26 results about "Feedback inhibition" patented technology

The invention belongs to the technical field of synthetic biology, and particularly relates to recombinant escherichia coli for efficiently producing O-acetyl-L-homoserine as well as a construction method and application of the recombinant escherichia coli. According to the invention, CRISPR-Cas9 gene editing is combined with metabolic modification, so as to precisely integrate OAH biosynthesis key nodes: introducing and overexpressing a mutant metX gene at a genome pseudogene site to enhance the expression of homoserine acetyltransferase, knocking out byproducts such as poxB and mgsA to synthesize genes, and carrying out high-yield synthesis of OAH. Corynebacterium glutamicum thrA (anti-feedback inhibition), aspB and bacillus subtilis acsA-acuA genes (construction of an acetic acid switch) are introduced, gene expression of gltA, tpiA and the like is optimized to increase supply of precursors such as acetyl CoA and the like, and finally a stable strain without plasmids, antibiotics and inducers is obtained. The production cost is effectively reduced, and the method has the advantage of high genetic stability and shows a good application prospect in OAH industrial production.

The invention provides a valine production strain as well as a construction method and application thereof, the strain is constructed by utilizing a CRIPSR / Cas9 system gene editing technology based on allele exchange, an artificial operon is designed and constructed, the artificial operon is constructed at a ygaY site, a strong promoter Ptrc is used for controlling ilvIH (G14N S17F)-pykF to carry out synchronous multi-copy expression, and the valine production strain can be used for producing valine. On the basis of the strain, an artificial operon is constructed at an ycgH site, a strong promoter Ptrc is used for controlling ilvC-ilvD to carry out synchronous multi-copy expression, meanwhile, feedback inhibition of valine on ilvIH is relieved, and an ilvIH natural promoter is replaced by the strong promoter Ptrc, so that more pyruvic acid flows to valine; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for efficiently synthesizing valine from the beginning by taking glucose as a substrate.

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

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

The invention discloses a genetically engineered bacterium for producing heparinogen and a construction method and application thereof, the genetically engineered bacterium takes Escherichia coli Nissle 1917 as a chassis bacterium, and the construction method comprises the following steps: (1) knocking out endonuclease I coding gene endA in a chassis bacterium genome; (2) knocking out a 6-phosphofructokinase I coding gene pfkA in a chassis bacterium genome; (3) carrying out overexpression on a 6-phosphoglucosamine synthetase encoding gene glmS and / or a UDP-glucose-6-dehydrogenase encoding gene kfiD from a chassis bacterium genome; (4) removing the feedback inhibition of the 6-phosphate glucosamine on the GlmS so as to promote the synthesis of UDP-N-acetylglucosamine; and (5) knocking out the 6-phosphate glucosamine deaminase coding gene nagB in the chassis bacterium genome. The engineering strain obtained by the invention can more efficiently synthesize the proheparin, the shake flask titer reaches 397.9 mg / L, which is increased by about 3 times compared with a wild strain, and the engineering strain has certain industrial application value.

The invention relates to the technical field of gene engineering, and discloses an ATP (adenosine triphosphate) transphosphoribosyl enzyme mutant as well as a coding gene, a recombinant vector, a transgenic cell and application of the ATP transphosphoribosyl enzyme mutant and the coding gene. The mutant is generated by mutating at least one amino acid of ATP (adenosine triphosphate) transphosphoribosyl enzyme with an amino acid sequence as shown in SEQ ID NO: 1; wherein the ATP transphosphoribosyl enzyme with the amino acid sequence as shown in SEQ ID NO: 1 is taken as a reference, and the mutation site of the mutant is the 214th site and / or the 215th site. The ATP transphosphoribosyl enzyme mutant can effectively relieve feedback inhibition of histidine, maintain high catalytic activity and remarkably improve the biosynthesis efficiency of histidine.

This invention provides an engineered bacterium for producing ectoine, its preparation method, and its application. The engineered bacterium is prepared using a method comprising the following steps: using *Corynebacterium glutamicum* as the starting strain, feedback inhibition by aspartate kinase LysC is relieved, the activity of the gene lysE encoding lysine efflux permease is reduced, and the ectoine synthesis gene cluster ectABC is expressed. Based on this, the activity of any one or at least two of the following—diaminobutyrate acetyltransferase EctA, pyruvate carboxylase Pyc, or aspartate kinase LysC—is enhanced, and / or the activity of any one or at least two of the following—maloquinone oxidoreductase Mqo or homoserine dehydrogenase Hom—is reduced to further increase ectoine yield.

The present application relates to a kind of acetolactate synthase and its application, belong to enzyme engineering and metabolic engineering technical field.The present application is based on the wild-type acetolactate synthase from Corynebacterium glutamicum (Corynebacterium glutamicum) Corynebacterium glutamicum ) ATCC13032, the encoding gene ilvBN Of it is carried out error-prone PCR, screening obtains acetolactate synthase mutant IlvBN M1 And IlvBN M2 , above-mentioned mutant removes L-valine, L-leucine and L-isoleucine on its feedback inhibition, under the condition that L-valine, L-leucine and L-isoleucine concentration is 0-30 mmol / L respectively, enzyme activity has no obvious change, can be widely used in the synthesis of L-valine, L-leucine and L-isoleucine.

The application is a kind of bionic nerve circuit model based on artificial synapse device. The circuit model is composed of n multi-stage connections of synapse-LIF artificial neuron devices. The composition of the synapse-LIF artificial neuron device includes synapse-like device and cytoplasm-like device. The synapse-like device includes artificial synapse device, voltage dividing resistor and voltage control switch. The cytoplasm-like device includes one comparator. Through different combinations of the four devices, namely excitatory synapse-like device, inhibitory synapse-like device, excitatory cytoplasm-like device and inhibitory cytoplasm-like device, the devices of six circuits, including feedforward excitation circuit, feedforward inhibition circuit, feedback inhibition circuit, lateral inhibition circuit, disinhibition circuit and mutual inhibition circuit, are formed. The application realizes the functions of perception afferent, information integration and motor control in the motor nervous system through the circuit model group, which is helpful for the repair or reconstruction of damaged motor conduction circuit.

The invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof in order to overcome the practical application problem of a genetic engineering method in the process of producing the bovine lactoferrin peptide at present. According to the invention, bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein are connected in series to form bovine lactoferrin peptide fusion protein, a pichia pastoris recombinant strain is constructed, and high-level expression of bovine lactoferrin peptide is realized. According to the method, positive charges of bovine lactoferrin are neutralized on the basis of calmodulin, toxicity of antibacterial peptide to a host is weakened, the tandem enhanced green fluorescent protein can emit green fluorescence under 488 nm exciting light, and real-time analysis of the expression level of bovine lactoferrin fusion protein is achieved. According to the method, by adopting a fusion expression technology, the toxicity to a host when the antibacterial peptide is produced by a genetic engineering method is overcome, so that the expression quantity is increased, and a foundation is laid for high-density fermentation production of the bovine lactoferrin peptide.

The invention relates to the technical field of bioengineering, and discloses a construction method and application of recombinant Escherichia coli engineering bacteria with high yield of ergothioneine, and the method comprises the following steps: by taking Escherichia coli as a chassis, integrating a mycobacterium smegmatis egtABCDE gene cluster at an ldhA site; a repressor protein gene metJ and a cysteine lyase gene yhaM are knocked out, a methionine adenosine transferase gene metK and a serine acetyltransferase gene cysE are overexpressed by using a strong promoter, and mutant metA, thrA, serA and hisG genes for relieving feedback inhibition are introduced, so that metabolic inhibition of methionine, cysteine and histidine pathways is systematically relieved; the invention also establishes a matched fermentation process. According to the invention, endogenous precursor supply and exogenous synthesis pathways are synergistically enhanced, the precursor supply bottleneck is solved, the yield of ergothioneine is increased, and the method is suitable for industrial production.

The present application relates to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase mutant and its application, and belongs to the technical field of enzyme engineering. The present application obtains key residues of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic acid synthase by combining protein sequence comparison and alanine scanning strategy, randomly mutates the key residues, and obtains a mutant MenD which is resistant to feedback inhibition of 1,4-dihydroxy-2-naphthalene carboxylic acid intermediate metabolite in the methionine quinone pathway through multiple rounds of screening W322I\R323F The ability of the recombinant bacillus subtilis to produce heptamethylnaphthoquinone is improved, and the yield is 1.22 times that of the control strain, and the final yield reaches 186.5 mg / L. The present application not only provides a new direction for the modification of the methionine quinone pathway of bacillus subtilis, but also will be beneficial to the industrial large-scale production of heptamethylnaphthoquinone.

The invention provides an engineering bacterium for producing Ectoine as well as a preparation method and application thereof, and the engineering bacterium is prepared by the preparation method comprising the following steps: taking corynebacterium glutamicum as a starting strain, relieving feedback inhibition of aspartate kinase LysC, reducing activity of gene lysE for coding lysine efflux permease, and obtaining the Ectoine-producing engineering bacterium. And expressing an Ectoin synthetic gene cluster ectABC. On the basis, the activity of any one or a combination of at least two of diaminobutyric acid acetyltransferase EctA, pyruvate carboxylase Pyc or aspartate kinase LysC is enhanced, and / or the activity of any one or a combination of two of malic acid quinone oxidoreductase Mqo or homoserine dehydrogenase Hom is reduced, so that the yield of Ectoine is further improved.

The application discloses a Saccharomyces cerevisiae de novo synthesis of naringenin engineering strain and a construction method and application thereof, and the engineering strain is constructed by modifying a genome of the Saccharomyces cerevisiae CEN.PK2-1C as follows: substrate competition pathway genes are knocked out, anti-feedback inhibition genes are overexpressed, and the like, so as to improve a metabolic flux j of a precursor substance tyrosine; tyrosine deaminase, 4-coumarate:CoA-ligase, chalcone synthase and chalcone isomerase are expressed heterogeneously, so as to realize de novo synthesis of naringenin. Finally, the yield of naringenin is further improved by high-efficiency expression of Sc4CL and HaCHS at multiple copy sites.

Provided are a method for improving the yield of L-valine and a modified bacterium, wherein the genome of the bacterium comprises a modification for reducing the feedback inhibition of L-threonine on L-threonine synthesis, and the modified bacterium can achieve production under anaerobic and aerobic conditions. In addition, a method for increasing the yield of L-valine by means of using the modified bacterium is also provided.

The application belongs to the technical field of genetic engineering, and particularly relates to a phosphoglycolate dehydrogenase mutant and application thereof in L-serine production. The mutant contains a mutation of the 370th amino acid compared with a protein with an amino acid sequence as shown in SEQ ID NO:1. The mutant protein releases feedback inhibition, and the catalytic activity is significantly improved compared with the parent enzyme, thereby promoting the production of L-serine catalyzed by the phosphoglycolate dehydrogenase, and providing a new synthetic route and an engineering bacterium for the production of L-serine.

The invention discloses a bacillus licheniformis recombinant strain with high yield of L-cysteine, and belongs to the technical field of synthetic biology and microbial metabolism engineering. The cysteine synthesis flux is enhanced through multiple knockout and combined overexpression. An efficient engineering strain is constructed by knocking out a serine hydroxymethyltransferase coding gene glyA and an L-serine ammonolysis enzyme coding gene sdaAA of a serine catabolism key pathway, knocking out a cysteine catabolism gene metC and combining gene overexpression to introduce a feedback inhibition removal mutant cysEf and a transporter eamA, and the efficient engineering strain is used for preparing a recombinant strain. Finally, the yield of the L-cysteine reaches 1.075 g / L in shake flask fermentation (the conversion rate is 18.69%), and the application potential of the bacillus licheniformis in sulfur metabolism engineering is highlighted.

The application discloses an engineered Trichoderma reesei strain with high yield of beta-glucosidase, and a construction method and application thereof. In view of the problems of low beta-glucosidase activity of a natural cellulase system of Trichoderma reesei in the prior art and easy accumulation of cellobiose to cause feedback inhibition, the application takes Trichoderma reesei RUT-C30 as a starting strain, and through three-step progressive operation, firstly, a beta-glucosidase gene expression cassette derived from Aspergillus niger is integrated into a URA5 gene locus; then, an expression cassette containing a URA5 back complement unit and a BGL gene is constructed and integrated into an ACE1 gene site; finally, a transcription activator XYR1 expression cassette is integrated into a PEP1 site. Through the above iterative modification, an engineered strain with stable genetic traits, capable of simultaneously high-level secretion of beta-glucosidase and filter paper enzyme is obtained. It is verified through experiments that the beta-glucosidase activity of the engineered strain can be up to 80.6 IU / mL, which is 268.3 times that of the starting strain.

This invention relates to a method based on argB A method for constructing a high-arginine-producing genetically engineered strain using mutants, and the application of this genetically engineered strain in the microbial fermentation production of arginine. The genetically engineered strain was obtained by performing the following gene editing on its genome, starting with Corynebacterium glutamicum ATCC 13032; this invention first involves knocking out the gene encoding the arginine biosynthesis repressor protein. argR and last Relieve transcriptional repression; knock out the gene encoding glutamate kinase. proB Blocking the competitive pathway for the synthesis of the byproduct L-proline; knocking out global nitrogen metabolism regulators. amtR To relieve nitrogen metabolism restriction and enhance nitrogen source supply; to knock out the gene encoding the mechanosensitive channel protein. yggB By altering cell membrane permeability and reducing the leakage of byproducts (such as glutamate), it indirectly promotes arginine accumulation; based on the knockout of the original proB, ldh Replace with Ptac- argB *(T94S, I158V, R273K) strains were constructed to reduce lactic acid byproducts while resisting feedback inhibition; finally, the L-arginine exporter gene was overexpressed via plasmid. lysE By combining the above modification strategies, a genetically engineered strain ARG6-Ptac-, which produces high levels of arginine, was constructed. argB * / pXMJ19- lysE This engineered strain exhibits excellent L-arginine production capacity in both shake flask and fermenter scales (10.3 g / L in shake flask and 105.2 g / L in fed-batch fermentation), and its genetic traits are stable, making it suitable for industrial production.

The application discloses a homoserine dehydrogenase mutant and application thereof. The amino acid sequence of the mutant has one mutation type of A381D, A381L, A381I, A381P, A381V, A384R, A384D, I397C, I397D, I397G, I397S, I397R, I397V, I397A compared with a wild-type homoserine dehydrogenase Hom from corynebacterium glutamicum. The enzyme activity of the mutant is significantly improved compared with a wild-type control through enzyme activity determination analysis of induced expression and purification. Meanwhile, the mutant can efficiently remove the feedback inhibition of L-threonine and L-isoleucine. The homoserine dehydrogenase mutant, a coding gene thereof and a host cell containing the coding gene can be applied in industry to produce L-homoserine and derivative amino acids.

The application belongs to the technical field of genetic engineering, and particularly relates to a phosphoglycolate dehydrogenase mutant, an engineering bacterium and application of the engineering bacterium in L-serine production. The mutant contains a mutation of the 375th amino acid compared with a protein with an amino acid sequence as shown in SEQ ID NO:1. The mutant protein releases feedback inhibition, and the catalytic activity is significantly improved compared with the parent enzyme, thereby promoting the production of L-serine catalyzed by the phosphoglycolate dehydrogenase, and providing a new synthetic route and engineering bacterium for the production of L-serine.

The invention provides an engineering bacterium for producing Ectoine as well as a preparation method and application thereof, and the engineering bacterium is prepared by the preparation method comprising the following steps: taking corynebacterium glutamicum as a starting strain, relieving feedback inhibition of aspartate kinase LysC, reducing activity of gene lysE for coding lysine efflux permease, and obtaining the Ectoine-producing engineering bacterium. And expressing an Ectoin synthetic gene cluster ectABC. On the basis, the activity of any one or a combination of at least two of diaminobutyric acid acetyltransferase EctA, pyruvate carboxylase Pyc or aspartate kinase LysC is enhanced, and / or the activity of any one or a combination of two of malic acid quinone oxidoreductase Mqo or homoserine dehydrogenase Hom is reduced, so that the yield of Ectoine is further improved.

The invention discloses a recombinant escherichia coli engineering strain for producing hydroxytyrosol and application, and belongs to the technical field of genetic engineering and biological engineering. According to the invention, by knocking out tyrR, ptsG, crr, pheA, feaB and pykA genes of escherichia coli, the competition between feedback inhibition and carbon metabolism is eliminated; the mutant aroGfbr and the mutant tyrC are subjected to overexpression, feedback and inhibition, and tyrosine precursor supply is strengthened; the method comprises the following steps: carrying out rational design on a key rate-limiting enzyme 4-hydroxyphenylacetic acid 3-hydroxylase to obtain an S462A / M293Y double mutant, and carrying out overexpression on an nadC gene to optimize cofactor balance, so that the hydroxytyrosol yield of the constructed engineering strain in a shake flask reaches 4.90 g / L; in a 5L bioreactor, the yield is further increased to 13.25 g / L, and the method can be widely applied to industrial production of hydroxytyrosol-containing products in the fields of food, health care products and medicines.

The invention relates to acetolactate synthase and application thereof, and belongs to the technical field of enzyme engineering and metabolic engineering. Wild acetolactate synthase from Corynebacterium glutamicum ATCC13032 is taken as a basis, error-prone PCR is performed on a coding gene ilvBN of the wild acetolactate synthase, acetolactate synthase mutants IlvBNM1 and IlvBNM2 are obtained through screening, the feedback inhibition effects of L-valine, L-leucine and L-isoleucine on the mutants are relieved, and the activity of the mutants is improved. Under the condition that the concentrations of the L-valine, the L-leucine and the L-isoleucine are respectively 0-30 mmol / L, the enzyme activity is not obviously changed, and the method can be widely applied to synthesis of the L-valine, the L-leucine and the L-isoleucine.

The invention discloses a recombinant strain for preparing L-isoleucine and application of the recombinant strain, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, acetohydroxy acid synthase and threonine dehydratase which are resistant to feedback inhibition are screened, and the expression intensity of threonine dehydratase ilvA is regulated, so that the yield and molar conversion rate of the strain L-isoleucine are improved; by balancing metabolism and growth of the L-isoleucine, the yield of the L-isoleucine is increased to 6.58 g / L, and the molar conversion rate reaches 94.3%; by further balancing supply and consumption of cofactors, the yield of the L-isoleucine is increased to 6.80 g / L, and the molar conversion rate reaches 96.6%. The recombinant escherichia coli constructed by the invention can efficiently catalyze L-threonine to synthesize L-isoleucine, lays a foundation for subsequent efficient de novo synthesis of L-isoleucine and biosynthesis of derivatives, and has potential value and significance for development of synthetic biology.

This invention provides a DAHP synthase mutant that removes prephenylacetic acid feedback regulation and its application. Through sequence alignment, phylogenetic analysis, and enzyme activity assays, the feedback inhibition mode of DAHP synthase (AroA) from Bacillus amyloliquefaciens was determined. Then, combined with protein stability calculations, binding energy analysis, and deep learning prediction, the DAHP synthase mutant AroA was obtained. M54K The DAHP synthase mutant AroA M54K When the methionine at position 54 of the DAHP synthase is mutated to tyrosine, and the prephenylacetic acid concentration is 50–600 μmol / L, the mutant AroA is produced. M54K The relative enzyme activities all reached over 90%, relieving the feedback inhibition of prephenylacetic acid and increasing the L-tyrosine yield to 525.58 mg / L. Further, the mutant AroA... M54K With prephenyl acid dehydrogenase mutant TyrA I309A / E330V Or TyrA M53I / A354V Co-expression increased L-tyrosine production to 980.21–1094.58 mg / L. This invention reveals the feedback inhibition regulation mechanism of DAHP synthase from Bacillus amyloliquefaciens, providing a theoretical and technical basis for the modification of aromatic amino acid synthesis pathways and the efficient production of L-tyrosine.