524 results about "Metabolic engineering" patented technology

The invention belongs to the technical application field of microbial metabolic engineering and synthetic biology, and particularly relates to a pichia pastoris engineering strain for high-yield sclareol as well as a construction method and application of the pichia pastoris engineering strain. The method comprises the following steps: constructing a biosynthetic pathway of sclareol in a host strain, and optimizing an intracellular mevalonic acid metabolic pathway and a central metabolic pathway to obtain an engineering strain A; the host strain is pichia pastoris; or overexpressing or knocking out a metabolism regulation factor in the obtained engineering bacterium A to obtain an engineering bacterium strain B; or, in the obtained engineering bacterium B, a synthetic pathway is targeted to peroxisome and optimized by adopting a cell region compartment strategy, and an engineering bacterium strain C is obtained. The invention provides a pichia pastoris chassis cell for synthesizing a diterpenoid sclareol and an engineering strain for synthesizing sclareol. By introducing and optimizing the expression of a sclareol synthesis pathway, the fermentation yields of sclareol in shake flask batch and bioreactor batch fed-batch fermentation are respectively 631.6 mg / L and 10.5 g / L.

The invention discloses construction and application of an L-malic acid producing issatchenkia orientalis strain, and belongs to the technical field of microorganisms and the technical field of microbial fermentation and metabolic engineering. Through a metabolic engineering means, a cytoplasm metabolic pathway of an issatchenkia orientalis strain is transformed, and a pyruvate decarboxylase coding gene, glycerol dehydrogenase and lactic dehydrogenase are knocked out; a pyruvate carboxylase gene derived from aspergillus flavus, a malate dehydrogenase gene derived from corynebacterium glutamicum and phosphoenolpyruvate carboxylase derived from escherichia coli are screened and integrated, so that a cytoplasm rTCA cycle synthesis pathway is enhanced; inserting a dicarboxylic acid external transporter gene from schizosaccharomyces cerevisiae at a neutral site GD21, and knocking out endogenous dicarboxylic acid internal transporter; the L-malic acid yield of the modified yeast strain in the fermentation process is remarkably increased and reaches 140.16 g / L. The engineering strain provided by the invention has good industrial application potential in efficient L-malic acid production.

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 recombinant pichia kudriavzevii strain and application thereof, pichia kudriavzevii is taken as an original strain, and a reduced TCA pathway is introduced and comprises the following steps: overexpressing fumarate reductase FRD in the strain; weakening or inactivating an ethanol pathway, comprising: knocking out any one or a combination of at least two of encoding genes of ethanol dehydrogenase ADH1, ADH3 or ADH6; enhancing the PPP pathway in the pentose phosphate pathway of the strain, including: overexpressing glucose-6-phosphate dehydrogenase ZWF1 and / or 6-phosphogluconate dehydrogenase GND1; the method for reducing the activity of the malate dehydrogenase MDH2 in the TCA circulation pathway comprises the step of knocking out the coding gene of the malate dehydrogenase MDH2 in the TCA pathway. The pichia kudriavzevii is modified by using genetic engineering and metabolic engineering methods, so that the yield and the yield of succinic acid are increased, and the pichia kudriavzevii has important application prospects in industrial production.

The invention relates to the technical production field of metabolic engineering and genetic engineering, and particularly provides a uracil production strain as well as a construction method and application thereof. According to the strain, a uridine kinase gene udk is knocked out on an E.coli W3110 genome, and meanwhile, a T7RNA polymerase gene is integrated; the uridine monophosphate kinase gene pyrH is weakened; the method comprises the following steps: overexpressing a carbamyl phosphate synthetase gene carAB, a ribose phosphate pyrophosphate kinase gene prsA, a pyrimidine-5 '-nucleoside enzyme gene ppnN, UMP phosphohydrolase genes umpH and umpG, a ribonucleoside hydrolase 1 gene rihA and a glutamine synthetase gene glnA; an orotic acid nucleoside monophosphate decarboxylase gene pyrF and an orotic acid ribosyltransferase gene pyrE of wild type bacillus subtilis B.subtilis 168 are introduced in a heterologous manner, so that an orotate nucleoside monophosphate decarboxylase gene pyrF and an orotate ribosyltransferase gene pyrE are obtained. The safe, efficient and low-cost production of the uracil is realized.

The invention discloses a rhodosporidium toruloides strain with high homologous recombination efficiency as well as a construction method and application thereof, and belongs to the field of oil-producing yeast engineering bacteria. According to the rhodosporidium toruloides strain, the rhodosporidium toruloides strain NP11-SaCas9-1 is used as an original strain, and an RAD52 gene is over-expressed. The modified yeast engineering bacterium provided by the invention can realize high homologous recombination through CRISPR system mediation, the homologous recombination efficiency reaches 90-100%, a multi-gene editing platform can be constructed on the basis, and the modified yeast engineering bacterium can be applied to the fields of synthetic biology, metabolic engineering, genetic engineering and the like, so that the modified yeast engineering bacterium is simpler, quicker, more convenient and more accurate in the genetic modification process. And the homologous recombination activity of the rhodosporidium toruloides can be improved by expressing the homologous recombination related protein Rad52, and the genetic engineering strategy can be used for modifying other unconventional yeasts.

The invention discloses a combined module for improving the yield of actinomycetes polyketone natural products and construction and application of recombinant bacteria containing the combined module, and belongs to the technical field of genetic engineering. In order to improve the yield of natural products such as polyketone, non-ribosome peptide and the like, the invention provides a combined module capable of improving the yield of actinomycetes natural products by combining and transforming a synthesis pathway of CoASH and a post-modification pathway of PPTase through a metabolic engineering strategy, and the module consists of CoASH cofactor synthesis module elements SCoaAR106A and SCoaD and a PPTase post-modification module element HPC3. By constructing a recombinant vector and recombinant bacteria containing the combined module, the yield of natural products such as actinomycetes polyketone, polyether and non-ribosomal peptide can be effectively improved by over-expression of the combined module.

The invention relates to an L-valine production strain as well as a construction method and application thereof, the strain is obtained by transformation through a metabolic synthesis path of L-valine, and the construction method comprises the following steps: by taking Cornebacterium glutamicum ATCC 13032 (C.glutamicum) as an original strain, carrying out editing transformation such as gene knockout, gene overexpression, gene heterologous expression, gene double-copy expression and the like on a genome of the original strain by utilizing a metabolic engineering means. The L-valine production strain disclosed by the invention can be used for synthesizing L-valine from the beginning by taking glucose as a carbon source, has the advantages of high production rate, short fermentation period, high strain stability and low cost, and lays a foundation for realizing large-scale production of L-valine. According to the fermentation tank fermentation method, the L-valine is produced in a mechanical stirring type fermentation tank by using the L-valine production strain Va1-09, 86.4 g / L of L-valine is produced by fermenting for 36 hours, and the method has a good industrial prospect.

The invention discloses a group of mitochondrial signal peptides and application thereof, and a group of signal peptides with a mitochondrial targeting function is obtained for the first time by screening endogenous mitochondrial key functional proteins of rhodotorula. The invention further realizes expression of a target gene through mitochondrial signal peptide mediation, realizes expression of a target product synthetic pathway gene in rhodotorula mitochondria, and successfully constructs synthetic pathways of terpene such as limonene, linalool, patchouli alcohol and kaurene, branched chain alcohol such as isobutanol and organic acid such as 3-hydracrylic acid. Further, carbon metabolic flow of pyruvic acid flowing to the target product is dredged, and synthesis of the mitochondrial region indoor target product is finally realized. The characterization of a group of rhodotorula mitochondrial signal peptides disclosed by the invention can realize the synthesis of mitochondrial regional chamber target products, and lays a foundation for the research on the realization of metabolic engineering transformation in subcellular organelles.

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 present disclosure relates to the seminal discovery of a generation and use of genetically engineered Vibrio sp. Provided is the use of the genetically engineered bacteria for the construction, maintenance, manipulation, and / or propagation of DNA constructs; protein expression; protein secretion; vectors and other metabolic tools; metabolic engineering; expression of cellular extracts for cell-free biology; shuttle vectors; cloning vectors; and for synthetic biology applications. The disclosure also relates to the use of the replication machinery of Vibrio sp. as a cloning or expression vector for replication of recombinant DNA constructs. The disclosure also relates to methods of use of the above.

The invention discloses a spermidine synthetase mutant and application of the spermidine synthetase mutant in synthesis of spermidine, and belongs to the technical field of genetic engineering and metabolic engineering. According to the method, the spermidine synthetase is mutated firstly, the efficiency of the spermidine synthetase is improved, and on the basis, the dissolved oxygen and fed-batch concentration are optimally controlled through fermentation, so that the yield of spermidine is improved. The corynebacterium glutamicum provided by the invention is an engineering bacterium with plasmids and without defects, and after the corynebacterium glutamicum is subjected to fed-batch fermentation in a 5L fermentation tank for 68 hours, the yield of spermidine reaches 25.1 g / L, and the sugar-acid conversion rate is 17.9%.

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 provides construction and application of a hyaluronic acid recombinant bacterium, and belongs to the fields of biochemical engineering, synthetic biology, genetic engineering and metabolic engineering, the recombinant corynebacterium glutamicum contains a hyaluronic acid synthase gene, a UDP-glucose dehydrogenase gene and a glutamide-6-phosphate fructose transaminase gene; the hyaluronic acid synthase gene, the UDP-glucose dehydrogenase gene and the glutamine-6-phosphate fructose transaminase gene are respectively connected with a sequence RBS1, a sequence RBS2 and a sequence RBS3; the recombinant bacterium provided by the invention also contains a dCas9 protein gene dCas9 and any one of genes sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the recombinant bacterium provided by the invention is high in hyaluronic acid yield and low in production cost.

The embodiment of the invention provides a recombinant pichia pastoris strain as well as a construction method and application thereof. The construction method comprises the following steps: by taking a pichia pastoris strain as a host strain, constructing a dual-synthesis path of phenylalanine ammonialyase and tyrosine ammonialyase in the host strain to obtain a first engineering strain; removing chorismic acid feedback inhibition of the first engineering strain based on metabolic engineering to obtain a second engineering strain; optimizing an aromatic amino acid synthesis route of the second engineering strain to obtain a third engineering strain; knocking out a branch metabolic pathway of the third engineering strain to obtain a fourth engineering strain; increasing precursor supply of the fourth engineering strain to obtain a recombinant pichia pastoris strain; according to the construction method provided by the embodiment of the invention, the synthesis path of p-coumaric acid can be systematically optimized, the recombinant pichia pastoris strain for efficiently producing p-coumaric acid by taking methanol as a carbon source is constructed, and compared with a host strain, the recombinant pichia pastoris strain has the advantage that the yield of p-coumaric acid is remarkably increased.

The invention relates to a method for producing glycosylglycerol. The method comprises the following steps: S1, culturing trichocephalus microcoleus to a logarithmic phase; and S2, adding glycerol and glucose into the culture environment of the coleus microcoleus to induce the coleus microcoleus to accumulate the glycosylglycerol. According to the invention, by optimizing the culture of the coleus microcoleus BL0902, the production of the glycosylglycerol by using the strain is realized, and on the basis, the strain is subjected to metabolic engineering transformation, so that the yield of the glycosylglycerol is greatly improved by the obtained engineering strain.

The invention relates to the technical production field of metabolic engineering and genetic engineering, and particularly provides a genetically engineered bacterium for producing 2 '-deoxyuridine as well as a construction method and application of the genetically engineered bacterium. According to the strain, deoxyuridine kinase tdk is knocked out on an E.coli W3110 genome, and T7 RNA polymerase is integrated; an aspartate transaminase gene aspC, a glutamine synthetase gene glnA, a 5 '-deoxynucleotidase gene yfbR and a uridine monophosphate kinase gene pyrH are subjected to overexpression; the method comprises the following steps: introducing a ribonucleoside diphosphate reductase gene nrdBCA from Escherichia phage T4 in a heterologous series connection manner; the CTP synthase gene pyrG * of Corynebacterium glutamicum is introduced in a heterologous mode, and the CTP synthase gene pyrG * of Corynebacterium glutamicum is introduced in a heterologous mode. The safe, efficient and low-cost production of the 2 '-deoxyuridine is realized.

The invention belongs to the field of application of microbial genetic engineering and metabolic engineering, and particularly relates to a method for improving the biosynthesis efficiency of sclareol. Specifically, in a host strain, gene knockout is realized through a CRISPR / Cas9 technology, or / and a DNA fragment is introduced, and a strain metabolic pathway is adjusted to obtain a corresponding engineering strain; carrying out biosynthesis by utilizing the obtained engineering strain to obtain sclareol; the gene knockout is realized by introducing donor DNA (Deoxyribose Nucleic Acid) containing upstream and downstream homologous arms of a coding frame; the DNA fragment sequentially comprises an upstream homologous arm, a promoter, a gene, a terminator and a downstream homologous arm from upstream to downstream, the host strain is selected from any one of saccharomyces cerevisiae. According to the method, the cell life is associated with product synthesis, and meanwhile, metabolic balance is coupled, so that the synthesis efficiency of a target product is improved, and a foundation is laid for industrial fed-batch fermentation.

The invention relates to engineering Escherichia coli for producing L-fucose and application of the engineering Escherichia coli, the production of the L-fucose is enhanced through a metabolic engineering strategy by utilizing a mode of combining genome integration and plasmid expression path genes, and the efficient production of the L-fucose is realized by optimizing a double-plasmid system and enhancing precursor supply. In addition, a hok / sok system and a cysC auxotroph system are introduced, and the plasmid-based engineering strain without antibiotic dependence is successfully constructed. The strain produces 7.99 g / L of L-fucose and 61.91 g / L of L-fucose through shake-flask culture and fed-batch culture under the condition that antibiotics are not added, the highest L-fucose yield reported so far is provided, and the use of antibiotics is completely not needed in the fermentation process.

The invention discloses a yarrowia lipolytica engineering strain with high yield of resveratrol as well as construction and application of the yarrowia lipolytica engineering strain, and belongs to the technical field of synthetic biology and metabolic engineering. The invention provides a yarrowia lipolytica engineering strain. A 4-coumaric acid-CoA ligase gene, a multi-copy resveratrol synthase gene, a transketolase 1 gene, a fusion gene of a histidine phosphate aminotransferase gene and a tyrosine ammonia lyase gene, a glycolytic pathway hexokinase gene, a 6-phosphofructokinase-1 gene and a phosphoglycerate kinase gene are integrated, so that the recombinant protein is obtained. According to the present invention, by using glucose as the substrate, the high yield of resveratrol can be achieved by using glucose as the substrate, the resveratrol yield during shake-flask culture fermentation can achieve 3.157 g / L, the resveratrol yield can be increased to 30.7 g / L during 5L-scale fed-batch fermentation, and the resveratrol yield can be increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L; the maximum value is reported by the yarrowia lipolytica system at present.

The invention discloses a method for efficiently producing ergothioneine by metabolic engineering modified escherichia coli and application, and belongs to the technical field of biology. By adopting the recombinant escherichia coli EGT-1-B * provided by the invention, glucose can be converted into ergothioneine; therefore, the raw materials are cheap and easy to obtain, additives and heavy metals which are harmful to human health are avoided, a good foundation is laid for later product purification, and the requirements of commercial application can be met. Furthermore, genes related to metabolism of histidine, methionine, cysteine and S-adenosylmethionine are further overexpressed in the recombinant escherichia coli EGT-1-B *, so that the content of precursor amino acid synthesized by the ergothioneine is increased, and meanwhile, after the gene yqaB is knocked out, the yield of the ergothioneine is further increased; the utilization rate of glucose to ergothioneine can be further improved, and the yield of ergothioneine can reach 140.29 mg / L.

The invention relates to the field of industrial biotechnology. The invention can be integrated in the biotechnological production of chemicals and fuels as it provides for a more sustainable alternative. In particular, the invention relates to the metabolic engineering of a microorganism for the production of an aromatic compound of interest.

The invention belongs to the field of metabolic engineering, and discloses a genetically engineered bacterium of high-yield beta-alanine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli W3110 as an original strain, and is obtained by performing the following gene editing on a genome of the escherichia coli W3110, the genetically engineered bacterium does not express proteins such as lactose operon repressor protein, pyruvate dehydrogenase, acetokinase and the like; protein such as aspartic acid-alpha-decarboxylase, phosphoenolpyruvate carboxylase, pyruvate carboxylase and the like is overexpressed; the phosphoenolpyruvate carboxykinase is replaced by phosphoenolpyruvate carboxykinase from Mannheim succinic acid producing bacteria. The genetically engineered bacterium BA-17 for high yield of beta-alanine, which is constructed by the invention, is fermented in a 5L fermentation tank for 60 hours, the yield of beta-alanine reaches 126.7 g / L, the conversion rate reaches 60.3%, and the production intensity reaches 2.11 g / L / h.

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 belongs to but not limited to the technical field of gene engineering, and discloses a method for improving the biosynthesis of yeast terpenoids and application of the method, and the method for improving the biosynthesis of the yeast terpenoids based on m7G methyltransferase complex genes TRM8-TRM82 comprises the following steps: overexpressing a TRM8 gene and a TRM82 gene; in a saccharomyces cerevisiae engineering strain genome for producing terpenoids, a promoter of a yeast endogenous TRM8 gene is replaced by a constitutive promoter PGK1p, and a promoter of a yeast endogenous TRM82 gene is replaced by a constitutive promoter TEF1p, so that the production efficiency of the saccharomyces cerevisiae terpenoids can be improved. The invention constructs a new wine brewing yeast engineering bacterium for producing the terpenoid lupeol as a representative compound, and the yield of the lupeol can be effectively improved. The new metabolic engineering method for improving the biosynthesis of the yeast terpenoids based on the TRM8-TRM82 has a remarkable effect.

The invention belongs to the technical field of synthetic biology genome editing, and relates to a chassis strain for gene cluster in-vivo traceless editing, a construction method and a gene cluster traceless editing method.The chassis strain is based on escherichia coli BL21 (DE3) and integrates a CRISPR-Cas9 system and a homologous recombination system (lambda Red) of a lambda bacteriophage source; and then knocking in an inducible sgRNA expression cassette (sgT) of a targeted editing vector to construct the vector. The strain has an efficient editing capability aiming at exogenous DNA, traceless editing of a target biosynthetic gene cluster is realized, and editing plasmids can be quickly eliminated, so that iterative editing is facilitated. The method is simple, convenient and rapid to operate and high in practicability, and provides powerful technical support for metabolic engineering and synthetic biology breeding transformation of streptomyces industrial strains, activation of synthetic gene clusters of recessive natural products and excavation of novel natural products.

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 recombinant escherichia coli capable of increasing the yield of fucosyllactose and a construction method and application of a fucosyltransferase mutant of the recombinant escherichia coli, and belongs to the field of synthetic biology and metabolic engineering. According to the invention, recombinant escherichia coli B20 and B21 are constructed based on key enzyme engineering transformation. Under the shake flask condition, the 3-FL production capacity of the recombinant strain is obviously improved to 6.20 g / L and 6.34 g / L from the initial 3.45 g / L, and the amplification reaches 79.7% and 83.8% respectively. The yields of 3-FL of the strain B20 and the strain B21 respectively reach 53.88 g / L and 54.64 g / L under the culture condition of a 3L fermentation tank. The recombinant escherichia coli for expressing the 2 '-FL is constructed, and the yield of the 2'-FL reaches 64.86 g / L in a 3L fermentation tank. The invention provides a new technical scheme for efficient microbial synthesis of the 3-FL.

This disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. This disclosure describes a cell metabolically engineered for production of a mixture of at least three different oligosaccharides. Furthermore, this disclosure provides a method for the production of a mixture of at least three different oligosaccharides by a cell as well as the purification of at least one of the oligosaccharides from the cultivation.