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 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 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.

Metabolic and evolutionary engineering of diploid yeast for the production of first-and second-generation ethanol

2020 ◽  
Author(s):  
Yang Sun ◽  
Qian Xue ◽  
Junyan Hou ◽  
Meilin Kong ◽  
Xiaowei Li ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Gene Cluster ◽  
Second Generation ◽  
Corn Starch ◽  
Xylose Fermentation ◽  
Strain Improvement ◽  
Limiting Factors ◽  
Evolutionary Engineering ◽  
Diploid Strain ◽  
Xylose Consumption

Abstract Background: Saccharomyces cerevisiae has been widely used in the fermentation of plant-derived sugars to produce ethanol, called first-generation (1G) bioethanol, but made an impact on global food markets. Significant efforts have been therefore to employ non-food lignocellulosic feedstocks for bioethanol production, known as second-generation (2G) bioethanol. However, S. cerevisiae cannot naturally utilize xylose, a major component in lignocellulosic hydrolysates, and it has low tolerance to common carboxylic acid inhibitors present in lignocellulosic hydrolyzates. Metabolic engineering and evolutionary engineering have shown great power in strain improvement, which were also adopted here to solve these limiting factors in developing 2G bioethanol.Results: An efficient expression of a six-gene cluster, including XYL1/XYL2/XKS1/TAL1/PYK1/MGT05196, was achieved in the evolved S. cerevisiae diploid strain A21Z, showing the ability to use mixed glucose and xylose. The engineered strain A21Z expressing the six-gene cluster displayed a high xylose consumption after 96 h, reaching 90.7% of the theoretical yield in ethanol production. To investigate its industrial characteristics, A31Z was obtained by direct evolution of A21Z under the treatment of industrial hydrolysate from wheat straw. Under different fermentation conditions with 1G and 2G feedstock candidates, A31Z showed a markedly improved xylose fermentation performance. A31Z could produce more ethanol and less glycerol compared to the control Angel from corn starch during 120 h, with a final ethanol production at 122.32 g/L. The ability to produce higher ethanol production was also found under the fermentation using carbon source from hydrolysis of Dried Distillers Grains with Solubles (DDGS) or whole corn.Conclusions: Here, we report an effective strategy to improve xylose fermentation with an evolutionary engineering in the industrial S. cerevisiae diploid strain A31Z. This study demonstrated that a constructed A31Z has the higher xylose consumption and efficient ethanol production in mixed glucose and xylose with acetate. A31Z also gave a good ethanol production in 1G and 2G industrial feedstocks, indicating its significant contribution in the transition stage from the 1st generation to the 2nd generation bioethanol.

Effect of nitrogen sources on oxidoreductive enzymes and ethanol production during D-xylose fermentation by Candida shehatae

1992 ◽  
Vol 38 (3) ◽  
pp. 258-260 ◽  
Author(s):  
Sanjay Palnitkar ◽  
Anil Lachke
Keyword(s):  
Ethanol Production ◽  
Ammonium Sulphate ◽  
Organic Nitrogen ◽  
Xylose Fermentation ◽  
Continuous Fermentation ◽  
Nitrogen Sources ◽  
Potassium Nitrate ◽  
Yield Coefficient ◽  
Xylose Utilization ◽  
Candida Shehatae

The effect on D-xylose utilization and the corresponding xylitol and ethanol production by Candida shehatae (ATCC 22984) were examined with different nitrogen sources. These included organic (urea, asparagine, and peptone) and inorganic (ammonium chloride, ammonium nitrate, ammonium sulphate, and potassium nitrate) sources. Candida shehatae did not grow on potassium nitrate. Improved ethanol production (Y(p/s), yield coefficient (grams product/grams substrate), 0.34) was observed when organic nitrogen sources were used. Correspondingly, the xylitol production was also higher with organic sources. Ammonium sulphate showed the highest ethanol:xylitol ratio (11.0) among all the nitrogen sources tested. The ratio of NADH- to NADPH-linked D-xylose reductase (EC 1.1.1.21) appeared to be rate limiting during ethanologenesis of D-xylose. The levels of xylitol dehydrogenase (EC 1.1.1.9) were also elevated in the presence of organic nitrogen sources. These results may be useful in the optimization of alcohol production by C. shehatae during continuous fermentation of D-xylose. Key words: xylose fermentation, Candida shehatae, nitrogen source, oxidoreductive enzymes.

Ascorbic acid and CoQ10 ameliorate the reproductive ability of superoxide dismutase 1-deficient female mice†

2019 ◽  
Author(s):  
Naoki Ishii ◽  
Takujiro Homma ◽  
Jaeyong Lee ◽  
Hikaru Mitsuhashi ◽  
Ken-ichi Yamada ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Superoxide Dismutase ◽  
Coenzyme Q10 ◽  
Knockout Mice ◽  
Knockout Mouse ◽  
Superoxide Dismutase 1 ◽  
Wild Type ◽  
Positive Effects ◽  
Reproductive Ability ◽  
Ascorbic Acid Supplementation

Abstract Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q10, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44–75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

Maintenance of ΔpH by a butanol-tolerant mutant of Clostridium beijerinckii

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 607-613 ◽  
Author(s):  
Fanqiang Wang ◽  
Shelby Kashket ◽  
Eva R. Kashket
Keyword(s):  
Phosphate Buffer ◽  
Sodium Citrate ◽  
Potassium Citrate ◽  
Clostridium Beijerinckii ◽  
Wild Type ◽  
Arginine Residues ◽  
Tolerant Mutant ◽  
Mutant Cells ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane ΔpH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The ΔpH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the ΔpH component of the protonmotive force) and supplemented with glucose and Mg2+. The ΔpH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a ΔpH of 1·4 when butanol was added to a concentration of 1·5 % (w/v), while the wild-type ΔpH was reduced to 0·1. The ΔpH of both strains was completely dissipated with 1·75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the ΔpH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0·8 %) prevented this ΔpH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in ΔpH occurred more slowly than in the mutant, and sparing of the ΔpH by 0·8 % butanol was less pronounced. The authors interpret these data to mean that the ΔpH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.

scholarly journals Direct Ethanol Production from Lignocellulosic Sugars and Sugarcane Bagasse by a RecombinantTrichoderma reeseiStrain HJ48

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jun Huang ◽  
Dong Chen ◽  
Yutuo Wei ◽  
Qingyan Wang ◽  
Zhenchong Li ◽  
...  
Keyword(s):  
Ethanol Production ◽  
Trichoderma Reesei ◽  
Sugarcane Bagasse ◽  
Consolidated Bioprocessing ◽  
Ethanol Yield ◽  
Genome Shuffling ◽  
Wild Type ◽  
Mutant Population ◽  
Ethanol Resistance ◽  
Lignocellulosic Sugars

Trichoderma reeseican be considered as a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production ofT. reeseiCICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing.

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