39 results about "Xylose Reductase" patented technology

The invention belongs to the technical field of biology, and particularly relates to a method for producing xylitol through two-step enzyme catalysis of hemicellulose hydrolysate, and the method comprises the following steps: taking hemicellulose hydrolysate as a raw material, adding arabinose dehydrogenase, NADH oxidase and coenzyme NADH, and carrying out first-step fermentation until no L-arabinose residue exists in fermentation liquor to obtain fermentation reaction liquor I; and adding xylose reductase and glucose dehydrogenase into the fermentation reaction liquid I, and carrying out second-step fermentation to obtain a fermentation product containing xylitol. According to the two-step enzyme catalysis method provided by the invention, xylitol is prepared, and high-purity xylose does not need to be used as a raw material; according to the present invention, the cheap hemicellulose hydrolysate can be directly adopted as the raw material, the xylose conversion rate and the xylitol yield can exceed 99%, the impurity arabitol content in the product is low, the other impurity saccharic acid is easy to separate, and the problems of complex process, large separation difficulty and high cost in the xylitol refining process are successfully solved.

The invention provides a mutant of xylose reductase and application of the mutant in synthesis of D-tagatose. According to the present invention, the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification on the basis of the sequence and structure-oriented semi-rational modification strategy so as to obtain the high activity xylose reductase mutant, and the xylose reductase mutant has the following characteristics that: the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification so as to obtain the xylose reductase mutant, according to the invention, the mutant of the xylose reductase is integrated into a redox pathway for synthesizing the D-tagatose by virtue of a genetic engineering means, and is expressed in bacillus subtilis 168, so that a recombinant engineering bacterium for synthesizing the D-tagatose by fermenting D-galactose is successfully constructed, the efficient synthesis of the D-tagatose is realized, and the yield of the D-tagatose is improved. And the method has obvious practical application value and economic benefit.

The invention belongs to the field of biochemical engineering, and particularly relates to a method for preparing xylitol through continuous catalysis of immobilized enzyme coupled ion exchange, which specifically comprises the following steps: co-immobilizing xylose reductase, glucose dehydrogenase and NADP + coenzyme, producing xylitol through enzyme catalysis by taking xylose and glucose as raw materials, and coupling an ion exchange reaction after the enzyme catalysis reaction to obtain the xylitol. Due to the arrangement of the ion exchange reaction, the byproduct gluconate can be separated, and the pH of the reaction liquid can be regulated and controlled by using alkaline anions exchanged in the ion exchange process, so that the normal operation of the reaction is further ensured; the xylitol and the by-product gluconate obtained by the reaction can be separated without an additional separation process. The method provided by the invention successfully solves the problems of complex process, high separation difficulty and high cost in the process of refining xylitol by a biological method.

The invention relates to the technical field of enzyme engineering, in particular to a xylose reductase XR21 mutant and application thereof. Specifically, the invention provides a novel xylose reductase mutant aiming at the defect of poorer thermal stability of the existing wild type xylose reductase, and particularly provides a xylose reductase mutant which is obtained by performing single or multiple site amino acid mutation on the 19th site, the 48th site, the 79th site, the 208th site and the 300th site of a xylose reductase sequence shown as SEQ ID NO: 2, compared with wild type xylose reductase, the obtained xylose reductase mutant has the advantages that the activity of catalyzing an epimer substrate arabinose into arabitol can be obviously reduced, and meanwhile, the selectivity to xylose is improved, so that the operation process of downstream separation and purification is simplified, and the biological catalysis requirement of green industry is met.

The present application belongs to the technical field of enzyme engineering, and particularly relates to a xylose-reducing enzyme mutant with improved xylose selectivity and poison tolerance and application thereof. Specifically, the present application obtains the xylose-reducing enzyme mutant with improved xylose selectivity and poison tolerance by performing site-directed saturation mutation and combined mutation screening on the xylose-reducing enzyme derived from Chaetomium exospilum, and particularly performing mutation on sites corresponding to Y49, I102, L107, L109, H111, V114, S166, S221, F225 and / or Y312 of the wild-type xylose-reducing enzyme shown in SEQ ID NO:2, so that the xylose-reducing enzyme mutant can efficiently and selectively produce xylitol with non-detoxified hemicellulose hydrolysate as raw material, and has a good industrial application prospect.

The invention relates to the technical field of enzyme engineering, in particular to a xylose reductase XR31 mutant and application thereof. Specifically, the invention provides a novel xylose reductase mutant aiming at the defect of poor thermal stability of the existing wild type xylose reductase (SEQ ID NO: 2), and particularly provides a xylose reductase mutant which is obtained by performing single or multiple site amino acid mutation on the 21st site, the 46th site, the 128th site, the 168th site and the 205th site of a xylose reductase sequence shown as SEQ ID NO: 2, compared with wild type xylose reductase, the obtained xylose reductase mutant has the advantages that the catalytic activity and the thermal stability on substrate xylose are obviously improved, the xylose reductase mutant can stably work under a high-temperature condition, and substrate conversion in industrial application is more efficiently realized.

The invention relates to a method for characterizing intracellular NADPH (Nicotinamide Adenine Dinucleotide Phosphate) generation amount of yarrowia lipolytica through xylose consumption, and belongs to the technical field of biology. According to the method, the yarrowia lipolytica overexpressing xylose reductase, xylitol dehydrogenase and xylulokinase is inoculated into a culture medium taking xylose as a unique carbon source for culture, and a linear relation between the xylose consumption and the NADPH generation amount is established, so that the generation level of the NADPH can be represented by measuring the xylose consumption in the culture medium. The invention provides an effective method for characterizing the intracellular NADPH level of the yarrowia lipolytica, the intracellular NADPH level can be characterized only by detecting the consumption of xylose, and the detection method is simple and efficient.

The present invention belongs to the field of bioengineering technology, and relates to a genetically engineered bacterium for producing 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid (AA-2G), and a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium for producing 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid (AA-2G) described in the present invention contains an α-galactosidase gene (agal), a xylose reductase gene (XR), a mannitol dehydrogenase gene (MDH), an L-galactose dehydrogenase gene (LGDH) with a S136A mutation, a D-arabinose-1,4-lactone oxidase gene (ALO), and a sucrose phosphorylase gene (SPase). The present invention provides a method for producing AA-2G using the recombinant genetically engineered bacterium. The genetically engineered strain realizes the synthesis of AA-2G by whole cells, greatly shortens the synthetic production pathway of AA-2G, and improves the yield of AA-2G.

The present invention belongs to the field of microbial technology, and relates to a genetically engineered bacterium for preparing ethanol based on hydrolyzed sugar from wood fiber and its application. The bacterium is a high xylose utilization and inhibitor tolerance engineered bacterium. The engineered bacterium is based on Saccharomyces cerevisiae S288C as a chassis strain, and co-expresses heterologous xylose reductase-xylitol dehydrogenase pathway-related genes, xylose transport-related genes, xylose isomerase-related genes and xylose isomerase genes, and over-expresses endogenous xylulose kinase-related genes, non-oxidative pentose phosphate pathway-related genes, reduced glutathione system genes, superoxide dismutase expression system-related genes, NADPH / NADP + The genes and transcription factors related to the cofactor circulation system were knocked out, the genes related to the competitive metabolic pathway were knocked out, and the bacteria were domesticated with sugar sources and inhibitors as pressure; the engineered bacteria can efficiently metabolize xylose and produce high-concentration ethanol in non-detoxified lignocellulose hydrolysate with high inhibitor concentrations and without any nutrient salt addition.

The invention belongs to the technical field of biology, and particularly relates to a method for producing xylitol by catalyzing hemicellulose hydrolysate through a biological enzyme, which comprises two stages: in the first stage, adding hemicellulose hydrolysate, xylose reductase, arabinose dehydrogenase and glucose dehydrogenase to form a fermentation system, adjusting the pH value of the system to 8.0-9.5, and fermenting, adding L-arabinose until no L-arabinose residue exists in the fermentation liquid to obtain a fermentation reaction liquid I; in the second stage, the pH value of the fermentation reaction liquid I is adjusted to 6.5-7.5, fermentation continues to be conducted, and a fermented product containing xylitol is obtained. According to the method for preparing the xylitol through the biological enzyme catalysis method, cheap hemicellulose hydrolysate can be directly adopted as a raw material, and high-purity xylose does not need to be used as the raw material; the xylose reductase, the arabinose dehydrogenase and the glucose dehydrogenase used in the method are wide in gene source, the three-enzyme one-pot catalytic process is simple and efficient, the conversion rate of xylose and the yield of xylitol both can exceed 99%, the content of impurity arabitol in the product is low, other impurity saccharic acid is easy to separate, and the method is suitable for industrial production. The problems of complex process, high separation difficulty and high cost in the xylitol refining process are successfully solved.

The present invention discloses a method for improving xylose metabolism capacity by regulating the expression of genes of the reductive TCA pathway through an oxygen-responsive metabolic switch, and belongs to the technical field of gene recombination and metabolic engineering. The method disclosed in the present invention is achieved by regulating the expression of genes related to the rTAC pathway using an oxygen-responsive metabolic switch that is low-expressed under aerobic conditions and high-expressed under anaerobic conditions. The engineered bacteria constructed by this method can achieve efficient metabolism of xylose during anaerobic fermentation using xylose as a single substrate or corn straw hydrolysate with glucose and xylose as the main carbon sources as a substrate. The method of improving xylose metabolism capacity of the present invention provides a technical reference for constructing engineered bacteria that can efficiently metabolize xylose-containing substrates such as corn straw hydrolysate under anaerobic fermentation production conditions.

The invention discloses a method for regulating and reducing TCA pathway gene expression through an oxygen response metabolic switch to improve xylose metabolic capability, and belongs to the technical field of gene recombination and metabolic engineering. The method disclosed by the invention is realized by regulating and controlling expression of rTAC pathway related genes by using an oxygen response metabolic switch which is low in expression under an aerobic condition and high in expression under an anaerobic condition. The engineering bacteria constructed by the method can realize efficient metabolism of xylose in an anaerobic fermentation process taking xylose as a single substrate or corn straw hydrolysate taking glucose and xylose as main carbon sources as a substrate. The method for improving the xylose metabolism capability provides technical reference for constructing engineering bacteria which efficiently metabolize corn straw hydrolysate and other xylose-containing substrates under the anaerobic fermentation production condition.

A method for producing an aqueous solution containing ethylene glycol and / or a salt of pyruvic acid, by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase (EC 4.1.2.55) to give an aqueous solution which contains glycol aldehyde and a salt of pyruvic acid, after which the glycol aldehyde is reduced using an NAD(P)H-dependent alcohol dehydrogenase (EC 1.1.1.1) or an NAD(P)H-dependent xylose reductase (EC 1.1.1.307) and subsequently optionally removing either the salt of pyruvic acid or the ethylene glycol from the solution.

The invention provides a strain for producing beta-carotene as well as a construction method and application of the strain, and belongs to the technical field of synthetic biology and genetic engineering. The construction method of the MYy107 strain disclosed by the invention comprises the following steps: step 1, integrating a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate diphosphate decarboxylase gene ERG19 and an isopentene diphosphate isomerase gene IDI1 on an RT08 bacterium, so as to obtain an RT10 strain; 2, a xylose reductase gene XYL1, a xylitol dehydrogenase gene XYL2, a xylose isomerase mutant XylA3 * and a xylulokinase gene Yl.Xk are integrated on the RT10 strain, and the MYy107 strain is obtained. According to the MYy107 strain constructed by the invention, the beta-carotene can be produced by using xylose and acetic acid at the same time, and the yield of the beta-carotene is remarkably improved and reaches 710.01 mg / L.

The invention discloses recombinant escherichia coli for producing D-tagatose by using redox enzyme driven by cofactor regeneration as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of biological engineering. The method comprises the following steps: introducing xylose reductase xyrB, glucose dehydrogenase Gox2015, galactitol dehydrogenase RlGDH mutant T193G / G98C and water-producing NADH oxidase SpNox into escherichia coli, so as to construct recombinant escherichia coli; according to the method disclosed by the invention, a way for synthesizing the D-tagatose by regenerating and driving oxidoreductase through cofactors is designed and constructed, and lactose in whey can be completely converted into the D-tagatose and sodium gluconate by combining the way with beta-galactosidase, so that high-valued utilization of a dairy product processing by-product whey is realized.

Method for producing an aqueous solution containing ethylene glycol and / or a salt of pyruvic acid by treating 2-keto-3-deoxy-D-xylonate (KDX) and / or 2-keto-3-deoxy-L-arabonate (KDA) in vitro with an aldolase (EC 4.1.2.55), thereby obtaining an aqueous solution containing glycolaldehyde and a salt of pyruvic acid, after which the glycolaldehyde is reduced by means of an NAD(P)H-dependent alcohol dehydrogenase (EC 1.1.1.1) or an NAD(P)H-dependent xylose reductase (EC 1.1.1.307) and subsequently optionally removing either the salt of pyruvic acid or the ethylene glycol from the solution.

The present invention discloses a method for synthesizing D-xylitol based on the NAD(H) cofactor circulation system and a special engineering bacterium thereof. The present invention provides a method for constructing an engineering strain for synthesizing D-xylitol, comprising the following steps: allowing a receptor bacterium to simultaneously express NAD(H)-dependent D-xylose reductase and NAD(H)-dependent formate dehydrogenase to obtain an engineering strain for synthesizing D-xylitol. The present invention will generate huge economic benefits for the production of D-xylitol and has great promotion and application value.

The invention discloses a culture medium for improving the utilization rate of pichia pastoris xylose and application of the culture medium. The culture medium comprises a YNB culture medium, xylose, methanol and histidine, wherein the content of the YNB culture medium is 13.4 g / L, the content of the xylose is 1-10 g / L, the content of the methanol is 7.91-31.64 g / L, and the content of the histidine is 0.04 g / L. Based on the problem that the xylose metabolism capability of a wild type K.phaffi.GS115 strain is not high, and culture medium optimization is needed to improve the xylose metabolism capability of the strain, methanol is added into a xylose culture medium to relieve the inhibition effect of high-concentration xylose on the strain and improve the xylose utilization rate of the strain; according to the method, the inhibition effect of high-concentration xylose on the wild type K.phaffi.GS115 strain is solved, the xylose consumption rate of the wild type K.phaffi.GS115 strain is increased, and the method has positive promotion significance on popularization of xylose microorganism application.

The present invention belongs to the field of genetic engineering technology, specifically relating to genetically engineered bacteria and their use in xylitol production. The genetically engineered bacteria include a host cell and a xylose reductase gene and a xylose transporter gene inserted into the host cell. The present invention introduces the xylose transporter gene and the xylose reductase gene into Vibrio natriuresis. The resulting genetically engineered bacteria can functionally express and obtain a recombinant xylose transporter, thereby transporting extracellular xylose into the cell and converting the xylose into xylitol using the xylose reductase. This invention is the first to construct a genetically engineered Vibrio natriuresis bacterium capable of utilizing xylose and producing xylitol, which is of great significance for subsequent research on the use of Vibrio natriuresis in xylitol production.

The present invention relates to a genetically engineered erythritol strain and a construction method thereof, as well as application of the strain in preparing erythritol by microbial fermentation. The present invention introduces xylose reductase, xylitol dehydrogenase and xylitol kinase into Yarrowia lipolytica to reshape the xylose metabolic pathway, strengthens the expression of transketolase TKL1, transaldolase TAL and erythrose reductase ER in the erythritol bioproduction pathway, reduces the production of byproducts mannitol and arabitol by knocking out mannitol dehydrogenase MDH and arabitol dehydrogenase ArDH, reduces the conversion of erythritol into other substances by knocking out erythritol dehydrogenase EYD, weakens the accumulation of erythritol, strengthens the expression of sugar uptake pathway genes hexokinase HK and transporters Stp1 and Stp2, and finally produces a high-yield erythritol bacterium that can utilize a mixed carbon source of glucose and xylose, with the yield increased from 50.17 g / L to 195.56 g / L. This provides an excellent strain source for the industrial production of erythritol synthesized using xylose as a substrate, and has great industrial application potential.

The application discloses a genetically engineered bacillus subtilis for producing porphyra-334, a kind of bacteriosporin amino acid, and a construction method and application thereof, and belongs to the field of microbial metabolic engineering and synthetic biology.The strain takes bacillus subtilis WB600 as a starting bacterium: 1) a competitive pathway gene ywjH is knocked out to strengthen precursor supply;2) a gene cluster NlmysABCD derived from a porphea lincolnii is integrated in multiple copies to enhance pathway flux;3) sporulation genes spo0A and spoIIIE and threonine degradation gene tdh are knocked out to block byproduct generation and redistribute metabolic resources;4) xylose metabolism genes xylAB are introduced.The yield of porphyra-334 of the engineered bacterium reaches 3.38 g / L in shake flask fermentation, and the yield is increased to 15.1 g / L in 50 L fermenter fed-batch culture.

This invention discloses a strain for the efficient synthesis of D-tagatose, its preparation method, and its applications. Through strategies such as the discovery of novel galactitol dehydrogenase and promoter engineering, this invention initially increased tagatose yield by 28.48%. Simultaneously, a highly efficient and sensitive tagatose biosensor was developed, which was used as a screening tool for xylose reductase. Ss XR molecularly modified the strain to obtain the F128M-Q219K double mutant, which achieved a tagatose yield of 5.39 g / L in the producing strain. Based on this, amino acid complementation optimization was performed on the high-yielding strain, and the final engineered strain achieved a tagatose yield of 44.61 g / L in a 5 L bioreactor. This study provides a new strategy and core technology support for the efficient biosynthesis of tagatose.

The present invention relates to a method for producing D-fructose comprising the step of reducing D-glucosone to D-fructose with a modified xylose reductase enzyme or a functional fragment thereof comprising a motif GX1GX2X3X4, wherein the modified xylose reductase enzyme has a lower D-glucose specificity and a lower D-xylose specificity compared to the unmodified xylose reductase enzyme, wherein the modified xylose reductase enzyme comprises at least one mutation within the motif GX1GX2X3X4 present in the unmodified xylose reductase enzyme, X1 being leucine or phenylalanine, X2 being leucine or cysteine, X3 being tryptophan and X4 being lysine or arginine, wherein X3 in the modified xylose reductase enzyme is tyrosine or phenylalanine.

The invention belongs to the field of biological catalysis, and particularly relates to a genetically engineered bacterium and application of the genetically engineered bacterium in preparation of xylitol through whole-cell catalysis of xylose, the genetically engineered bacterium comprises a host cell and a target gene inserted into the host cell, and the host cell is vibrio natrieti; the target genes comprise a xylose reductase gene and a glucose dehydrogenase gene. According to the invention, heterologous xylose reductase and glucose dehydrogenase genes are introduced into vibrio natriticus, and the dosage ratio of the xylose reductase to the glucose dehydrogenase genes is adjusted in a genome integration form, so that the vibrio natriticus genetically engineered bacterium is constructed. Compared with escherichia coli, the whole-cell biocatalyst disclosed by the invention is short in preparation period, high in catalytic activity and relatively high in production efficiency.

Provided herein are recombinant microorganisms having two or more copies of a nucleic acid sequence encoding a xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is an exogenous nucleic acid. Optionally, the recombinant microorganism comprises at least one nucleic acid sequence encoding a xylulokinase and / or at least one nucleic acid sequence encoding a xylose transporter. The provided recombinant microorganism is capable of growing on xylose as a carbon source.

This invention discloses a genetically engineered Bacillus subtilis strain for producing Mycosporine-glycine, its construction method, and its applications. Starting with Bacillus subtilis WB600, this strain significantly enhances Mycosporine-glycine synthesis by knocking out the competing pathway gene ywjH and expressing the AvmysABC gene cluster from Anabaena polymorpha at multiple sites. Further knocking out the sporulation genes spo0A and spoIIIE and introducing the xylose metabolism gene clusters xylA-xylB, combined with a mixed carbon source fermentation strategy, significantly increased the yield. The engineered strain achieved a Mycosporine-glycine yield of 3.64 g / L in shake-flask fermentation and 15.8 g / L in a 50 L fed-batch fermenter.

Method for producing an aqueous solution containing L-sorbose by reducing D-glucose, which is dissolved in an aqueous solution, to D-sorbitol in vitro by treatment with an NAD(P)H-dependent oxidoreductase (xylose reductase), after which the D-sorbitol is oxidized to L-sorbose in vitro with an NAD(P)+-dependent oxidoreductase (mannitol dehydrogenase), after which the two oxidoreductases are separated.

The present invention belongs to the field of biocatalysis, specifically relating to genetically engineered bacteria and their use in whole-cell catalysis of xylose to produce xylitol. The genetically engineered bacteria comprise a host cell, Vibrio natriuresis, and target genes inserted into the host cell. The host cell is Vibrio natriuresis; the target genes include xylose reductase and glucose dehydrogenase genes. The present invention introduces heterologous xylose reductase and glucose dehydrogenase genes into Vibrio natriuresis, and adjusts the gene dosage ratio of xylose reductase and glucose dehydrogenase through genomic integration to construct a genetically engineered Vibrio natriuresis. Compared to Escherichia coli, the whole-cell biocatalyst of the present invention has a shorter preparation cycle, higher catalytic activity, and higher production efficiency.

The application discloses a method for synchronously and efficiently metabolizing mixed sugar based on a Pichia pastoris mixed flora and application thereof. The Pichia pastoris mixed flora comprises at least one Pichia pastoris engineering strain for xylose metabolism and one Pichia pastoris engineering strain for oligosaccharide metabolism. By means of functional division of the strains, the interference of glucose signal on the xylose metabolic pathway is physically and genetically isolated, and the synchronous metabolism of xylose and cellulosic oligosaccharide is realized. The metabolic burden of each strain is reduced, the exclusive pathway of each strain can be optimized, the pathway competition and the imbalance of auxiliary factors are avoided, the overall fermentation rate and the sugar conversion rate are improved. The mixed flora system has a certain buffering capacity to environmental fluctuations and is more stable than a single strain. The strategy is particularly suitable for complex lignocellulose hydrolysate, and can effectively solve the problem of asynchronous utilization of multiple fermentable sugars in the lignocellulose hydrolysate and improve the total utilization rate of raw materials. In the process of simultaneous saccharification and fermentation of the mixed flora, the utilization rate of xylose and cellulosic oligosaccharide is improved, and the integrated biorefinery cycle is shortened.

The invention belongs to the technical field of biology, and particularly relates to a genetically engineered bacterium and application of the genetically engineered bacterium in preparation of xylitol through a biological method. According to the invention, several expression control technologies such as theophylline switch, Cre / LoxP and the like are utilized, so that the expression products of the genetically engineered bacteria can be controlled by judging whether theophylline is added or not, the expression products are arabinose-1-dehydrogenase and then xylose reductase, after arabinose is consumed by the arabinose-1-dehydrogenase, xylose is converted into xylitol by utilizing the xylose reductase, and the xylose is converted into xylitol by utilizing the xylose reductase. The generation of byproducts in the process of preparing the xylitol from the hemicellulose hydrolysate is prevented.