scholarly journals Improving Xylose Fermentation in Saccharomyces cerevisiae by Expressing Nuclear-Localized Hexokinase 2

2020 ◽  
Vol 8 (6) ◽  
pp. 856 ◽  
Author(s):  
Liyuan Zheng ◽  
Shan Wei ◽  
Meiling Wu ◽  
Xuehao Zhu ◽  
Xiaoming Bao ◽  
...  
Keyword(s):  
Glucose Sensor ◽  
Rna Binding ◽  
Xylose Fermentation ◽  
Rna Binding Proteins ◽  
Ethanol Yield ◽  
Xylose Utilization ◽  
Hexokinase 2 ◽  
Xylose Consumption ◽  
Glucose Signaling ◽  
Engineered Yeast

Understanding the relationship between xylose and the metabolic regulatory systems is a prerequisite to enhance xylose utilization in recombinant S. cerevisiae strains. Hexokinase 2 (Hxk2p) is an intracellular glucose sensor that localizes to the cytoplasm or the nucleus depending on the carbon source. Hxk2p interacts with Mig1p to regulate gene transcription in the nucleus. Here, we investigated the effect of nucleus-localized Hxk2p and Mig1p on xylose fermentation. The results show that the expression of HXK2S14A, which encodes a constitutively nucleus-localized Hxk2p, increased the xylose consumption rate, the ethanol production rate, and the ethanol yield of the engineered yeast strain by 23.5%, 78.6% and 42.6%, respectively. The deletion of MIG1 decreased xylose utilization and eliminated the positive effect of Hxk2p. We then performed RNA-seq and found that the targets of Hxk2pS14A on xylose were mainly genes that encode RNA-binding proteins. This is very different from the known targets of Mig1p and supports the notion that the Hxk2p-Mig1p interaction is abolished in the presence of xylose. These results will improve our understanding of the interrelation between the Snf1p-Mig1p-Hxk2p glucose signaling pathway and xylose utilization in S. cerevisiae and suggests that the expression of HXK2S14A could be a viable strategy to improve xylose utilization.

scholarly journals Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast

2020 ◽  
Author(s):  
Roksolana Vasylyshyn ◽  
Olena Kurylenko ◽  
Justyna Ruchala ◽  
Nadiya Shevchuk ◽  
Neringa Kuliesiene ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Xylose Fermentation ◽  
Xylose Utilization ◽  
Wild Type ◽  
Xylose Consumption ◽  
Lignocellulosic Hydrolysates ◽  
Xylose Transport ◽  
Positive Effects ◽  
Arginine Residues ◽  
Ogataea Polymorpha

Abstract Background Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. Results In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae , were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha . Conclusions The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulted strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

scholarly journals Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast

2020 ◽  
Author(s):  
Roksolana Vasylyshyn ◽  
Olena Kurylenko ◽  
Justyna Ruchala ◽  
Nadiya Shevchuk ◽  
Neringa Kuliesiene ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Xylose Fermentation ◽  
Xylose Utilization ◽  
Wild Type ◽  
Xylose Consumption ◽  
Lignocellulosic Hydrolysates ◽  
Xylose Transport ◽  
Positive Effects ◽  
Arginine Residues ◽  
Ogataea Polymorpha

Abstract Background Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. Results In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae , were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha . Conclusions The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulted strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

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

2012 ◽  
Vol 78 (16) ◽  
pp. 5708-5716 ◽  
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.

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

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.

scholarly journals BAG3 directly stabilizes Hexokinase 2 mRNA and promotes aerobic glycolysis in pancreatic cancer cells

2017 ◽  
Vol 216 (12) ◽  
pp. 4091-4105 ◽  
Author(s):  
Ming-Xin An ◽  
Si Li ◽  
Han-Bing Yao ◽  
Chao Li ◽  
Jia-Mei Wang ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Cancer Cells ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Aerobic Glycolysis ◽  
Ductal Adenocarcinoma ◽  
Aberrant Expression ◽  
Hexokinase 2 ◽  
Pancreatic Cancer Cells

Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents.

scholarly journals Deletion ofFPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

2013 ◽  
Vol 79 (10) ◽  
pp. 3193-3201 ◽  
Author(s):  
Na Wei ◽  
Haiqing Xu ◽  
Soo Rin Kim ◽  
Yong-Su Jin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Enzymatic Reactions ◽  
Xylose Metabolism ◽  
Xylose Consumption ◽  
Content Type ◽  
Ethanol Yields

ABSTRACTAccumulation of xylitol in xylose fermentation with engineeredSaccharomyces cerevisiaepresents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producingS. cerevisiaestrains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, whenFPS1was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed thatFPS1deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion ofFPS1decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion ofFPS1resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD+/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export inS. cerevisiaeand present a new gene deletion target,FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

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