263 results about "Metabolic engineering" patented technology

The invention relates to a construction method of escherichia coli engineering bacteria with high yield of N-acetylglucosamine. According to the method, the metabolic network of escherichia coli is directionally modified through metabolic engineering, the problems of'product re-decomposition ', by-product accumulation, poor genetic stability and the like of natural strains are solved, and efficient industrial production of GlcNAc is realized.

The invention provides a genetically engineered bacterium for producing O-succinyl-L-homoserine as well as a construction method and application of the genetically engineered bacterium. In a chassis bacterium genome, the expression of a 2-ketoglutaric acid decarboxylase encoding gene sucA is enhanced, and the expression of a succinyl-coenzyme A synthetase encoding gene sucD is weakened, so that the supply of succinyl-coenzyme A is increased; the method comprises the following steps: increasing the NADPH (Nicotinamide Adenine Dinucleotide Phosphate) reducing capacity and ATP (Adenosine Triphosphate) energy supply of a chassis bacterium, increasing DNA (Deoxyribose Nucleic Acid) in combination with a transcription dual regulatory factor ompR to improve the stress resistance of escherichia coli under high osmotic pressure, and introducing an overexpression plasmid containing a homoserine transsuccinylase coding gene metA to construct the genetically engineered bacterium for producing O-succinyl-L-homoserine. The engineering strain obtained through a systematic metabolic engineering modification strategy can realize effective accumulation of OSH, the shake flask yield of OSH reaches 19.8 g / L, the fed-batch fermentation yield of a 5L fermentation tank reaches 110.5 g / L, the sugar-acid conversion rate reaches 52.6%, and a foundation is laid for subsequent construction of high-yield OSH engineering bacteria.

The invention relates to the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides a cell for the production of a negatively charged, preferably sialylated, oligosaccharide wherein the cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase. The invention further provides the use of said cells in culture or incubation. Also described are methods of producing negatively charged, preferably sialylated oligosaccharides using the cells, and purification of the negatively charged, preferably sialylated oligosaccharides.

The invention belongs to the technical field of metabolic engineering and biological manufacturing, and particularly relates to an engineering bacterium for producing gallic acid (GA) and application of the engineering bacterium in production of gallic acid. According to the method, key metabolic nodes in the gallic acid production process are systematically regulated, for example, the hydroxylase activity is improved, the pyruvic acid flow direction is regulated, the aromatic amino acid precursor supply is improved, and the dynamic balance between the engineering bacterium growth and the gallic acid product synthesis is realized, so that the gallic acid yield and the carbon yield are remarkably improved. Experiments show that the gallic acid yield of the engineering bacteria prepared by the invention reaches 12.5 g / L in a shake flask experiment, and reaches 109.8 g / L in a 3 L fermentation tank; and the carbon yield is increased to 0.64 g / g glucose, which is the highest level reported in the current literature.

The invention relates to recombinant escherichia coli for de novo synthesis of piceatannol as well as a construction method and application thereof, and belongs to the field of synthetic biology and metabolic engineering. According to the invention, an L-tyrosine production strain is used as an original strain, five key genes required for synthesizing piceatannol are combined and connected to a plasmid vector, and a recombinant plasmid is introduced into the original strain, so that an engineering strain for heterologous synthesis of piceatannol is successfully constructed. According to the invention, the yield of piceatannol is further obviously improved through copy number optimization.

Escherichia coli is modified by means of metabolic engineering, a genetically engineered bacterium for high yield of O-succinyl-L-homoserine is obtained, and a construction method of the genetically engineered bacterium comprises the following steps: weakening a synthetic route of an organic acid metabolic byproduct of a chassis strain E.coli W3110 [delta] metI [delta] metJ [delta] thrB [delta] metB; wherein the synthetic route of the organic acid metabolic byproducts is selected from at least one of a formic acid synthetic route, a lactic acid synthetic route and an ethanol synthetic route. The constructed genetically engineered bacterium is applied to microbial fermentation production of the O-succinyl-L-homoserine, effective accumulation of the O-succinyl-L-homoserine is achieved, the sugar acid conversion rate reaches 46.8%, and a foundation is laid for subsequent construction of high-yield O-succinyl-L-homoserine engineering bacteria.

The invention discloses application of a gene GlZn2Cys661 in regulation and control of synthesis of ganoderic acid. The nucleotide sequence of the gene GlZn2Cys661 is as shown in SEQ ID NO. 1. The gene expression is regulated and controlled by constructing an overexpression or silent vector, so that the synthesis of the ganoderic acid is promoted or inhibited, and an effective method is provided for regulating and controlling the yield of the ganoderic acid. The invention relates to the technical field of bioengineering, and discloses an application of a gene GlZn2Cys661 in regulation and control of ganoderic acid synthesis. The invention provides a key gene target and a core engineering strain for efficiently producing ganoderic acid through a metabolic engineering means, and has important application value.

The invention belongs to the technical field of biology, and particularly discloses a high-density fermentation method and application of recombinant spider silk protein, and the high-density fermentation method comprises the following steps: firstly, transforming an expression vector carrying spider silk protein genes into modified pichia pastoris cells to obtain a strain; the activated strain is subjected to expanding culture, high-density fermentation culture and purification, and recombinant spider silk protein is obtained; wherein the transformation means of the pichia pastoris cell is to integrate Vitreoscilla hemoglobin Vhb gene into the pichia pastoris cell by utilizing a gene recombination technology, and the expression of the Vitreoscilla hemoglobin Vhb gene is improved by carrying out metabolic engineering transformation on the pichia pastoris cell for expression and optimizing the regulation and control of staged fermentation parameters, so that the expression efficiency of the Vitreoscilla hemoglobin Vhb gene is improved. The problems that in existing recombinant protein fermentation, oxygen dependence is high, product degradation is serious, purity is low, and large-scale stability is poor are solved, the yield and purity of recombinant spider silk protein are guaranteed, and large-scale production of the recombinant spider silk protein is facilitated.

The invention relates to the field of microbial metabolism engineering, and discloses a method for increasing the yield of 2 '-fucosyllactose (2'-FL) through the synergistic effect of various alpha-1, 2-fucosyltransferases. According to the method, two or more alpha-1, 2-fucosyltransferases with different sources are co-expressed in microbial cells, and the complementary enzymatic characteristics of the alpha-1, 2-fucosyltransferases are utilized, so that the synthesis efficiency of the 2 '-FL is remarkably improved, and the yield of the 2'-FL is effectively improved. The microbial cell is escherichia coli C43 (DE3) (E. coli C43 lacZ waaF with lacZ and waaF genes knocked out, the serial number is SL), a pETDuet-1 plasmid is constructed, and genes manB, manC, gmd, wcaG and zwf are overexpressed on a pETDuet-1 vector, so that the microbial cell can be used for preparing the microbial cell. In a 5L fermentation tank, after the final strain SL07 is used for final fermentation for 68 hours, the yield of 2 '-FL reaches 148g / L, and the production intensity reaches 2.18 g / L / h. The embodiment shows that the engineering strain for co-expressing the alpha-1, 2-fucosyltransferase from different sources can obviously improve the yield of the 2 '-FL.

The invention relates to a strong promoter P7 suitable for streptomyces and application thereof, and relates to the field of genetic engineering and microbial metabolism engineering. The nucleotide sequence of the strong promoter P7 is as shown in SEQ ID No.1, and the strong promoter comprises a plasmid vector of the strong promoter; a host cell comprising the plasmid vector; the invention also discloses application of the strong promoter, the plasmid vector and the host cell in starting expression of a target gene. Compared with the prior art, the characterization of the strong promoter P7 provides an effective tool element for streptomyces strong promoter engineering and high-efficiency gene expression, and has important significance on streptomyces silent gene characterization, high-efficiency gene expression, metabolite synthesis, metabolic pathway reconstruction and the like. The strong promoter can be applied to common streptomyces type strains, and has important significance on high yield of important proteins including enzymes and important metabolites from actinomycetes.

The invention belongs to the field of metabolic engineering, and discloses a genetically engineered bacterium for producing myo-inositol as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli as an original strain, and is obtained by performing the following gene editing on a genome of the escherichia coli: non-expressed lactose operon repressor protein, glucose phosphate dehydrogenase, acetokinase, glucose phosphate isomerase and glycerol repressor protein; and overexpressing inositol-1-phosphate synthase, inositol monophosphate, glucokinase, glucose permease and glycerol kinase. Compared with the prior art, the genetically engineered bacterium disclosed by the invention has remarkable advantages in the aspects of key enzyme expression rate, metabolism specificity, fermentation period and the like, and an efficient, stable, economical and feasible solution is provided for industrial production of myo-inositol.

The present disclosure provides a recombinant Escherichia coli strain modified by metabolic engineering means and a method for producing L-homoserine by using the same. The strain, designated as Escherichia coli having a strain number of 13-XA, is deposited in China General Microbiological Culture Collection Center (CGMCC) with an accession number of CGMCC No. 25099, dated Jun. 16, 2022. With respect to the chromosome DNA thereof, one or more genes associated with fatty acid metabolism are knocked out or attenuated, and / or a promoter is replaced for enhancement; one or more genes associated with the L-homoserine metabolic pathway are knocked out or attenuated, and / or one or more genes associated with the L-homoserine metabolic pathway are overexpressed or enhanced, and / or one or more genes associated with the L-homoserine metabolic pathway are mutated.

The invention relates to the field of synthetic biology, metabolic engineering and fermentation engineering, and discloses a genetically engineered bacterium and application thereof in production of alpha-ketoglutaric acid.The genetically engineered bacterium comprises the steps that the genetically engineered strain with escherichia coli BW25113 or corynebacterium glutamicum ATCC13032 as a host is constructed; overexpression type isocitrate dehydrogenase genes are integrated through chromosomes, alpha-ketoglutarate dehydrogenase complex genes are knocked out, NADPH invertase is overexpressed through plasmids, and exogenous AKG transporter protein is introduced, so that metabolic flux directional regulation and control and product efflux enhancement are realized; meanwhile, a staged fermentation process is adopted and comprises dynamic gradient adjustment of inorganic phosphorus concentration and two-stage pH control. According to the method, the AKG yield of 78.2 g / L can be obtained under the 5L tank scale, the sugar acid conversion rate reaches 0.68, and the method has the advantages of being high in yield, low in byproduct and controllable in cost. The obtained AKG is suitable for a plurality of fields such as medical intermediates, functional foods and cell culture additives, and has wide industrial application prospects.

The invention belongs to the technical field of bioengineering, and discloses a genetic engineering strain for enhancing schizochytrium limacinum DHA oil production based on carbon flux guidance and fatty acid transport, a method and application thereof. The gene engineering strain is obtained by introducing an optimized coding gene ACLY and / or Fat1p coding gene related to guiding carbon flux and fatty acid transport into a schizochytrium limacinum body; wherein the gene sequence of the gene ACLY is SEQ ID No. 9, and the gene sequence of the gene Fat1p is SEQ ID No. 10. According to the invention, a schizochytrium limacinum engineering bacterium for enhancing DHA oil production based on carbon flux guidance and fatty acid transfer is constructed by means of metabolic engineering. The schizochytrium limacinum engineering bacterium is combined with fatty acid transfer based on carbon flux guiding, can be used for grease synthesis by guiding carbon flux, and can prevent grease from being degraded through fatty acid transfer.

The invention relates to aspartate kinase for relieving feedback inhibition of L-threonine and application of aspartate kinase, and belongs to the field of enzyme engineering and metabolic engineering. The mutant is obtained by carrying out E253K and / or K507E mutation on the basis of wild type aspartate kinase as shown in SEQ ID NO.1, the enzyme activity of the mutant is not obviously changed under the condition that the concentration of L-threonine is 0-12 mmol / L, and the feedback inhibition effect of L-threonine on the mutant is relieved. The method can be widely applied to synthesis of essential amino acids including L-threonine, L-tryptophan, L-isoleucine, L-lysine, L-leucine, L-valine, L-methionine, L-phenylalanine and the like.

The invention belongs to the technical field of microbial genetic transformation, and particularly relates to a method for high-yield production of pleurotus ostreatus transformants, a genetic transformation system and application of the genetic transformation system. Through optimization of protoplast preparation conditions, the number of the obtained protoplast is 4.95 * 10 < 8 > CFU.mL <-1 >, and the regeneration rate of the protoplast in an OM-YEPS regeneration culture medium is 12.7%. A method for producing high-yield pleurotus ostreatus transformants is researched by using a PEG-mediated protoplast transformation method, a set of efficient and stable genetic transformation and screening method is established, about 200-300 positive transformants with stably expressed exogenous genes are obtained, and the transformation efficiency is 65.38%. The genetic transformation system of the high-yield Pleurotus ostreatus transformant, constructed by the invention, lays a solid technical foundation for subsequent gene function verification, metabolic engineering transformation and molecular breeding with excellent characters, and further accelerates the biological research and industrial application process of Pleurotus ostreatus and even other macro fungi.

The invention provides a 2, 5-dimethylpyrazine production strain and a construction method and application thereof.According to the strain, key feedback inhibition is relieved through a perfect metabolic engineering strategy and strategies of knocking out a competitive pathway, introducing heterologous aminoacetone oxidase, deleting L-threonine efflux protein and the like, competitive enzyme conversion of aminoacetone is reduced through a system, and the 2, 5-dimethylpyrazine production strain is obtained. Spontaneous transformation of aminoacetone in Escherichia coli is enhanced, metabolism of L-threonine is expanded to synthesis of 2, 5-dimethylpyrazine, the yield of 2, 5-dimethylpyrazine is further increased, the constructed strain does not contain plasmids and has the advantages of clear genetic background, sustainable transformation and the like, and the 2, 5-dimethylpyrazine is produced through fermentation. The method is simple to operate, can be used for directly synthesizing 2, 5-dimethylpyrazine from the beginning by using a cheap carbon source, and has a very good application prospect.

The present disclosure provides a P450 cytochrome enzyme for andrographolide synthesis and its application, belonging to the field of bioengineering. The present disclosure uses Saccharomyces cerevisiae CEN.PK2-1D as a host, and implements knockout of ROX1 and GAL80 genes on the genome, and integrative expression of GGPP synthase encoding gene and CPS diterpene synthase encoding gene at ROX1 site; and implements free expression of ApCPR and CYP71A8 and CYP71D10 both with truncated signal peptides, successfully constructing recombinant S. cerevisiae, and achieving de novo synthesis of 3,15,19-Trihydroxy-8(17),13-ent-labdadiene-16-oic acid. Compared with the blank, a response value of a product peak reaches 1.9*106, and this strategy provides necessary reference for analyzing biosynthetic pathway of andrographolide and using metabolic engineering to synthesize andrographolide and related derivatives thereof.

The invention relates to a method for efficiently synthesizing lysine by corynebacterium glutamicum based on multi-level metabolism reconstruction, and belongs to the technical field of metabolic engineering. The method is not limited to single gene or single path transformation, but carries out system integrated regulation and control from multiple levels of lysine main synthesis pathway, central carbon metabolism redistribution, by-product synthesis inhibition and lysine transport and tolerance mechanism, and constructs a ''flux-energy-tolerance'' synergistically matched integral metabolism network; the method is a fundamental technical framework for realizing 240g / L high yield. On the premise that genetic stability of engineering bacteria and controllability of the fermentation process are guaranteed, stable and efficient synthesis of lysine under the high-density fermentation condition is achieved, the final fermentation concentration of lysine stably reaches 240 g / L or above, meanwhile, high carbon source conversion efficiency and low by-product generation level are both considered, and the method is suitable for industrial production. Therefore, the technical bottleneck that the existing lysine biological manufacturing technology is limited to the platform stage of yield of about 200 g / L for a long time is broken through.

The present application relates to the engineered microorganism capable of synthesizing calycosin or its derivative and its construction method and application. The present application carries out metabolic engineering modification on the Saccharomyces cerevisiae capable of synthesizing daidzein, screens the isoflavone-3'-hydroxylase capable of efficiently converting the intermediate product into calycosin, optimizes the enzyme combination, obtains the engineered Saccharomyces cerevisiae capable of efficiently synthesizing calycosin and calycosin glycoside, and realizes the calycosin glycoside yield of 47.60 mg / L after 72 h fermentation through the mode of adjusting metabolic flux and optimizing culture conditions, which lays the foundation for the industrialized production of calycosin glycoside.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a glucosamine-6-phosphate synthetase mutant and application thereof. A plurality of glucosamine-6-phosphate synthase mutants capable of remarkably improving the yield of N-acetylglucosamine are screened by performing single-point mutation or combined mutation on amino acid residues near a substrate binding pocket of the glucosamine-6-phosphate synthase or on the surface of protein of the glucosamine-6-phosphate synthase. According to the technical scheme, a foundation is laid for producing glucosamine by further transforming escherichia coli through metabolic engineering.

This invention belongs to the field of microbial metabolic engineering and synthetic biology, specifically relating to a strain for high-yield... R,R -2,3-Butanediol ( R,R Salt-tolerant Jeju Bacillus DT01 IM3 DLB6 (-2,3-BDO), production strain constructed from this strain and its application in fermentation production. R,R Applications of -2,3-BDO. This invention screens a high-yielding strain through heavy ion beam irradiation mutagenesis. R,R Jeju Bacillus DT01 IM3 DL B6 with -2,3-BDO, preservation number CGMCC No. 37311. This strain exhibits excellent salt tolerance and low endotoxin properties. The production strain constructed from it has extremely high product synthesis capabilities, enabling high-concentration, high-intensity industrial fermentation production.

The present application belongs to the field of microbial metabolic engineering and synthetic biology technology application, in particular to a high methanol tolerance yeast chassis strain, a high 3-hydroxypropionic acid yield engineering bacterium and application. The high methanol tolerance yeast chassis strain takes wild type Pichia pastoris or fatty acid producing Pichia pastoris as a starting strain, is modified according to at least one of the following conditions (a)-(h), and a high methanol tolerance strain is obtained, wherein (a) MIOX gene knockout; (b) DMA2 gene knockout; (c) MET17 gene knockout; (d) BUB2 gene knockout; (e) CLB4 gene knockout; (f) UBP12 gene knockout; (g) ZFP gene knockout; (h) AOX1 gene mutation. The high methanol tolerance yeast chassis strain is further modified to obtain a high 3-hydroxypropionic acid yield engineering bacterium. The present application combines rational and non-rational metabolic engineering strategies, realizes efficient biosynthesis of fatty acids and 3-hydroxypropionic acid with methanol as the only carbon source, and lays an important foundation for low-carbon green biological manufacturing of chemicals.

The invention relates to the field of microbial genetic engineering and synthetic biology, and particularly discloses a yersinia pseudotuberculosis temperature-sensitive promoter and application thereof. The screened and identified temperature-sensitive promoter element fills the blank of efficient constitutive promoter resources of yersinia pseudotuberculosis. The promoters can be used as universal gene expression regulation modules to be applied to metabolic engineering and synthetic biology research of yersinia pseudotuberculosis and other prokaryotic hosts, and are beneficial to high-efficiency expression of target protein or biosynthesis of high-value products. According to the method, the expression of the target gene can be regulated and controlled by changing the culture temperature without adding chemical inducers, so that the operation is convenient, the cost is saved, and the method has outstanding advantages in large-scale fermentation production.

This invention discloses a nicotinamide phosphoribosyltransferase mutant, a recombinant strain, and a method for constructing the same. The mutants are I457V, G344A, Q378A, T283G, or F182A, obtained by single-point mutation of the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO:1. This invention utilizes a method derived from... Schizotequatrovirus By rationally designing and site-directed mutagenesis of KVP40 nicotinamide phosphoribosyltransferase, combined with metabolic engineering to block the NMN degradation pathway and enhance substrate synthesis and product transport, the yield of β-NMN by the recombinant strain during shake-flask fermentation reached 4.82 g / L. Through optimization of fed-batch fermentation conditions, the highest yield of β-NMN during fed-batch fermentation in shake flasks reached 15.60 g / L.

The invention belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to yarrowia lipolytica engineering bacteria for producing zeaxanthin as well as a construction method and application of the yarrowia lipolytica engineering bacteria. Specifically, the yarrowia lipolytica is subjected to genetic engineering modification based on a genetic engineering technology, a zeaxanthin biosynthetic pathway is constructed by expressing beta-carotene hydroxylase in an original strain, and meanwhile, a zeaxanthin biosynthetic pathway is constructed through a multiple metabolic engineering modification strategy. The method comprises the steps of key enzyme PaCrtZ multi-copy integration, oxidation-reduction balance regulation and control, upstream mevalonic acid pathway enhancement through membrane-free organelles and the like, and the yield of zeaxanthin is further increased. Meanwhile, by optimizing the culture conditions and applying the optimal fermentation conditions to perform tank fermentation, the yarrowia lipolytica can continuously generate more zeaxanthin, so that the yarrowia lipolytica has excellent practical application value and application prospect.

The present application relates to the field of agricultural biotechnology, in particular to a bidirectional promoter derived from Myceliopthora thermophila and application thereof. The bidirectional promoter derived from Myceliopthora thermophila has a unique bidirectional transcription activity feature, and can simultaneously and efficiently initiate the transcription and expression of two independent genes in the same regulation region in opposite directions. The present application provides a more favorable promoter element for the metabolic engineering of the industrial filamentous fungus Myceliopthora thermophila, significantly simplifies the construction process of a multi-gene co-expression system, and realizes the synergistic expression of two functional genes through a single promoter element, which has a wide application space and market prospect in the production of enzyme preparations, biological medicine products and the like.

The invention provides a construction method of a multi-gene synergistic expression vector and application of the multi-gene synergistic expression vector in improvement of oil tea squalene synthesis, and belongs to the technical field of plant genetic engineering and metabolic engineering. MAPK, PP1 and HMGR gene segments are obtained through an RT-PCR technology, the MAPK, PP1 and HMGR gene segments are cloned to expression cassettes containing independent promoters and terminators respectively, and after Insulator isolation elements are inserted, the expression cassettes are integrated to a pCAMBIA1300 vector through a modular assembly technology. Wherein the HMGR gene contains S47A and T208A mutation sites so as to enhance the enzyme activity. The vector constructed by the invention can cooperatively express a target gene in plant cells, so that accurate regulation and control on the phosphorylation state of HMGR are realized. When the method is applied to tobacco or camellia oleifera callus, the squalene content is effectively increased, the method has the advantages of multi-target regulation and control, efficient and flexible construction, high universality and the like, and an efficient tool is provided for anabolism enhancement of natural products.

The invention discloses a BPS mutant HsamBPS12Mutant and application thereof, and belongs to the field of protein engineering.The BPS mutant HsamBPS12Mutant is modified into a mutant with a chalcone synthase (CHS)-like function on the basis of benzophenone synthase (BPS) in hypericum plants through precisely designed directional mutation of 12 sites, and an effective strategy is provided for modifying natural BPS to obtain new catalytic activity. The obtained mutant retains part of BPS activity, obtains CHS-like catalytic ability, has the characteristic of dual functions, and expands the application boundary of BPS enzymes in metabolic pathway engineering. The mutant can be applied to the fields of plant metabolism engineering, synthetic biology, natural product biosynthesis, efficient production of medicinal components and the like, and has good biotechnology application potential and industrial value.

The present application relates to the field of genetic engineering, in particular to L-tryptophan genetically engineered bacteria under the influence of regulatory factors and a construction method and application thereof, the Escherichia coli is reformed by using metabolic engineering means to improve the yield of L-tryptophan, mainly by inactivating the regulatory factor RpoS in the Escherichia coli, and simultaneously carrying out site-directed mutagenesis on FlhD and Crp, optimizing the metabolic flow of the Escherichia coli, and improving the activity of the regulatory factor capable of promoting L-tryptophan, further improving the ability of the Escherichia coli to produce L-tryptophan and the efficiency of the whole metabolic pathway, effectively improving the production of L-tryptophan, so that the yield of L-tryptophan is doubled compared with the original strain.