86 results about "Xylose Reductase" patented technology

Described are recombinant yeast which ferment xylose to ethanol and which maintain their ability to do so when cultured for numerous generations in non-selective media. The preferred yeast contain multiple copies of integrated genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase fused to promoters which are non-glucose inhibited and which do not require xylose for induction. Also described are preferred methods for integrating multiple copies of exogenous DNA into host cells by transforming cells with replicative/integrative vectors, and then replicating the cells a number of times under selective pressure to promote retention of the vector in subsequent generations. The replicated vectors thus serve to integrate multiple copies of the exogenous DNA into the host cells throughout the replication/selection phase. Thereafter the selective pressure can be removed to promote loss of the vector in subsequent generations, leaving stable integrants of the exogenous DNA.

The present invention relates to a method for preparing an ethanol producing, xylose utilizing strain of Saccharomyces cerevisiae comprising genes for overexpression of xylose reductase, xylitol dehydrogenase and xylulokinase, wherein in addition to said genes for production o phosphoacetyltransferase, and acetaldehyde dehydrogenase are introduced and optionally overexpressed.

A new xylose reductase encoding gene from Neurspora crassa was heterologously expressed in E. coli as a His-tag fusion protein and subsequently purified in high yield. This xylose reductase was shown to have a high turnover rate and catalytic efficiency, high stability at room temperature, broad pH profile, and a preference of NADPH over NADH. This enzyme is utilized in production of xylitol and other sugar alcohols such as sorbitol and also in the metabolic enhancement of organisms used for fermentation of plant biomass into ethanol.

Disclosed are xylose-fermenting recombinant yeast strains expressing xylose reductase, xylitol dehydrogenase, and xylulokinase and having reduced expression of PHO13 or a PHO13 ortholog, as well as methods of fermenting xylose to obtain ethanol using the recombinant yeast strains.

The invention discloses recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose and a construction method. The construction method comprises the steps of replacing a promoter of a saccharomyces cerevisiae xylulokinase gene XKS1 with a promoter PFBA1 by virtue of a homologous recombination method, introducing xylose reductase XYL1 and a xylitol dehydrogenase XYL2 expression cassette, increasing the activities of transketolase TKL1 and transaldolase TAL1 so as to obtain recombinant bacteria 1, introducing a farnesyl-diphosphate farnesyltransferase gene ERG9, a squalene monooxygenase gene ERG1 and a dammarenediol synthase gene DS into the recombinant bacteria 1 so as to obtain recombinant bacteria 2, and introducing a nicotinamide adenine dinucleotide-hydroxymethylglutaryl coenzyme A reductase gene NADH-HMGr, farnesyl diphosphatesynthase ERG20 and a protopanoxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 into the recombinant bacteria 2, so as to obtain recombinant bacteria 3. According to the recombinant saccharomyces cerevisiae, dammarenediol and protopanoxadiol can be artificially synthesized by virtue of xylose.

The invention discloses recombinant zymomonas mobilis capable of producing ethanol by using xylose and a fermentation method thereof. The recombinant zymomonas mobilis GX1 capable of producing the ethanol by using the xylose is preserved in China General Microbiological Culture Collection Center, and has a preservation number of CGMCC 3438. The controlling elements of Escherichia coli rrnB and related genes of xylose metabolism are fused to build plasmids with artificially fused operons for the first time; and recombinant zymomonas mobilis strains capable of producing the ethanol by using the xylose are obtained through a method of electrotransformation and acclimatization. The recombinant zymomonas mobilis can efficiently express exogenous genes, can efficiently produce the ethanol by using glucose and the xylose to ferment together at the temperature of between 30 and 34 DEG C, and can produce the ethanol by using corncob hydrolysate containing the xylose and the glucose. The strains have important significance for producing clean energy by effectively using waste lignocellulose and solving the current energy crisis.

Provided is a construction method of an escherichia coli genetically engineered bacteria producing succinic acid by xylose metabolism and the method for producing succinic acid by fermentation using the bacteria. The ATP biosynthesis pathway of escherichia coli is modified by means of molecular biology and the enzyme activity related to the pathway is over-expressed; thus the total ATP amount within escherichia coli cells is effectively increased such that the recombinant escherichia coli is able to grow using xylose metabolism, and the succinic acid synthesis efficiency is thereby significantly improved.

The invention discloses a gene engineering strain and an application thereof. The gene engineering strain comprises host cells and target genes transferred into the host cells, wherein the target genes include xylose reductase genes and NADPH (Nicotinamide Adenine Dinucleotide Phosphate) regenerate enzyme genes. The gene engineering strain is applied to the preparation of xylitol which is prepared through the following steps of: inoculating the engineering strain to a culture solution to culture until the OD600 is equal to 0.6-0.8, adding an inducer for inducible expression, and separating and purifying the culture solution after the inducible expression is finished. Compared with the prior art, the activity of the xylose reductase in the engineering strain is greatly improved and is 5 times as high as that of a neurospora crassa; and the gene engineering strain disclosed by the invention also contains an NADPH regeneration system, so that the xylose conversion ratio of the gene engineering strain is as high as 80.32%.

The invention discloses a recombinant saccharomyces cerevisiae engineering strain and application thereof. GAP gene fragments are cloned from Pichiapastoris GS115, and induced promoters of a PYES2 carrier are replaced by constitutive GAP promoters. Then an xylose reductase gene xyl1 fragment and an xylitol dehydrogenase gene xyl2 fragment are connected together and introduced into a PYES2 carrier containing constitutive GAP promoters to constitute a PYES2- GAP-xyl1-xyl2 plasmid. The plasmid is introduced into saccharomyces cerevisiae. The engineering strain of the present invention can co-express xylose reductase and xylitol dehydrogenase, has a high activity and high expression level of xylose reductase and xylitol dehydrogenase. In addition, the invention can efficiently utilize pentose and hexose degraded from cellulose and hemicellulose in straws, so as to increase output of fuel ethanol, reduce production costs and reduce environmental pollution. Therefore, the engineering strain can be used for producing fuel ethanol.

Disclosed is a method for producing ethanol from xylose using recombinant Saccharomyces cerevisiae. In accordance with the method, NADH as a cofactor of xylose reductase required for the ethanol production, and NAD⁺ as a cofactor of xylitol dehydrogenase are coupled with each other and utilized, and acetaldehyde dehydrogenase mediating production of acetic acid as a byproduct is removed, and ethanol can thus be produced at a high yield and high production efficiency.

The invention discloses a preparing method of xylitol. Xylose, glucose, xylose reductase, glucose dehydrogenase, coenzyme, CaCO3 and water form a biological catalysis system, so that xylose is catalyzed to be subjected to a reduction reaction to generate xylitol. Specifically, xylose reductase and NADPH serve as a catalysis system to catalyze xylose to be subjected to the reduction reaction to generate xylitol; glucose dehydrogenase serves as a regenerated catalyst of NADPH, glucose is catalyzed to be subjected to an oxidation reaction to generate gluconic acid, and NADP+ is catalyzed to be subjected to a reduction reaction to generate NADPH. Dual enzymes are adopted to produce xylitol, and after the reaction is conducted for 24 h, xylose and glucose are completely consumed; in products, the converting rate of xylitol is 98%, the concentration of xylitol can reach 146.23 g/L, the converting rate of gluconic acid is 95%, and the concentration of gluconic acid can reach 185.05 g/L.

The invention discloses construction and application of xylitol high-temperature and high-yield engineered strains. Xylose reductase genes from different sources are efficiently expressed in heat-resistant yeast Kluyveromyces marxianus through a gene engineering method, so that the strain can be used for producing a large amount of xylitol by efficiently using and fermenting xylose under higher temperature (which is higher than 42 DEG C) through a xylose reductase metabolism path. Heat-resistant engineered yeast strains YZJ015 and YZJ017 capable of using xylose to carry out fermentation are constructed, and the preservation numbers are respectively CGMCC No.7819 and 7820. The yeast strains can be applied to produce xylitol with higher yield and higher production rate under the temperature of 42-45 DEG C by fermentation through different xylose concentrations. Furthermore, the strain YZJ017 (CGMCC No.7820) can also be used for preparing xylitol from glycerinum and xylose through fermentation.

The invention discloses a method for preparing xylitol by utilizing whole-cell catalysis. The method comprises the following steps: (1) culturing recombinant Escherichia coli containing xylose reductase genes XR and glucose dehydrogenase genes GDH in an LB culture medium until the OD is 0.6-0.8, adding IPTG (Isopropyl-beta-d-Thiogalactoside) to induce expressions of the xylose reductase and glucose dehydrogenase, and centrifuging to obtain whole cells; and (2) enabling an aqueous solution containing xylose and glucose, the whole cells obtained in the step (1) and CaCO3 to form a biological catalysis system, and catalyzing the xylose to carry out a reduction reaction so as to produce the xylitol. The method disclosed by the invention can be effectively applied to comprehensive utilization of straws, the reaction system is applied to treating crop straw hydrolyte, and the substrate of the hydrolyte can be completely consumed within 20 hours. According to the method disclosed by the invention, extraction of enzymes in cells and addition of exogenous cofactors can be saved, and the process and cost for producing the xylitol by biological catalysis can be greatly simplified.