216results about "Carbon-nitrogen lyases" patented technology

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.

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 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 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 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 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 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.

The invention provides a tyrosine ammonia lyase mutant and application thereof in production of p-coumaric acid. The tyrosine ammonia lyase mutant is obtained by mutating wild type tyrosine ammonia lyase with an amino acid sequence as shown in SEQ ID No.1, and a mutation site is selected from at least one of Q108S, Y340Q and P375H. The catalytic activity center of wild type tyrosine ammonia lyase is analyzed, and site-directed mutagenesis is carried out on five catalytic activity center sites, so that the mutant shows different enzyme catalytic activity when the mutation sites are different, the mutation sites are the same but the mutation targets are different; q108S, Y340Q and P375H single mutations can significantly improve the enzyme catalytic activity of the tyrosine ammonia lyase mutant, and the tyrosine ammonia lyase mutant having any two mutation sites, especially having three mutation sites, also has better enzyme catalytic activity than the wild type, and has great application potential.

A method for preparing glycine by using threonine relates to a fermentation process in which threonine is decomposed into glycine and acetaldehyde by aldolase. Glycine and acetyl coenzyme A can be produced in a fermentation process, in which acetaldehyde is reduced into acetyl coenzyme A or an acetyl coenzyme A derivative by acetylating acetaldehyde dehydrogenase; or threonine is dehydrogenated by threonine dehydrogenase to obtain 2-amino-3-ketobutyric acid, which is then ligated by 2-amino-3-ketobutyrate CoAligase to obtain acetyl coenzyme A. Coenzyme A can be converted into an acetyl coenzyme A derivative under different fermentation conditions.

The invention relates to the technical field of bioengineering, and discloses a phenylalanine ammonia lyase mutant and application thereof. In order to improve the synthesis efficiency of the L-4, 4 '-biphenyl alanine derivative, single-point or multi-point combined substitution is carried out on the 96th amino acid, the 97th amino acid and the 410th amino acid of the phenylalanine ammonia lyase sourced from Lactobacillus bacterium to obtain mutants, and the mutants are single mutants F96V and L97V, and double-site combined mutants F96V / I410V and L97V / I410V. Compared with phenylalanine ammonialyase from other sources, the phenylalanine ammonialyase mutant provided by the invention can accept a large-volume (E)-4-phenylcinnamic acid substrate, has higher catalytic conversion rate, wider substrate range and higher yield, is lower in production cost, and is suitable for industrial production. And the large-volume biphenyl alanine compound can be asymmetrically prepared with extremely high stereoselectivity.

Aspects of the disclosure relate to aromatic amino acid ammonia lyases (ALs), phenylalanine ammonia lyases (PALs), and tyrosine ammonia lyase (TALs), including engineered enzymes, and their use in catalyzing chemical reactions.

The invention provides a composition comprising a Plasmodium species multimeric protein capable of forming a nanoparticle and at least one antigen of interest, wherein the multimeric protein comprises a monomer chosen from Plasmodium species pyridoxal 5'-phosphate synthase (PLP), chaperone 60 protein (Cpn60), and caseinolytic protease (Clp). In embodiments, the invention provides the composition wherein the monomers assemble to form a multimeric nanoparticle and / or which comprises an adjuvant. The invention further provides a nucleic acid encoding the multimeric protein and the at least one antigen of interest, E. coli comprising the nucleic acid, and the use of such in a method for producing the inventive multimeric protein and the at least one antigen of interest. Also provided are a method and use of the inventive multimeric protein and the at least one antigen of interest for immunizing a subject against Plasmodium species.

The invention discloses efficient synthesis of guanidinoacetic acid by a carbamyl phosphoric acid synthesis-enhanced multi-enzyme cascade system, and belongs to the field of biological catalysis engineering. The invention provides a dominant mutant E31K / G351N of L-arginine: glycine amidino transferase, the catalytic efficiency of guanidinoacetic acid is improved by 34.6% compared with that of wild type, an eight-enzyme synergistic arginine circulation system is subsequently constructed based on a mutant strain, 18.35 g / L GAA (56.75 mM) is realized by a 5L fermentation tank, the arginine conversion rate reaches 261.12%, and the yield of the glycine amidino transferase is greatly improved. The highest level of catalytic synthesis of guanidinoacetic acid by escherichia coli is publically reported at home and abroad at present. According to the research, an efficient technical route is provided for GAA industrial production, and a universal strategy framework is established for rational design of a multi-enzyme system and multi-gene co-expression optimization.

This invention provides compositions and treatment methods for reducing hyperphenylalaninemia. [Solution] A genetically modified bacterium is provided, comprising: a) one or more genes encoding phenylalanine ammonia lyase (PAL), which are operably linked to a promoter that is not naturally associated with the PAL gene and can be directly or indirectly induced; b) one or more genes encoding a phenylalanine transporter, which are not naturally associated with the phenylalanine transporter gene and can be operably linked to a promoter that is directly or indirectly induceable; and c) one or more genes encoding mutant fumarate and nitrate reductase (FNR), which are not naturally associated with the FNR gene and can be operably linked to a promoter that is directly or indirectly induceable.

The present invention relates to the field involved in the production of phenylbutanone or phenylbutanone derivative compounds, such as frambinone or zingerone, and in particular strains genetically modified to express a benzalacetone reductase.

The invention discloses a Rogowski eutrophy alkali-producing engineering strain for producing p-coumaric acid as well as a construction method and application of the Rogowski eutrophy alkali-producing engineering strain. The preparation method comprises the following steps: by taking a chemoautotrophic microorganism Ropriavidus alcaligenes H16 as an original strain, firstly, by knocking out a synthetic route of a strain poly-3-hydroxybutyrate (PHB), introducing a tyrosine ammonia lyase gene from Rhodotorula glutinis to construct a basic engineering strain capable of producing p-coumaric acid, and then, carrying out fermentation on the basic engineering strain to obtain the p-coumaric acid. Then, endogenous 3-deoxy-7-phosphoheptanone acid synthase gene aroG1 and chorismate synthase gene aroC are overexpressed, and the total copy is increased, so that the p-coumaric acid synthetic pathway flux of the engineering strain is enhanced; and introducing NAD (P) transhydrogenase gene pntAB from Escherichia coli to improve the supply of cofactors, so as to finally obtain the eutrophy alcaligenes Rosei engineering strain capable of synthesizing p-coumaric acid by using CO2.

The present application relates to the technical field of synthetic biology, in particular to a multi-module synergistic optimization of brown cyanidin and brevifolin production of saccharomyces cerevisiae engineering bacteria and its construction method and application. The saccharomyces cerevisiae engineering bacteria includes naringenin synthesis pathway, optimization pathway of shikimic acid, synergistic pathway of phenylalanine, 6-OH luteolin synthesis pathway, brown cyanidin and brevifolin synthesis pathway, multiple copies of SbF6H and MpalOMT2, NADPH optimization pathway, and SAM cycle optimization pathway. The saccharomyces cerevisiae engineering bacteria is used for producing brown cyanidin and brevifolin, and improving the yield of the two.

The present invention concerns a microorganism comprising at least one heterologous gene coding for a L-arginine:glycine amidinotransferase (AGAT) and at least one heterologous L-threonine 3-dehydrogenase gene (tdh) and at least one heterologous glycine C-acetyltransferase (2-amino-3-oxobutanoate coenzyme A ligase) gene (kbl) and a method for the fermentative production of guanidinoacetic acid (GAA) using such microorganism. The present invention also relates to a method for the fermentative production of creatine.

The invention provides recombinant Escherichia coli for synthesizing heme and derivatives thereof, and the recombinant Escherichia coli is used for knocking out genes such as pyruvate oxidase poxB, pyruvate formate lyase pflB and porphyrinoperoxidase yfeX and enhancing genes such as phosphoenolpyruvate carboxylase ppc, glycerol transporter glpF and 3-phosphoglycerol dehydrogenase glpD, so that the heme and the derivatives thereof can be synthesized. After genes such as exogenous 5-aminolevulinic acid synthase hemA and ferrous chelating enzyme hemH are introduced, the heme and the derivatives thereof can be synthesized by taking glycerol as a raw material, and the heme has an application prospect in the field of biological manufacturing.

The invention provides genetically engineered saccharomycetes capable of simultaneously producing baicalein and baicalin as well as a construction method and application of the genetically engineered saccharomycetes. According to the invention, by weakening or deleting a glucose 5-phosphate dehydrogenase gene (ZWF1) and phenylpyruvate decarboxylase genes ARO10 and PDC5 in a saccharomyces cerevisiae genome and enhancing the expression of a pre-benzoic acid dehydratase gene Pha2, the metabolic flow of a shikimic acid pathway is increased, the aldrin pathway is blocked, and the yield of the shikimic acid pathway is increased. Therefore, the yield of L-phenylalanine serving as a starting material for synthesizing baicalein and baicalin is increased; meanwhile, exogenous genes PAL, 4CL, CHS, CHI, FNSII, F6H, CPR and F7GAT are integrated onto the genome by virtue of a gene recombination technology, so that baicalein and baicalin are produced from the beginning by taking glucose and glycerol as a mixed carbon source without adding precursor substances such as exogenous L-phenylalanine or malonyl CoA and the like.

The invention relates to the technical field of medicinal chemistry, and particularly discloses application of echinacoside in preparation of a medicine for improving autism social and cognitive impairment, and the application comprises the following steps: constructing a recombinant saccharomyces cerevisiae strain, and knocking out a precursor competition pathway gene; performing metabolic flow reprogramming fermentation on the recombinant strain; centrifuging the fermentation liquor at 7000-9000 r / min and pretreating the fermentation liquor with an 8-12 kDa ultrafiltration membrane; carrying out dual-targeting affinity chromatography purification on the pretreatment liquid; the preparation method comprises the following steps: preparing echinacoside into a liposome containing pH sensitive poly-histidine; and adding a freeze-drying protective agent into the suspension to carry out gradient pre-freezing and vacuum freeze-drying. According to the application, a mode of combining recombinant saccharomyces cerevisiae strain construction and metabolic flow reprogramming fermentation is adopted, targeted enrichment of echinacoside is realized through affinity chromatography coupled with autism brain region specific double ligands, then the echinacoside is prepared into the liposome containing pH sensitive poly-histidine, the double ligands are modified, and the effects of precise targeting and efficient effect are achieved.

The present invention relates to a microorganism transformed to be capable of producing guanidinoacetic acid (GAA) and containing at least one gene encoding a protein having the function of NADH-dependent dehydrogenase, and a method for the fermentative production of GAA using such a microorganism. The present invention also relates to a method for the fermentative production of creatine.

A method for the incorporation of formaldehyde into biomass is described, comprising the following enzymatically catalyzed steps: (1) condensation of pyruvate and formaldehyde to 4-hydroxy-2-oxobutanoic acid (HOB); (2) amination of the resulting 4-hydroxy-2-oxobutanoic acid (HOB) to produce homoserine; (3) conversion of the resulting homoserine to threonine; (4) conversion of the resulting threonine to glycine and acetaldehyde or acetyl-CoA; (5) condensation of the resulting glycine with formaldehyde to produce serine; and (6) conversion of the resulting serine to produce pyruvate, which can then be used as a substrate in step (1).

Provided herein are phenylalanine-degrading enzyme variants for use in degrading phenylalanine and in treating phenylketonuria (PKU). More specifically, provided herein are phenylalanine-degrading enzyme variants that exhibit increased thermal stability and physicochemical resistance as measured by residual phenylalanine-degrading activity following challenge.

A process for preparing L-glufosinate by the enzymatically catalyzed reaction of homoserine and methylphosphinic acid is provided. The process according to the present disclosure does not include a step of producing O-acyl-L-homoserine, and O-acyl-L-homoserine is not used in the process.

Provided herein are phenylalanine-degrading enzyme variants for use in degrading phenylalanine and in treating phenylketonuria (PKU). More specifically, provided herein are phenylalanine-degrading enzyme variants that exhibit increased thermal stability and physicochemical resistance as measured by residual phenylalanine-degrading activity following challenge.