16 results about "Aspartate decarboxylase" patented technology

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 provides a beta-alanine producing strain as well as a construction method and application thereof. According to the bacterial strain, an acetaldehyde dehydrogenase gene adhE, a lactic dehydrogenase gene ldhA, an acetokinase gene ackA, a pyruvate dehydrogenase gene poxB, a DNA binding transcription inhibition factor coding gene lacI, an aspartate kinase thrA, a pantothenic acid synthase gene panC, an alanine synthesis transaminase gene cycA and an alanine synthesis transaminase gene yfbQ are knocked out from an E.coli W3110 genome, and the bacterial strain is obtained. A phosphoenolpyruvate carboxylase gene ppc, a pyridine nucleotide transhydrogenase coding gene pntAB, an aspartic acid transaminase gene aspC, an aspartic acid decarboxylase gene panD derived from pseudomonas aeruginosa and a beta-alanine transporter gene NCgl0580 derived from corynebacterium glutamicum are integrated at the same time, and the bacillus subtilis is used for producing beta-alanine and has the advantage of high fermentation yield.

The application discloses an L-aspartate alpha-decarboxylase with improved substrate tolerance, and the L-aspartate alpha-decarboxylase is derived from Bacillus subtilis and is subjected to site-directed mutation, i.e., phenylalanine at the 4th site is changed into tryptophan, isoleucine at the 33rd site is changed into alanine, and isoleucine at the 88th site is changed into tryptophan, so as to obtain an enzyme mutant; the mutant is recombined into E. coli, and after expression is induced, the mutant catalyzes the substrate L-aspartate to generate beta-alanine. When the substrate addition concentration of the whole cell catalytic system is 60 g / L, the conversion rates of the three enzyme mutants T4W, I33A and I88M can reach about 90%, and the yields are 1.2, 1.3 and 1.2 times of that of the unmutated strain respectively, and the substrate tolerance is obviously improved; the combined mutation also has a certain improvement on the substrate tolerance, and the finding has important research value for industrial preparation of beta-alanine.

The present application relates to the field of microorganism, and in particular to a method for producing vitamin B5 by using high-activity aspartate decarboxylase.The present application screens L-aspartate alpha-decarboxylase from Bacillus licheniformis, which has significantly higher activity than other sources of PanD in catalyzing the production of beta-alanine. The present application uses PanD from B. licheniformis to construct an engineered bacterium for fermentative production of vitamin B5, thereby eliminating the metabolic bottleneck of beta-alanine in biosynthesis of vitamin B5. Compared with the chemical method for producing vitamin B5 with high pollution, the present application has the advantages of renewable raw materials, easy treatment and resource utilization of waste residue, waste water and waste gas, etc., thereby being applicable to industrial production of vitamin B5 in practice and having important application value.

The application relates to the technical field of enzyme catalysis. The application discloses an L-aspartate alpha-decarboxylase mutant, which is obtained after the 54th amino acid residue in the amino acid sequence of L-aspartate alpha-decarboxylase panD is mutated into X; the amino acid sequence of the L-aspartate alpha-decarboxylase panD is shown in the sequence table SEQ ID NO. 1. The method has the advantages of green environmental protection, low cost and the like.

The invention provides a genetic engineering strain for efficiently producing beta-alanine, an escherichia coli strain is modified by adopting a genetic engineering means, and a multi-plasmid system is constructed, so that a metabolic pathway is optimized and the product synthesis efficiency is improved. The method comprises the following steps: firstly, by constructing a recombinant plasmid pET-PanD, introducing aspartic acid decarboxylase (L17F / G24R mutant) with mutation so as to promote efficient conversion of aspartic acid to beta-alanine; meanwhile, phosphoenolpyruvate carboxylase (PPC) is overexpressed through the recombinant plasmid pRSF-PPC, generation of oxaloacetic acid (OAA) is increased, and then the synthesis path of carbon flow to beta-alanine is improved. Besides, NADH dependent type aspartate dehydrogenase (AspDH) from pseudomonas aeruginosa is overexpressed by adopting a pBAD-AspDH plasmid, so that the dependence on NADPH is reduced, and the conversion efficiency is further improved.

The invention provides a production method of p-hydroxyacetophenone, which comprises the following steps of: co-expressing phenylalanine amino mutase, L-aspartic acid-beta-decarboxylase, monoamine oxidase and catalase in escherichia coli, converting L-tyrosine into (R)-3-amino-3-(4-hydroxyphenyl) propionic acid through TAM (Transcriptional Amplification Molecule), and converting the (R)-3-amino-3-(4-hydroxyphenyl) propionic acid into (R)-3-amino-3-(4-hydroxyphenyl) propionic acid. According to the method, (R)-3-amino-3-(4-hydroxyphenyl) propionic acid is converted into (R)-4-(1-aminoethyl) phenol through ADC, (R)-4-(1-aminoethyl) phenol is converted into p-hydroxyacetophenone through AOD, and generated hydrogen peroxide is removed through CAT. According to the method, the reaction process is simple, no coenzyme needs to be added, the selected enzyme has the advantages of being high in activity, high in optical specificity and the like, and the p-hydroxyacetophenone is produced through conversion of the recombinant bacterium, so that the method is high in production efficiency, environmentally friendly, low in cost and good in industrial application prospect.

The present application provides: a microorganism with enhanced activity of aspartate 1-decarboxylase derived from Tribolium castaneum; a composition for producing beta-alanine and / or a beta-alanine-derived compound, the composition comprising the microorganism; and a method for producing beta-alanine and / or a beta-alanine-derived compound, the method comprising a step for culturing the microorganism. The productivity of beta-alanine and / or a beta-alanine-derived compound is excellent.

The invention discloses a preparation method of an L-aspartic acid-alpha-decarboxylase mutant and D-pantothenic acid, and belongs to the technical field of gene engineering. The L-aspartic acid-alpha-decarboxylase mutant is obtained by mutation of an amino acid sequence as shown in SEQ ID NO.2, and the mutation is that the 87th amino acid is mutated from I to W. According to the L-aspartic acid-alpha-decarboxylase mutant obtained through mutation, when L-aspartic acid is catalyzed to be subjected to decarboxylation to generate beta-alanine, the substrate inhibition effect of L-aspartic acid on L-aspartic acid-alpha-decarboxylase can be relieved, and high enzyme activity is still kept in the presence of high-concentration L-aspartic acid.

The present application relates to the field of microorganisms and specifically relates to highly active aspartate decarboxylases for the production of vitamin B5. In the present application, the L-aspartate α-decarboxylase derived from Bacillus licheniformis was screened, the PanD with significantly higher activity which catalyze the production of β-alanine than other orgins of PanD. The engineering bacterium for the fermentative production of vitamin B5 was constructed by applying PanD derived from B. licheniformis. The bottleneck of β-alanine metabolism in the biosynthesis of vitamin B5 was lifted. Compared with the highly polluting chemical method for the production of vitamin B5, the biological method for the production of vitamin B5 of the present application has the advantages of renewable raw materials, easy treatment and resource utilization of waste residue, waste water and waste gas, and thus can be used in practice for the industrial production of vitamin B5, which is of significant application value.

The invention provides an engineering bacterium for producing D-pantothenic acid as well as a construction method and application of the engineering bacterium. The engineering bacterium expresses an acetolactate synthase large subunit gene ilv2, an acetolactate synthase small subunit gene ilv6, a keto acid reductoisomerase gene ilvC, a dihydroxy acid dehydratase gene ilvD, a hydroxymethyltransferase gene ecm31, a keto pantoic acid reductase gene panE, an L-aspartic acid decarboxylase gene panD and a pantothenic acid synthase gene panC. According to the method, mitochondria is selected as a targeting compartment, and modular combination optimization approach positioning is adopted, so that the yield of D-pantothenic acid is greatly increased, compared with the yield of an original strain without spatial tissue optimization, the yield is increased by more than one time, the metabolic flux bottleneck in a traditional cytoplasm synthesis mode is broken through, and efficient synthesis of D-pantothenic acid is realized.

The present invention relates to a method for producing malonic acid by Saccharomyces cerevisiae through the alanine pathway, belonging to the field of bioengineering. In the present invention, Saccharomyces cerevisiae is used as the starting strain, and the aspartate decarboxylase gene (PAND) from Tribolium castaneum and the β-alanine pyruvate aminotransferase gene (BAPAT) from Bacillus cereus are integrated into the delta site of the Saccharomyces cerevisiae BY4741 genome to construct a malonic acid synthesis pathway in Saccharomyces cerevisiae, and the promoters of cytoplasmic aspartate aminotransferase and succinic semialdehyde dehydrogenase upstream and downstream of this pathway are replaced with strong constitutive promoters to further increase the malonic acid yield. This application provides a new route for the synthesis of malonic acid in Saccharomyces cerevisiae host. After shake flask fermentation of the recombinant Saccharomyces cerevisiae, the malonic acid yield is 4.4 mg / L, and in a 5-L fermenter, the highest malonic acid yield is 44.31 mg / L.

The invention discloses recombinant escherichia coli for full biosynthesis of malonic acid through a fumaric acid pathway and application, and belongs to the field of bioengineering. The method comprises the following steps: by taking escherichia coli BL21 (DE3) as a host, co-expressing an aspartic acid ammonialyase gene aspA from escherichia coli and an aspartic acid-alpha-decarboxylase gene panD from corynebacterium glutamicum; the preparation method comprises the following steps: co-expressing a succinate semialdehyde dehydrogenase gene yneI derived from escherichia coli and a heterologous gene beta-alanine pyruvate transaminase gene pa0132 derived from pseudomonas aeruginosa, and co-expressing a beta-alanine pyruvate transaminase gene bauA derived from pseudomonas aeruginosa and a malonyl-coenzyme A reductase gene mcr-C derived from orange filamentous bacteria; a malonic acid full-biosynthesis pathway of fumaric acid-malonic semialdehyde is constructed, so that the yield of malonic acid reaches 1414 mg / L after the constructed recombinant escherichia coli is fermented for 48 hours at a shake flask level.

The invention provides a spatial regulation and control system, a beta-alanine production strain and a construction method and application thereof, the spatial regulation and control system comprises a recombinant protein RIDD-RGG-RGG and a recombinant protein aspC-RIAD-RIAD-panD. According to the strain, adhE, ldhA, ackA, poxB, lacI, thrA, panC, cycA and yfbQ genes are knocked out on a wild escherichia coli genome, and the recombinant protein aspC-RIAD-RIAD-panD is obtained. A beta-alanine transporter gene NCgl0580 derived from corynebacterium glutamicum is introduced in a heterologous manner, a phosphoenolpyruvate carboxylase gene ppc is over-expressed, a spatial dynamic regulation and control system containing an aspartate transaminase gene aspC and an aspartate decarboxylase gene panD is integrated, and the synthesis efficiency and the production intensity of the obtained strain are remarkably improved.

The invention discloses L-aspartic acid alpha-decarboxylase with improved substrate tolerance. L-aspartic acid-alpha-decarboxylase from bacillus subtilis is subjected to site-specific mutagenesis, wherein phenylalanine at a No.4 site is mutated into tryptophan, phenylalanine at a No.6 site is mutated into tryptophan, and phenylalanine at a No.6 site is mutated into tryptophan; the isoleucine at the No.33 site is mutated into alanine; the isoleucine at the No.88th site is tryptophan; and transforming recombinant plasmids of the mutant into escherichia coli, carrying out induced expression, and then catalyzing a substrate L-aspartic acid to generate beta-alanine. When the substrate addition concentration of a whole-cell catalytic system is 60g / L, the conversion rates of the three enzyme mutants T4W, I33A and I88M can reach about 90%, the yields of the three enzyme mutants T4W, I33A and I88M are respectively 1.2, 1.3 and 1.2 times of those of non-mutated strains, and the substrate tolerance of the three enzyme mutants T4W, I33A and I88M is obviously improved; the tolerance of the combined mutation to the substrate is also improved to a certain extent, and the discovery has important research value for industrial preparation of beta-alanine.

This invention relates to the field of microbial metabolic engineering, and particularly to a recombinant *Halomonas* strain that synthesizes P3HB3HP from glucose, its construction method, and its applications. This invention constructs a synthetic pathway in *Halomonas* containing enzymes such as aspartate decarboxylase and 4-aminobutyrate aminotransferase. The resulting recombinant *Halomonas* strain can synthesize P3HB3HP with a high 3HP ratio from glucose alone via the β-alanine pathway, while maintaining a high P3HB3HP yield. The recombinant *Halomonas* strain provided by this invention can produce P3HB3HP using inexpensive carbon sources, significantly reducing substrate costs and achieving high cell density, high yield, high 3HP ratio, and stable P3HB3HP synthesis.