarcane bagasse whole slurries using robust xylose-capable Saccharomyces cerevisiae

10.22541/au.163319410.08076047/v1 ◽

2021 ◽

Author(s):

Thapelo Mokomele ◽

Bianca Brandt ◽

Johann Görgens

Keyword(s):

Saccharomyces Cerevisiae ◽

Sugarcane Bagasse ◽

Initial Screening ◽

Fed Batch ◽

Xylose Consumption ◽

Microbial Inhibitors ◽

Ethanol Yields ◽

Pre Treatment ◽

Whole Slurry ◽

Simultaneous Saccharification

The pre-treatment of lignocellulose material toward cellulosic bioethanol production releases microbial inhibitors that severely limit the fermentation ability of Saccharomyces cerevisiae. This study evaluated to what degree robust xylose capable strains may improve the fermentability of non-detoxified sugarcane bagasse (SCB) slurries derived from steam explosion (StEX), and further compared this to slurries derived from ammonia fibre expansion (AFEX) pre-treatment. Initial screening in separate hydrolyses and co-fermentation processes using StEx-SCB hydrolysates identified S. cerevisiae TP-1 and CelluXTM4 with higher xylose consumption (≥ 88%) and ethanol concentrations (≥ 50 g/L). Subsequent fermentations compared StEx and AFEX pre-treated SCB material under industrially relevant fed-batch pre-hydrolysis simultaneous saccharification and co-fermentation (PSSCF) conditions, which resulted in only 3 g/L differences in ethanol titres for StEx and AFEX PSSCF fermentations. The study achieved non-detoxified whole-slurry co-fermentation using StEx pre-treated SCB, with higher ethanol yields than previously reported, by utilising robust xylose-capable strains.

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Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01419-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5708-5716 ◽

Cited By ~ 96

Author(s):

Sun-Mi Lee ◽

Taylor Jellison ◽

Hal S. Alper

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Directed Evolution ◽

Ethanol Yield ◽

Xylose Isomerase ◽

Mutant Enzyme ◽

Xylose Utilization ◽

Xylose Consumption ◽

Content Type ◽

Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

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Pulsed addition of HMF and furfural to batch-grown xylose-utilizing Saccharomyces cerevisiae results in different physiological responses in glucose and xylose consumption phase

Biotechnology for Biofuels ◽

10.1186/1754-6834-6-181 ◽

2013 ◽

Vol 6 (1) ◽

pp. 181 ◽

Cited By ~ 26

Author(s):

Magnus Ask ◽

Maurizio Bettiga ◽

Varuni Duraiswamy ◽

Lisbeth Olsson

Keyword(s):

Saccharomyces Cerevisiae ◽

Physiological Responses ◽

Xylose Consumption

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Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene

Applied and Environmental Microbiology ◽

10.1128/aem.02636-08 ◽

2009 ◽

Vol 75 (11) ◽

pp. 3818-3822 ◽

Cited By ~ 51

Author(s):

Akinori Matsushika ◽

Hiroyuki Inoue ◽

Seiya Watanabe ◽

Tsutomu Kodaki ◽

Keisuke Makino ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Yield ◽

Xylitol Dehydrogenase ◽

Wood Chips ◽

Bioethanol Production ◽

Xylose Consumption ◽

Acid Hydrolysate ◽

A Genome ◽

Dehydrogenase Gene ◽

Flocculent Yeast

ABSTRACT The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP+-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

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Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

FEMS Yeast Research ◽

10.1111/j.1567-1364.2012.00808.x ◽

2012 ◽

Vol 12 (5) ◽

pp. 582-597 ◽

Cited By ~ 63

Author(s):

Gionata Scalcinati ◽

José Manuel Otero ◽

Jennifer R.H. Vleet ◽

Thomas W. Jeffries ◽

Lisbeth Olsson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Evolutionary Engineering ◽

Xylose Consumption

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Efficient anaerobic consumption of D-xylose by E. coli BL21(DE3) via xylR adaptive mutation

BMC Microbiology ◽

10.1186/s12866-021-02395-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jung Min Heo ◽

Hyun Ju Kim ◽

Sang Jun Lee

Keyword(s):

Consumption Rate ◽

Xylose Isomerase ◽

Fold Increase ◽

Adaptive Mutation ◽

Growth Media ◽

Missense Mutations ◽

Xylose Consumption ◽

E Coli ◽

Fermentation Technology ◽

Xylose Transporter

Abstract Background Microorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both D-glucose and D-xylose under anaerobic conditions, glucose is consumed first, whereas D-xylose is consumed very slowly. Results When BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR gene (which encodes the XylR transcriptional activator), respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption. Conclusions Anaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.

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Improving Acetic Acid and Furfural Resistance of Xylose-Fermenting Saccharomyces cerevisiae Strains by Regulating Novel Transcription Factors Revealed via Comparative Transcriptomic Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.00158-21 ◽

2021 ◽

Vol 87 (10) ◽

Author(s):

Bo Li ◽

Li Wang ◽

Ya-Jing Wu ◽

Zi-Yuan Xia ◽

Bai-Xue Yang ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Transcription Factors ◽

Ethanol Yield ◽

Regulation Mechanism ◽

Lignocellulosic Hydrolysate ◽

Inhibitor Tolerance ◽

Xylose Consumption ◽

Content Type ◽

Beneficial Effects

ABSTRACT Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of Saccharomyces cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) were revealed during mixed glucose and xylose fermentation. Carbohydrate metabolism pathways were significantly enriched in response to Aa, while pathways of xenobiotic biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, other pathways were activated in response to Aa_Fur, i.e., cofactor and vitamin metabolism and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rates by nearly 50%, while the ethanol yield was enhanced by nearly 8% under acetic acid and furfural stress conditions. Co-overexpression of Haa1p and Tye7p resulted in a 59% increase in xylose consumption rate and a 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed acetic acid and furfural. IMPORTANCE Inhibitor tolerance is essential for S. cerevisiae when fermenting lignocellulosic hydrolysate with various inhibitors, including weak acids, furans, and phenols. The details regarding how xylose-fermenting S. cerevisiae strains respond to multiple inhibitors during fermenting mixed glucose and xylose are still unknown. This study revealed the transcriptional regulation mechanism of an industrial xylose-fermenting S. cerevisiae strain in response to acetic acid and furfural. The transcription factor Haa1p was found to be involved in both acetic acid and furfural tolerance. In addition to Haa1p, four other transcription factors, Hap4p, Yox1p, Tye7p, and Mga1p, were identified as able to improve the resistance of yeast to these two inhibitors. This study underscores the feasibility of uncovering effective transcription factors for constructing robust strains for lignocellulosic bioethanol production.

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Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

Biotechnology for Biofuels ◽

10.1186/s13068-021-02073-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Jung-Hoon Bae ◽

Mi-Jin Kim ◽

Bong Hyun Sung ◽

Yong-Su Jin ◽

Jung-Hoon Sohn

Keyword(s):

Saccharomyces Cerevisiae ◽

Directed Evolution ◽

Lignocellulosic Biomass ◽

Xylose Fermentation ◽

Xylose Isomerase ◽

Carbon Substrate ◽

Yeast Fermentation ◽

Xylose Utilization ◽

Secretory Expression ◽

Xylose Consumption

Abstract Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

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