403results about "Carbon-nitrogen lyases" patented technology

The invention relates to a recombinant escherichia coli THRS and a method for synthesizing L-threonine by using the recombinant escherichia coli THRS. The invention belongs to the technical field of bioengineering, and provides a recombinant escherichia coli THRS which overexpresses encoding genes of ppc, aspC, aspA, thrAmutBC, asd, rhtC, rhtA, asdtm and thrAmut, knockout an encoding gene of fnr and knockout an encoding gene of iclR, the recombinant escherichia coli THRS takes glucose as a substrate for fermentation, and the yield and conversion rate of L-threonine are obviously improved; the L-threonine strain has the advantages that the L-threonine strain does not contain exogenous plasmids, all modification sites are integrated onto a genome, higher stability is achieved, antibiotics do not need to be added into a fermentation culture medium of the strain, and the produced L-threonine product is free of antibiotic residues and higher in safety.

The invention relates to the technical field of biosynthesis, and particularly discloses a pyridone compound as well as a biosynthesis method and application thereof. On the basis of related technologies of synthetic biology, non-ribosome peptide synthetase is modified, non-ribosome peptide synthetase mutants are heterologously expressed in various hosts, a pyridone compound is found and obtained for the first time, the chemical molecular formula of the pyridone compound is determined to be C5H4N2O3, and the pyridone compound is named as 5-aminopyridine-2, 3, 6-triketone or 5-hydroxy-3-iminopyridine-2, 3, 6-triketone or 5-hydroxy-3-iminopyridine-2, 3, 6-triketone or 5-hydroxy-3-iminopyridine-2, 3, 6-triketone. The novel pyridone compound can be used as a raw material or an intermediate to be applied to development and preparation of novel antibiotics, drugs and dyes.

The embodiment of the invention provides a tyrosine ammonia lyase mutant and application thereof in synthesis of p-coumaric acid. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, and L248M + F497I + M4T + V482L. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO. 2 is taken as a template, a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

The embodiment of the invention provides a method for obtaining a tyrosine ammonialyase mutant based on high-throughput screening, the tyrosine ammonialyase mutant and application of the tyrosine ammonialyase mutant. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO.2: A245G + A247T, and D192V + A245G + A247T. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO.2 is taken as a template (basis), a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield, which indicates that the mutant has more significant application prospects in catalysis of L-tyrosine to generate p-coumaric acid.

The invention discloses application of threonine dehydratase in regulation and control of growth of Didymeria segeticola and application of threonine dehydratase serving as a bactericide target in regulation and control of growth of Didymeria segeticola. The threonine dehydratase gene Td disclosed by the invention is derived from Didymeria segeticola, and a knockout mutant is obtained by constructing a knockout gene segment of the Td gene and introducing the knockout gene segment into a D.segeticola protoplast. Experiments find that the Td gene plays a role in growth of D.segeticola, in-vivo pyruvic acid content, in-vivo ATP (adenosine triphosphate) content and sensitivity to wuyiencin. Molecular docking analysis, molecular dynamics simulation and trace thermophoresis experiments prove that the threonine dehydratase can be used as a bactericide target, and the bactericide wuyiencin is obtained through screening. The threonine dehydratase disclosed by the invention can be used as a bactericide target and a key protein of a disease pathogenic mechanism, and has a wide application prospect in prevention and treatment of plant pathogenic fungal diseases.

This application relates to the field of biosynthesis technology, and specifically discloses a pyridone compound, its biosynthesis method and application. Based on synthetic biology-related technologies, this application modifies non-ribosomal peptide synthetase, heterologously expresses non-ribosomal peptide synthetase mutants in a variety of hosts, and for the first time discovers and obtains a pyridone compound, and determines its chemical formula as C5H4N2O3, named 5-amino-pyridine-2,3,6-trione or 5-hydroxy-3-imino-pyridine-2,6-dione. This novel pyridone compound can be used as a raw material or intermediate in the development and preparation of novel antibiotics, drugs and dyes.

The invention discloses a Sarcabomyces lipolytica strain for synthesizing (2S)-naringenin and application of the Sarcabomyces lipolytica strain, and belongs to the technical field of engineering and bioengineering. According to the invention, a key enzyme CHS is subjected to semi-rational transformation, so that the yield of the strain (2S)-naringenin expressing the mutant V196A / T132C is increased by 60%. According to the method, the modified CHS is further integrated into a yeast genome, conversion of p-coumaric acid to (2S)-naringenin is promoted by strengthening a shikimic acid pathway and enhancing supply of cofactors, so that the yield of the (2S)-naringenin reaches 776mg / L after the constructed strain is fermented in a 24-pore plate for 96 hours, and the yield of the constructed strain can reach 8.65 g / L after the strain is fermented in a 5L fermentation tank; the method lays a foundation for the subsequent biosynthesis of flavonoid compounds, and has potential value and significance for the development of synthetic biology.

Provided herein, inter alia, is an engineered non-plant cell that produces a hyoscyamine alkaloid product, a precursor of a hyoscyamine alkaloid product, or a derivative of a hyoscyamine alkaloid product. Also described is a method for producing a scopolamine alkaloid, a precursor of a scopolamine alkaloid product, or a derivative of a scopolamine alkaloid product using a cell.

The invention relates to the technical field of genetic engineering, and discloses a genetically engineered bacterium for producing L-arginine as well as a construction method and application of the genetically engineered bacterium. Escherichia coli is taken as a starting strain, and the following treatment is performed: overexpressing genes gdhA and gdh of glutamate dehydrogenase, promoting generation of glutamic acid, overexpressing and coding arginine transporter gene argO to enhance extracellular transport of arginine, introducing exogenous argJ to replace a linear synthesis pathway in large intestines, improving the utilization rate of acetyl coenzyme A and accelerating the synthesis efficiency of arginine; a glutamine synthetase gene glnA is over-expressed, and glutamine synthesis is enhanced; the pckA is overexpressed to promote ATP synthesis; meanwhile, speA and adiA genes for coding the arginine decarboxylase are deleted, so that further synthesis of putrescine from L-arginine is inhibited, and the L-arginine accumulation capability of the strain is improved. Through the combined action of the treatment, a genetically engineered bacterium with extremely high L-arginine production yield is finally obtained, and the shake flask fermentation yield can reach 13.7 g / L.

Certain C. glutamicum strains overexpress genes coding for enzymes having the function of aspartate-semialdehyde dehydrogenase, aspartate aminotransferase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, diaminopimelate decarboxylase, aspartatokinase and diaminopimelate dehydrogenase. The C. glutamicum strains also express NAD(P)(+) transhydrogenase subunit alpha PntA and NAD(P)(+) transhydrogenase subunit beta PntB of Corynebacterium urealyticum. A method is developed for the fermentative production of L-lysine using such C. glutamicum strains.

The present invention relates to a modified bacterium with anti-tumor activity, which comprises an essential gene expression cassette under the control of a strictly hypoxia inducible promoter and is deficient in at least one gene involved in or regulating the endogenous anti-oxidative stress response pathway or a functional expression product thereof. The present invention also relates to a pharmaceutical composition comprising the modified bacterium and anti-tumor use thereof.

The invention relates to a recombinant thallus, a construction method of the recombinant thallus and application of the recombinant thallus in production of xanthoxylin WGX-50, and relates to the technical field of bioengineering. A recombinant plasmid containing a phenylalanine ammonialyase gene PAL with a nucleotide sequence as shown in SEQ ID NO.2 and a cinnamic acid coenzyme A ligase gene CNL with a nucleotide sequence as shown in SEQ ID NO.1 and a recombinant plasmid containing a tyramine N-hydroxycinnamoyl transferase gene THT with a nucleotide sequence as shown in SEQ ID NO.3 are introduced into the recombinant plasmid. A phenylalanine ammonialyase gene PAL, a cinnamic acid coenzyme A ligase gene CNL and a tyramine N-hydroxycinnamoyl transferase gene THT are overexpressed in escherichia coli, so that a biological synthesis path of xanthoxylin WGX-50 is formed by taking 3, 4-dioxomethyl phenylethylamine as a substrate for the first time, a foundation is laid for large-scale industrial production, and the method has a wide application prospect. The method has important economic value and social benefit.

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a genetically engineered bacterium for stably and efficiently producing L-isoleucine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes E. coliTHRD as a host and contains an RNA (Ribonucleic Acid) polymerase beta-subunit mutant coding gene rpoBM; compared with a parent RNA polymerase beta-subunit, the RNA polymerase beta-subunit mutant has the advantage that the amino acid residue at the 618th site of the amino acid sequence is sequentially replaced with leucine from the N terminal to the C terminal. The genetically engineered bacterium disclosed by the invention can tolerate L-isoleucine and accumulate less alpha-ketobutyric acid, and has a very good industrial application prospect.

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 provides a lysine artificial operon and application thereof. The gene sequence of the artificial operon comprises a promoter, a lysC gene for coding aspartate kinase, an asd gene for coding aspartate semialdehyde dehydrogenase, a dapA gene for coding dihydropicolinic acid synthetase and a dapB gene for coding dihydropicolinic acid reductase, the ddh gene is used for coding diaminopimelate dehydrogenase; the lysA gene is used for coding diaminopimelate decarboxylase; except for the first lysC gene, sequences of RBS ribose binding sites of other genes are RBS ribose binding sites translated after gene transcription by intercepting 20-26nt DNA sequences at the upstream of a coding region ATG (adenosine triphosphate); the RBS ribose binding site is connected in front of the corresponding gene.

The invention relates to the field of bioengineering, in particular to a fusion enzyme mutant and application thereof in fluororesveratrol synthesis. The invention provides a mutant composition. The mutant composition comprises a mutant of p-coumaroyl-CoA ligase and a mutant of stilbene synthase, the mutant of the p-coumaroyl-coenzyme A ligase is characterized in that on the basis of wild type p-coumaroyl-coenzyme A ligase, the mutant has mutation at the 113th site and / or the 537th site; the mutant of the stilbene synthase comprises mutation at the 170th site on the basis of wild type stilbene synthase. According to the invention, the fusion enzyme is subjected to directed evolution by applying a semi-rational CAST / ISM strategy and an irrational error-prone PCR strategy for the first time, a fusion enzyme mutant At4CL (F537G / F113G)-VvSTS (V170A) with improved catalytic activity is obtained, and finally, a series of fluorine-containing resveratrol is efficiently synthesized.

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 relates to the technical field of microbial metabolism engineering, in particular to a corynebacterium glutamicum producing strain for efficiently producing D-pantothenic acid as well as a construction method and application of the corynebacterium glutamicum producing strain. The construction method specifically comprises the following steps: (1) by taking wild corynebacterium glutamicum ATCC 13032 as a chassis, weakening ilvA, and constructing DPA1 through mutation expression; (2) knocking out the quinone pyruvate oxidoreductase and inserting a panBCE gene expression cassette into the quinone pyruvate oxidoreductase to construct DPA2; (3) introducing and using a pEC-xk99E overexpression plasmid to screen and express Escherichia coli W3110, Bacillus subtilis 168, and endogenous acetohydroxy acid synthase, acetolactate isomerized reductoisomerase and 2-hydroxy acid dehydratase, and carrying out a step (2) of carrying out a step (3) of screening and expressing the Escherichia coli W3110, Bacillus subtilis 168, and endogenous acetohydroxy acid synthase, acetolactate isomerized reductoisomerase and 2-hydroxy acid dehydratase in the DPA2; and (4) under the control of a strong promoter, carrying out tandem overexpression on the screened ilvBNCD gene by using pEC-xk99E, constructing DPA3, and enhancing a pantothenic acid main synthesis pathway and an upstream pathway pulling force, thereby obtaining the corynebacterium glutamicum for efficiently producing D-pantothenic acid. The strain provided by the invention can produce D-pantothenic acid with high yield and high conversion rate by utilizing a carbon source, and has important industrial application value.

The invention discloses a method for catalytic synthesis of malonic acid by using whole cells, and belongs to the field of bioengineering. According to the invention, escherichia coli BL21 (DE3) is taken as a host, an aspartic acid ammonia lyase gene aspA and an aspartic acid-alpha-decarboxylase gene panD, a succinate semialdehyde dehydrogenase gene yneI and a beta-alanine pyruvate transaminase gene, a beta-alanine pyruvate transaminase gene bauA and a malonyl-coenzyme A reductase gene mcr-C are expressed in modules, and recombinant escherichia coli is constructed. The malonic acid is produced by adopting a whole-cell catalysis method, so that the production period is shortened, the production efficiency of the malonic acid is improved, and the yield of the malonic acid can reach 51.0 g / L after the malonic acid is catalyzed for 24 hours.

The invention relates to the technical field of synthetic biology and microbial engineering, in particular to recombinant escherichia coli for de novo synthesis of forsythiaside A. The invention provides recombinant escherichia coli for de novo synthesis of forsythiaside A. A preparation method of the recombinant escherichia coli comprises a step of improving an escherichia coli starting strain, and the escherichia coli starting strain is escherichia coli MG1655. In the fermentation process of the recombinant escherichia coli, effective accumulation of forsythiaside A in fermentation liquor can be achieved, and a foundation can be laid for subsequent construction of high-yield forsythiaside A engineering bacteria.

The present invention is in the field of genetically modified enzyme and microorganism comprising such a modified enzyme for the production of raspberry ketone or zingerone. The modified enzyme is a polyketide synthase (PKS) issued or derived from a wild type PKS. The modified PKS has the capability to produce vanillylidene acetone from feruloyl-CoA and / or 4-hydroxybenzalacetone from 4-coumaroyl-CoA in a more effective way as compared to wild type BAS or wild type PmPKS, in particular in recombinant bacteria strains.

Genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with hyperphenylalaninemia are disclosed.

The application provides a recombinant Pichia pastoris strain and a construction method and application thereof. The construction method comprises the following steps: taking a Pichia pastoris strain as a host strain, constructing a double synthesis pathway of phenylalanine ammonia lyase and tyrosine ammonia lyase in the host strain to obtain a first engineering strain; relieving the branch acid feedback inhibition of the first engineering strain based on metabolic engineering to obtain a second engineering strain; optimizing the aromatic amino acid synthesis pathway of the second engineering strain to obtain a third engineering strain; knocking out the branch metabolic pathway of the third engineering strain to obtain a fourth engineering strain; increasing the precursor supply of the fourth engineering strain to obtain the recombinant Pichia pastoris strain. The construction method can systematically optimize the synthesis path of p-coumaric acid, and the recombinant Pichia pastoris strain for efficiently producing p-coumaric acid with methanol as a carbon source is constructed. Compared with the host strain, the yield of p-coumaric acid of the recombinant Pichia pastoris strain is significantly improved.

The invention provides a strain for producing L-isoleucine as well as a construction method and application thereof. The strain is obtained by modifying a chassis strain escherichia coli XX12 by utilizing a metabolic engineering modification method, ldhA, adhE and pflB genes are deleted, the transcriptional levels of aspC, pykF, pntAB, ppK, ppc, ilvAfbr, ilvIHfbr and ygaZH genes are up-regulated, the transcriptional level of a leuA gene is down-regulated, a bcd gene derived from B.subtilis 168 and ppnK and cysK genes derived from Cornebacterium glutamicum ATCC 13032 are heterologously expressed, and the strain has the advantages that the strain can be used for producing L-isoleucine; the method has the advantages of no need of adding resistant substances, good L-isoleucine synthesis capability, short fermentation period by using glucose as a carbon source, and good economic benefit and industrial application value.

The invention discloses a method for producing p-coumaric acid through double-enzyme cascade catalysis, which comprises the following steps: taking phenol, pyruvic acid and ammonium chloride as substrates, and carrying out cascade enzyme catalytic reaction on a tyrosine phenol lyase product and a tyrosine ammonia lyase product to synthesize p-coumaric acid. According to the method, phenol, pyruvic acid and ammonium chloride are used as substrates, a tyrosine phenol lyase product and a tyrosine ammonia lyase product are used as catalysts, the tyrosine phenol lyase product can convert the substrates into L-tyrosine, and the tyrosine ammonia lyase product can directly use the L-tyrosine as the substrate to convert the L-tyrosine into p-coumaric acid. Therefore, the tyrosine phenol lyase product and the tyrosine ammonia lyase product can realize cascade enzyme catalytic reaction in the same reactor, phenol with lower cost can be adopted as a conversion substrate, separation and purification of an intermediate product L-tyrosine are avoided, and the whole process flow is simple and efficient.

The invention discloses a construction method of a cyanobacteria-corynebacterium glutamicum artificial photosynthetic mixed bacteria system. The construction method comprises the following steps: step S1, amplifying by taking plasmid pUC-tho as a template to obtain a theophylline inducible promoter Ptho; step S2, carrying out amplification by taking a Synechocystis sp. PCC 6803 genome as a template so as to obtain a strong promoter Pcpc560, and carrying out amplification on the strong promoter Pcpc560 so as to obtain a strong promoter Pcpc560; step S3, carrying out amplification by taking the plasmid pUC-PAL as a template to obtain an arabidopsis thaliana phenylalanine lyase gene pal; step S4, carrying out amplification by taking the plasmid pUC-cscB as a template to obtain an escherichia coli W sucrose transporter gene cscB; step S5, carrying out amplification by taking the integrated plasmid Pcp3031 as a template, so as to obtain a terminator Trbcl; and step S6, fusing the strong promoter Pcpc560, the arabidopsis phenylalanine lyase gene pal and the terminator Trbcl, and respectively connecting with an expression vector pSI-SPE to obtain the expression vector containing the gene pal. According to the invention, a'cyanobacteria-corynebacterium glutamicum 'artificial photosynthetic mixed bacteria system is constructed by simulating a mutualistic symbiotic relationship ubiquitous in nature, and negative carbon biosynthesis of cis-muconic acid is realized.

The present invention relates to a recombinant Escherichia coli comprising at least one therapeutically active ingredient, preferably a therapeutic enzyme, in its outer membrane vesicles (OMV). The invention also relates to the use of this recombinant EcN to penetrate the intact intestinal epithelial barrier through OMV to deliver said therapeutically active ingredient, preferably a therapeutic enzyme, into the circulation. The OMV containing the therapeutic enzyme can stably catalyze various detoxification reactions in circulation, and is particularly used for clinically treating metabolic disorders (such as hyperuricemia) and the like.

The invention provides a crocodile source Aeromonas daka argH gene deletion strain, a crocodile source Aeromonas daka argH gene back-filling strain and a construction method and application of the crocodile source Aeromonas daka argH gene deletion strain and the crocodile source Aeromonas daka argH gene back-filling strain, an argH gene recombinant suicide plasmid pRE112-delta argH is constructed by adopting a seamless cloning method, an Aeromonas daka strain is jointed with E.coli WM3064 containing the pRE112-delta argH to obtain the argH gene deletion strain delta argH, and a corresponding gene back-filling strain C delta argH is further constructed. Through biological characteristic analysis, the argH gene deletion reduces synthesis of arginine of Aeromonas dakara and biofilm formation amount, reduces growth rate, proteolytic activity and resistance to crocodile-derived antibacterial peptide, but enhances hemolytic activity, and significantly improves the survival rate of Caenorhabditis elegans, so that the Aeromonas dakara has a good application prospect in treatment of caenorhabditis elegans, and the Aeromonas dakara has a good application prospect in treatment of caenorhabditis elegans. Important reference is provided for further researching the regulation effect of the argH gene in a bacterial pathogenic mechanism and developing related prevention and control strategies.

The invention relates to the technical field of synthesis of natural dyes by a biological method, in particular to an antibiotic-demand-free ornamental blue high-yield strain as well as construction and application of the antibiotic-demand-free ornamental blue high-yield strain. Corynebacterium glutamicum is used as an initial strain, a tricarboxylic acid circulating gene is regulated and interfered, and the copy number of exogenous blue pigment synthetase BpsA in a genome is adjusted, so that the ornamental blue high-yield strain is obtained. The strain does not need to use antibiotics in the fermentation process, the yield is stable, the product production capacity equivalent to the level when antibiotics are used can still be achieved through fermentation under the antibiotic-free condition, and the growth performance of the strain is kept. The method for producing the ornamental blue by using the strain provided by the invention ensures that the ornamental blue product is natural and green, also improves the efficient production capacity of the strain, solves the contradiction between green environmental protection and stable high yield of the ornamental blue dye, is green and environment-friendly, and has a remarkable industrial application prospect.