243 results about "Cofactor" patented technology

The invention belongs to the technical field of biology, and discloses an exogenous cofactor enhanced yarrowia lipolytica strain, a method and application in efficient synthesis of EPA (eicosapentaenoic acid) and lipids.The chassis strain of the yarrowia lipolytica strain is yarrowia lipolytica Po1f, desaturase is partially prolonged in an overexpression fatty acid synthesis route in the strain, and the EPA and the lipids.The yield of the EPA and the lipids.The yield of the EPA and the lipids.The yield of the EPA and the lipids. The desaturase gene mainly comprises a delta 9 elongase gene, a delta 8 desaturase gene, a delta 5 desaturase gene and a delta 17 desaturase gene, and G6PDH and malic enzyme are overexpressed at the same time on the basis. The strain provided by the invention improves the EPA and lipid yield of yarrowia lipolytica; the method is suitable for constructing a genetic engineering strain with high yield of EPA and lipid, provides a basis for producing grease rich in EPA from yarrowia lipolytica, and solves the problems of non-sustainability, high cost and the like caused by obtaining EPA from fish.

The invention provides an engineering strain for producing mannitol from methanol and / or formaldehyde and fructose and a method for producing mannitol by using the engineering strain. According to the method, a reducing agent is methanol or formaldehyde, a catalyst is dehydrogenase capable of utilizing the methanol or the formaldehyde, a reduction-state cofactor NADH is provided while the methanol or the formaldehyde is oxidized by the dehydrogenase, and D-fructose is converted into D-mannitol by coupling mannitol dehydrogenase. According to the method and the technology, methanol dehydrogenase and / or formaldehyde dehydrogenase, formate dehydrogenase and mannitol dehydrogenase are over-expressed in recombinant escherichia coli, so that a novel method and a novel technology for synthesizing D-mannitol through multi-enzyme coupling catalysis are constructed. The invention establishes a preparation method for co-producing D-mannitol, methanol (or formaldehyde) and reduced NADH from cheap raw materials, and the preparation method has important significance for green and efficient production of D-mannitol.

The invention provides a 7beta-hydroxysteroid dehydrogenase mutant and application thereof in synthesis of ursodesoxycholic acid by a biological enzyme method, through a genetic engineering technology and computer-aided design, phosphoric acid coordination residues of protein are modified, the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant with higher activity under an NADH (Nicotinamide Adenine Dinucleotide Hydroxide) system is constructed, and the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant can be used for synthesizing ursodesoxycholic acid. The modification strategy is suitable for other proteins for modifying cofactor preference and improving enzyme activity, and has remarkable industrial value when being used for efficiently catalyzing CDCA to be converted into UDCA through a one-pot one-step method.

A method of synthesising a 1N-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. The substrate has a structure of: Formula (I). The 1N-methyl derivative of pseudouridine has a structure of: Formula (II). R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group. R2 is H or a hydroxyl group. R3 is a methyl group or an ethyl group. The cofactor is a source of a methyl group or an ethyl group. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Enzymatic synthesis of 1N-methyl derivatives of pseudouridine may be more efficient and produce less waste than chemical syntheses. Also provided are methyltransferases and buffers useful in the method; and polynucleotides, expression vectors, and microorganisms useful for producing the methyltransferases.

Heterologous synthesis of a nitrogen-fixing system in a non-diazotrophic organism is a long-sought-after goal because of the significance of nitrogenase for areas related to agronomy, energy and environment. Two simplified nitrogenase analogs from Azotobacter vinelandii in Escherichia coli were successfully heterologously synthesized. One analog comprise the reductase component (NifH) and, in place of the catalytic component (NifDK), a cofactor maturase (NifEN); whereas the other analog comprised only NifEN. Metal, EPR and activity analyses verified the cluster composition and functional competence of the heterologously expressed NifH and NifEN. Moreover, NMR, nanoSIMS and growth experiments illustrated the abilities of both systems to mimic the nitrogenase in reducing N2 and incorporating the reduced N into the cellular mass. These results firmly established NifEN / NifH or NifEN as a simplified nitrogenase analog that could be engineered or expanded to facilitate future transgenic expression of nitrogenase and development of nitrogenase-based biotechnological applications.

This invention relates to a combined module for increasing the yield of polyketide natural products from actinomycetes, and the construction and application of recombinant bacteria containing this module, belonging to the field of genetic engineering technology. To improve the yield of natural products such as polyketides and nonribosomal peptides, this invention utilizes a metabolic engineering strategy to combine and modify the CoASH synthetic pathway and the PPTase post-modification pathway, providing a combined module that can increase the yield of natural products from actinomycetes. This module consists of the CoASH cofactor synthesis module element SCoaA. R106A Composed of SCoaD and PPTase post-modification module element HPC3, recombinant vectors and recombinant bacteria containing this module were constructed. It was found that overexpression of this module can effectively increase the yield of natural products such as actinomycete polyketides, polyethers, and non-ribosomal peptides.

The invention relates to the technical field of synthetic biology, in particular to a squalene high-yield saccharomyces cerevisiae engineering bacterium and a construction method and application thereof.Saccharomyces cerevisiae CEN.PK.2-1D is used as an original strain, pentose phosphate pathway genes ZWF1, GND1, TAL1 and TKL1 and IDP1 genes of TCA circulation are integrated in a genome of the original strain through homologous recombination, and the squalene high-yield saccharomyces cerevisiae engineering bacterium is obtained. Constructing a dual-channel cofactor supply system to improve the intracellular NADPH level and the NADPH / NADP + ratio; according to the saccharomyces cerevisiae genetic engineering strain Sc-Sq04 constructed by the method disclosed by the invention, the yield of squalene obtained by shake flask fermentation reaches 741.08 + / -28.9 mg / L by further integrating all genes of an MVA way, ERG20 and ERG9 genes, and a new thought is provided for transforming saccharomyces cerevisiae to synthesize terpene compounds and promoting industrial application.

The invention provides a method for improving fermentation yield and sugar-acid conversion rate of 5-hydroxytryptophan, alpha-ketoglutaric acid ferrous salt, alpha-ketoglutaric acid zinc salt, alpha-ketoglutaric acid cobalt salt and alpha-ketoglutaric acid manganese salt are added into a fermentation culture medium according to a specific proportion, alpha-ketoglutaric acid directly participates in TCA circulation, circulation operation can be accelerated, generation of NADH and FADH2 is increased, the yield of 5-hydroxytryptophan is increased, and the yield of 5-hydroxytryptophan is increased. The ATP synthesis efficiency is improved, and more energy is provided for cell growth; the alpha-ketoglutaric acid can balance amino acids required in the growth process of escherichia coli, promote the generation of proteins and nucleic acids and accelerate the growth of thalli; the alpha-ketoglutaric acid metal salt can improve the utilization efficiency of microelements by thalli; ferrous ions are cofactors of 5-hydroxytryptophan hydroxylase and can accelerate the hydroxylation process of tryptophan. Through the synergistic effect of the added components, the yield of the 5-hydroxytryptophan and the sugar-acid conversion rate are improved.

A method for contamination control when growing yeasts is provided. Bacterial contamination is controlled by using urea as the primary nitrogen source while simultaneously limiting the amount of nickel available to contaminating bacteria. Bacteria require nickel as a cofactor for urease enzymes in order to use urea for growth while yeasts do not require nickel as a cofactor for any enzymes. Nickel is limited by using metals in heat exchangers that do not leach nickel. Ethyl carbamate is limited by using a carbon / nitrogen ratio that consumes all urea during fermentation. After fermentation completes, yeast is recycled using centrifugation, enabling use of high concentrations of yeast to reduce fermentation time from 48 hours to 12 hours and to eliminate bacterial contamination from growth on free amino nitrogen.

The present disclosure relates to multi-enzyme pathways for the in vitro cell-free biosynthesis of the cofactor, coenzyme A ("CoA") from lower-cost substrates, such as D-pantothenate, L-cysteine, and ATP, and the use of these pathways in CoA-dependent cell-free systems for the biobased production of chemicals, such as acetyl-CoA, hexanoyl-CoA, malonyl-CoA, and cannabigerolic acid (CBGA).

The invention provides a preparation method and application of a preparation for RNA cell-free synthesis and belongs to the technical field of molecular biology, the preparation method of the preparation for RNA cell-free synthesis mainly comprises the steps that escherichia coli serves as a chassis strain, a pET-28a carrier is utilized, a recombinant strain for expressing T7RNA polymerase is constructed, and the T7RNA polymerase has enzyme activity at the temperature of 50-60 DEG C; inducing, culturing and collecting thalli, carrying out ultrasonic lysis, and treating a lysis solution to prepare a preparation for RNA cell-free synthesis; the preparation provided by the invention naturally contains various cofactors, and RNA polymerase does not need to be extracted and purified, so that the production cost is reduced.

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and / or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

This invention discloses an enzyme preparation for catalytic synthesis of epiinositol and a method for synthesizing epiinositol, belonging to the field of genetic engineering technology. The enzyme preparation includes squalinositol dehydrogenase, NADH oxidase, formate dehydrogenase, and muscle inositol dehydrogenase; the amino acid sequence of squalinositol dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of muscle inositol dehydrogenase is shown in SEQ ID NO. 8. The enzyme activities of the squalinositol dehydrogenase mutant and the muscle inositol dehydrogenase mutant obtained by mutation in this invention are significantly increased, thus further improving the yield and conversion rate of epiinositol during catalytic synthesis. Furthermore, the enzyme preparation of this invention also contains NADH oxidase and formate dehydrogenase, achieving in-situ regeneration of NADH, thereby effectively reducing the amount of cofactors and NADH used in the catalytic synthesis of epiinositol, and thus reducing production costs.

The invention discloses a difunctional alcohol dehydrogenase derived from Geobacillus thermodenitrifis, the difunctional alcohol dehydrogenase takes NAD < + > as a cofactor, can specifically catalyze a continuous oxidation reaction of omega-hydroxy fatty acid, and catalyzes the omega-hydroxy fatty acid to synthesize alpha, omega-binary fatty acid through single enzyme catalysis without an aldehyde intermediate. The alcohol dehydrogenase has dual catalytic functions of alcohol oxidation and aldehyde oxidation, the oxidation process from hydroxymethylene to carboxyl can be completed in one step, the reaction steps are simplified, accumulation and separation of intermediate aldehyde are avoided, the conversion efficiency is improved, and the alcohol dehydrogenase is alpha, omega-dibasic fatty acid and is a novel alcohol dehydrogenase. The invention provides a simple and efficient biocatalysis way for biosynthesis of dodecanedioic acid, especially dodecanedioic acid, and has important industrial application value.

The invention provides a slow-release organic-inorganic compound fertilizer based on synergistic interaction of a carrier immobilized enzyme and a cofactor and a preparation method of the slow-release organic-inorganic compound fertilizer, and belongs to the technical field of organic fertilizers. According to the application, heat-insulation enzyme-loaded particles are added during pile building, a semi-coke-perlite low-heat-conduction framework forms a cold island in a reactor core, and a starch-polypeptide-sodium tripolyphosphate cross-linked membrane is used for firmly immobilizing enzyme, so that heat inactivation is prevented, fibrin is rapidly cracked, free ammonia is adsorbed, and the pile temperature is promoted to rapidly rise; then, dynamic humidity regulating particles are put, reversible water release-water absorption of montmorillonite is combined with glycerin, and a sorbitol hydrophilic film automatically and stably controls water content and supplements easily-degradable carbon, so that over-wet anaerobic or over-dry enzyme loss is avoided; the enzyme cofactor powder added in the later period slowly releases manganese, calcium and copper to serve as enzyme complement, alanyl glutamine serves as a molecular chaperone to repair damaged conformation, and copper and zinc are chelated to passivate heavy metal; the three agents sequentially realize'keep-alive, stable-alive ', prolong the high activity period of enzyme, accelerate lignin degradation, and reduce nitrogen volatilization, odor and heavy metal risks at the same time.

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.

This invention relates to the field of synthetic biology, and more particularly to a microbial cell factory for producing catechols. The invention uses *Pseudomonas putida* strains with the pcaHG, catA, and catA2 genes knocked out as substrate bacteria, and introduces exogenous AroY and / or kpdB genes, or increases the expression of endogenous genes VanAB and PobA. Through a strategy of cofactor regulation and key enzyme overexpression, lignin-derived monomers are efficiently converted into catechols, achieving a conversion rate of 98.5% and a maximum yield of 14.1 mM, a significant improvement compared to the previous conversion rate of 64%.

In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the wildtype 7β-HSDH of SEQ ID NO:2, a decreased substrate inhibition and / or an altered cofactor usage, and the mutant has, in comparison with the wildtype 7β-HSDH of SEQ ID NO:2, 1 to 15 amino acid additions, substitutions, deletions and / or inversions in the sequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ ID NO:2.

The invention relates to the technical field of in-vitro detection, in particular to a kit for carbon dioxide detection and a detection method. By adjusting the pH value of a reaction system, reducing the pH value to 6.0-7.0, properly increasing the PEP feeding amount and reducing the PEPC enzyme dosage, the reaction rate can be remarkably inhibited under the condition, the PEP consumption rate is reduced, and the airborne stability can be improved. Furthermore, an activating agent acetyl coenzyme A is added in a reaction system and can be in synergistic effect with cofactors such as Mg < 2 + > and the like at a detection temperature, so that the activity of PEPC enzyme is obviously activated, and the reactivity during sample detection is improved, thereby improving the analysis sensitivity and test repeatability of the reagent and achieving a relatively ideal level; if the activators acetyl coenzyme A and GDP are added at the same time, the reactivity during sample detection can be remarkably improved, the analysis sensitivity and test repeatability of the reagent are remarkably improved, and the airborne stability and the performance of the reagent both reach the optimal level under the condition.

The present invention relates to a biocatalytic synthesis of esters that can also be implemented on an industrial scale. In particular, the invention relates to a process for the production of esters, via a hemiacetal intermediate, consisting of the following steps: – a step of bringing into contact, in a reaction medium, • a mixture of a C1– Cn alcohol and a C1– Cn aldehyde with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD(P)+ and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde, or alternatively • a C1– Cn alcohol with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD(P)+ and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal, the starting alcohol, and the formed aldehyde, or alternatively • a C1– Cn aldehyde with an enzymatic catalytic system comprising a reduced cofactor selected from NAD(P)H or NADH and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the starting aldehyde and the formed hemiacetal, where n may be any number, and – a step of isolating the corresponding ester.

Provided are engineered polypeptides having dehydrogenase activity that exhibit increased catalytic efficiency for nicotinamide-based unnatural cofactors relative to wild-type or parent polypeptides that encode a dehydrogenase that utilizes nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. Also provided are cell-free and whole cell biotransformation systems for converting a substrate into a product using a redox reaction with a re-cycled unnatural cofactor that utilizes an engineered polypeptide that has dehydrogenase activity and which exhibits increased catalytic efficiency for nicotinamide-based unnatural cofactor.

The invention provides an L-serine reaction system taking THFA in methylobacterium extorquens cells as a cofactor. The L-serine reaction system comprises a THFA solution from methylobacterium extorquens, EcSHMT, glycine, formaldehyde and PLP. The invention also provides a production method of L-serine. The method comprises the following steps: culturing methylobacterium extorquens to obtain a THFA solution; the EcSHMT is obtained; the method comprises the following steps: adding EcSHMT, glycine, formaldehyde and PLP into a THFA solution to construct a reaction system as shown in the specification, and carrying out enzymatic reaction to obtain the L-serine. The method has the advantages that a brand new L-serine production method with THFA in methylobacterium extorquens cells as a cofactor is provided, the extracted THFA has high activity, L-serine production without additional THFA can be realized by utilizing an enzymatic reaction system, the production cost of L-serine is greatly reduced, and the method is suitable for industrial production. And meanwhile, relatively high L-serine yield is ensured.

The invention relates to gene modification, in particular to a method for improving the catalytic capacity of engineering P450 peroxygenase hydrogen peroxide and application of the method. The invention relates to a method for improving hydrogen peroxide catalytic capacity of P450 enzyme, which comprises the following steps of: identifying a channel of the P450 enzyme by using Caver Web 1.0 software by taking a heme cofactor of the P450 enzyme as a starting point; the method comprises the following steps: selecting a target channel with the channel length of 10-30, the curvature of 0-2 and the diameter of 0-3 as a target channel, then selecting amino acid of which the side chain faces the channel at an inlet or a bottleneck of the target channel as key amino acid, and then performing specific mutation on one or more key amino acids, further, at least one channel capable of improving the hydrogen peroxide catalytic capability of the P450 enzyme is obtained, so that the catalytic capability of the P450 enzyme is improved under the catalysis of hydrogen peroxide. The catalytic activity of a plurality of mutants obtained by the invention is compared with that of a wild enzyme, and the channel with high catalytic activity is an H2O2 channel. The application of the method can improve the hydrogen peroxide utilization capability of the P450 enzyme, and further improves the activity of the catalytic substrate of the P450 enzyme.

In an example of a method, a deoxyribonucleic acid sample is exposed to tagmentation in the presence of a tagmentation buffer including a divalent cation cofactor and a transposase enzyme to generate a tagmented DNA fragment complex. A chelator mixture is added to the tagmented DNA fragment complex. The chelator mixture includes a chelator of the divalent cation cofactor at a weight ratio that is at least 1:1 with the divalent cation cofactor; and has a pH ranging from 8 to 9. The tagmented DNA fragment complex is incubated in the chelator mixture at a temperature of at least 55° C. for at least about 60 seconds, so that the transposase enzyme dissociates from a tagmented DNA fragment of the tagmented DNA fragment complex.

The invention discloses a methanol dehydrogenase mutant with improved cofactor affinity, particularly relates to a methanol dehydrogenase mutant with improved cofactor NADP < + > affinity, and belongs to the technical field of enzyme directed evolution and protein engineering. According to the present invention, the 195th site and the 196th site of the methanol dehydrogenase from the bacillus stearothermophilus DSM 2334 are modified so as to improve the affinity of the methanol dehydrogenase to the cofactor NADP < + >, and the 2424th site, the 243 site and the 246th site are modified on the basis so as to construct the methanol dehydrogenase mutant having high catalytic activity on the NADP < + > and low activity on the NAD < + >. V37A mutation is introduced, so that the mutant SM7 which takes NADP < + > as a cofactor and has catalytic activity is further improved. The methanol dehydrogenase mutant provided by the invention can provide a good catalytic element for bioconversion of methanol, and has a wide application prospect.

This application discloses a recombinant bacterial strain that highly expresses nonspecific peroxygenase, its construction method, and its applications, relating to the field of microbial engineering technology. The strain of this invention uses *Escherichia coli* BL21(DE3) as the host. First, the key gene *hemA* for heme synthesis is integrated into the genome using the CRISPR-transposon system. Then, a chassis engineered strain A2 with 2 copies of optimal integration is screened and co-transformed with the rDcaUPO-A161C expression plasmid and the molecular chaperone plasmid pG-KJE8. The intracellular heme level of this strain is increased by 2.84 times compared to the original strain, and the total enzyme activity of rDcaUPO-A161C is cumulatively increased by 8.9 times and the specific enzyme activity is increased by 6.21 times compared to the wild type. This fundamentally solves the problems of insufficient cofactors, low folding efficiency, and poor activity in heterologous expression of nonspecific peroxygenases, and can be used for the efficient production of nonspecific peroxygenases.

The invention belongs to the technical field of traditional Chinese medicine preparation, and provides a traditional Chinese medicine decoction piece synthetic biological component improving method which comprises the following steps: (1) constructing an anabolism pathway of a target active component, and screening to obtain engineering bacteria or engineering cells for efficiently expressing a key enzyme; (2) optimizing a synthesis path through modular metabolic engineering, wherein the synthesis path comprises cooperative regulation and control of a precursor supply module, a cofactor regeneration module and a product transfer module; (3) establishing a dynamic regulation culture system, and inducing and regulating biosynthesis of a target product through environmental factors; (4) carrying out structural modification on the synthetic product by adopting a biotransformation technology; and (5) directionally integrating the obtained active ingredients and the traditional Chinese medicine decoction piece matrix. By constructing a modular metabolism and dynamic regulation and control system, efficient biosynthesis of complex traditional Chinese medicine components is achieved, the content of effective components is increased, and the bonding strength of the active components and a decoction piece matrix is improved through an ultrasonic directional integration technology.

Bispecific antibodies whose FIX activation-inhibiting activity is not elevated and whose FVIII cofactor function-substituting activity is elevated have been successfully discovered.

This invention relates to the fields of molecular biology and biotechnology, and provides the ZmCNX2 gene, a cofactor 2 protein gene for nitrate reductase and xanthine dehydrogenase in maize, and its applications. The nucleotide sequence of the ZmCNX2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the cofactor 2 protein encoded by the ZmCNX2 gene for nitrate reductase and xanthine dehydrogenase is shown in SEQ ID NO:2. This invention found that mutants of the ZmCNX2 gene in maize exhibit a pre-harvest germination phenotype; compared with wild-type Arabidopsis seeds, the germination of transgenic Arabidopsis seeds was significantly delayed. These results indicate that deficiency of the ZmCNX2 gene can lead to maize seeds lacking dormancy and germinating directly on the ear, while overexpression of the ZmCNX2 gene in Arabidopsis can delay seed germination, further verifying the central role of this gene in the regulation of seed dormancy and germination.

The present invention provides light chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of ACE910 (Emicizumab), novel light chains showing FVIII cofactor function-substituting activity, and heavy chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of novel light chain-containing bispecific antibodies.