363 results about "Directed evolution" patented technology

The invention provides an expansible ring enzyme mutant and an application of the expansible ring enzyme mutant in synthesis of G-7-ADCA. A series of mutants with penicillin G ring expansion activity are obtained through ancestor enzyme sequence reconstruction, directed evolution and the like, and the mutants can directly expand the ring of the substrate penicillin G to generate G-7-ADCA. Therefore, the expansive ring enzyme mutants have important application value in industry.

The invention discloses a halohydrin dehalogenase mutant with improved dehalogenation efficiency and application of the halohydrin dehalogenase mutant, wild halohydrin dehalogenase comes from radioactive agrobacterium tumefaciens AD1, the wild halohydrin dehalogenase is modified by means of directed evolution, the halohydrin dehalogenase mutant with improved dehalogenation efficiency is screened out, and mutations of the mutant comprise at least one of A83W, P84G and F86W. The halohydrin dehalogenase mutant provided by the invention can efficiently and accurately degrade halohydrin pollutants 2, 3-dichloro-1-propanol and 3-chloro-1, 2-propylene glycol, the toxicity of the halohydrin pollutants is reduced, a high-added-value product is generated, and the dehalogenation efficiencies of the mutant A83W / P84G / F86W on the two substances within one hour can respectively reach 42.2% and 98.6%, which are 6 times and 5.2 times of those of a wild type; the halohydrin dehalogenase mutant provided by the invention can realize efficient dehalogenation at normal temperature in a water phase, does not need additional temperature control equipment, has the characteristics of high efficiency, low energy consumption and high selectivity, has the industrial application advantages of greenness, low carbon, economy and convenience, and is beneficial to pollutant dehalogenation and resource utilization.

The invention discloses a carbonyl reductase mutant as well as a preparation method and application thereof, and belongs to the technical field of enzyme engineering. According to the invention, on the basis of carbonyl reductase BcKRED from Bacillus cereus, a series of dominant mutants are obtained through a method of directed evolution in the field of enzyme engineering. The carbonyl reductase dominant mutant obtained by the invention has high catalytic activity, thermal stability and chiral selectivity reversed with wild-type carbonyl reductase. A green and efficient way is provided for industrial production of chiral alcohol by taking carbonyl reductase as a biocatalyst, and the method has a wide application prospect.

The invention discloses a darkness-induced enhanced luminescence expression vector as well as a construction method and application thereof, and the enhanced luminescence expression vector is constructed by taking a vector pDES as a skeleton vector. A core element formed by sequentially connecting a promoter PsDIN6, a mutated nnLuz gene mnnLuz, a HisspS gene of neon lamp bacteria, an H3H gene, a CPH gene and an NPGA gene of aspergillus nidulans is inserted between an attR1 element and an attR2 element of a vector pDES from the 5'end to the 3 'end, and the vector is obtained. A luciferase Luz gene of luminous fungus neon lamp bacteria is subjected to random mutation by using a directed evolution technology, a luciferase mutant gene mLuz of which the luminous efficiency is 5.7 times higher than the original luminous efficiency is obtained through screening, and an expression vector constructed by the luciferase mutant gene mLuz is instantaneously expressed on tobacco leaves, so that the tobacco leaves do not emit light in daytime and emit bright fluorescence in darkness.

The invention relates to a diacylglycerol acyltransferase mutant and application thereof in synthesis of triacylglycerol from saccharomyces cerevisiae, and belongs to the technical field of enzyme engineering. Diacylglycerol acyltransferase is a rate-limiting step of a synthetic route of saccharomyces cerevisiae triacylglycerol (TAG), and at present, a research on a DGA1 mutant for efficiently synthesizing TAG is lacked, and a research on the aspect of a DGA1 catalytic mechanism is also lacked. The DGA1 mutant capable of effectively improving TAG synthesis is obtained by screening in combination with a directed evolution technology, and a result shows that 282-site mutation of diacylglycerol acyltransferase can significantly increase the accumulation amount of TAG. Meanwhile, the catalytic mechanism of the DGA1 is preliminarily explored by combining protein structure prediction, molecular docking and molecular dynamics simulation, and a foundation is laid for deeply analyzing the catalytic mechanism of the DGA1. The recombinant saccharomyces cerevisiae is also constructed based on the mutant, so that the TAG synthesis is improved, and meanwhile, the relative proportion of C18: 1 in total fatty acids is remarkably increased.

The invention discloses a 3-sterone-delta1-dehydrogenase mutant and application of the 3-sterone-delta1-dehydrogenase mutant in preparation of a steroid drug intermediate. The mutant is obtained by performing single mutation or multiple mutation on 551, 468, 48 and 157 sites of an amino acid sequence of 3-sterone-delta1-dehydrogenase as shown in SEQ ID NO: 1. The 3-ketosterone-delta1-dehydrogenase is subjected to molecular modification through directed evolution and semi-rational design, the mutant with improved enzyme activity is screened in a high-throughput mode, the mutant can effectively improve the conversion rate of a substrate 11alpha, 17alpha-dihydroxyprogesterone, and the substrate conversion rate of the mutant ISM-2 is improved to 92.4% compared with 36.7% before mutation.

The invention relates to screening and application of a Candida antarctica lipase B mutant. Specifically, the Candida antarctica lipase B mutant provided by the invention is obtained by carrying out rational design and directed evolution combined strategy mutation on the basis of a wild Candida antarctica lipase B gene sequence, and the amino acid sequence of the mutant is shown as SEQ ID NO.3. The invention also relates to a preparation method of the Candida antarctica lipase B mutant. The mutant has higher enzyme activity and thermal stability, the hydrolytic catalytic activity of the mutant is improved to 2.1 times of the original hydrolytic catalytic activity, and the half-life period of the mutant enzyme at 50 DEG C is prolonged by 4 times. On the basis, pichia pastoris is used as a host to express the mutant, and a strain with hydrolytic activity improved by 52% is obtained through high resistance screening. The lipase is prepared into an immobilized enzyme to be used in a synthetic reaction of chlorogenic acid derivatives, the highest conversion rate of chlorogenic acid reaches 74% in 24 hours, and the immobilized enzyme has a good industrial application prospect.

The invention relates to the technical field of gene engineering, in particular to a glycosyl transferase mutant, a sucrose synthase mutant and application of the glycosyl transferase mutant and the sucrose synthase mutant in synthesis of rebaudioside M. Wild glycosyl transferase and wild sucrose synthase are modified according to a directed evolution theory to obtain a glycosyl transferase mutant and a sucrose synthase mutant. Rebaudioside M is obtained by catalyzing rebaudioside D through a glycosyl transferase mutant and sucrose synthase mutant co-expression strain, and the catalytic efficiency is superior to that of a wild enzyme. The rebaudioside M is obtained by completely catalyzing 110 g / L of rebaudioside D through the co-expression strain of the glycosyl transferase mutant and the sucrose synthase mutant, only 20 h is needed, the conversion rate reaches 97%, the yield of the rebaudioside M reaches 122 g / L, the raw material conversion rate and the yield of the rebaudioside M are effectively increased, and meanwhile the synthesis time is shortened.

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

Provided herein are cells, compositions, and methods for identifying enzyme mutants having increased activity in cleaving synthetic polymers. The methods employ high-throughput cell surface display and directed evolution for rapidly assessing millions of enzyme mutants.

The invention provides a top-down directional evolution method for growth-promoting microbiome of rice. The top-to-bottom rice growth-promoting microbiome directional evolution method comprises the following steps: step 1, selecting a microbiome with a growth-promoting effect on rice seedlings, combining Bottleneck + Migration into an LB culture medium for iterative culture, and after 15 rounds of non-selective iteration, transferring each round of iteration into a new LB culture solution; 2, respectively inoculating the flora subjected to 15 rounds of non-selective iteration in the step 1 into a Hoagland nutrient solution; 3, the nutrient solution samples with the flora added in the step 2 are respectively and equivalently added into rice seedling culture dish samples; 4, observing the growth vigor of the rice once every five days, and detecting morphological indexes such as leaf length and root length of the rice; 5, repeating the steps 1-4 on the inoculated flora screened in the step 4, and circulating for 5 times; the method has the advantage of screening out the microbiome capable of remarkably promoting the growth of the rice seedlings.

The invention relates to a T7RNA polymerase mutant for synthesizing single-stranded DNA as well as a screening method and application of the T7RNA polymerase mutant, and belongs to the technical field of enzyme engineering. The invention provides a method for directed evolution and screening of a T7RNA polymerase mutant, which comprises the following steps: knocking out exonuclease and mismatch repair protein from a chassis strain genome, knocking in single-stranded annealing protein, resistance gene containing deletion mutation and a single-stranded DNA synthesis template, and constructing to obtain a genetically engineered bacterium for screening; a T7RNA polymerase mutant library is constructed, recombinant plasmids with the mutant library are transferred into engineering bacteria, T7RNA polymerase mutants synthesize single-stranded DNA to repair deletion mutation of resistance genes, the transformed engineering bacteria can grow on a resistance plate, the T7RNA polymerase mutants obtained through multiple rounds of screening can efficiently synthesize the single-stranded DNA through in-vitro verification, and the T7RNA polymerase mutant can be used for preparing the single-stranded DNA. A brand new tool is provided for synthesis of single-stranded DNA, and the method has the potential of being applied to the fields of gene editing, evolutionary engineering and the like.

Described herein are platforms, systems, media, and methods for a machine learning-driven iterative drug discovery process. Some aspects comprise: receiving a first input data set comprising first binding interaction information between a target molecule and a library of compounds; processing the first input data set using a machine learning module to generate a model representation of binding interactions, wherein the representation is configured to predict binding between the target molecule and an input compound; determining an updated library of compounds using the model representation of binding interactions, wherein the updated library of compounds comprises one or more new compounds predicted to bind the target molecule; receiving a second input data set comprising second binding interaction information between the target molecule and the updated library of compounds; and processing the second input data set using the machine learning module to update the model representation of binding interactions, wherein the predictive accuracy of the updated model representation is improved.

The invention provides a halohydrin dehalogenase mutant and an application of the halohydrin dehalogenase mutant in synthesis of a chiral gamma-amino alcohol compound. Halogenated alcohol dehalogenase mutants enabling 2-substituted oxetane to be subjected to ring opening are obtained through the technologies of gene mining, directed evolution and the like, and the enzymes can directly perform ring opening on a substrate 2-aryl oxetane under the action of a nucleophilic reagent to generate a series of chiral gamma-amino alcohol compounds which are important precursors for synthesizing drugs. Therefore, the halohydrin dehalogenase mutants have important application value in the industry.

The invention discloses efficient synthesis of ursodesoxycholic acid by improving the positive enzyme activity of 7 beta-hydroxysteroid dehydrogenase through directed evolution, and belongs to the field of biological catalysis engineering. According to the invention, the 7beta-hydroxysteroid dehydrogenase mutant TEAE / F152L / W101N with improved positive reaction activity and improved affinity with a substrate is obtained. Compared with TEAE, the forward enzyme activity of the mutant enzyme is improved by 2.9 times, the reverse activity of the mutant enzyme is reduced by 3.6 times, the catalytic efficiency kcat / Km of the mutant enzyme to 7K-LCA is improved by 4.3 times, and compared with a wild type mutant TEAE / F152L / W101N, the enzyme activity of the mutant enzyme is improved by 206.23 times, and the catalytic efficiency of the mutant enzyme to 7K-LCA is improved by 58.56 times. The invention provides a universal technical platform for directed evolution of hydroxysteroid dehydrogenase, provides an excellent mutant enzyme for biological efficient synthesis of ursodesoxycholic acid, and lays a solid research foundation for efficient and low-cost preparation of ursodesoxycholic acid.

The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

The invention discloses a high temperature resistant beta-mannase mutant and a preparation method thereof, wild type beta-mannase comes from Coprinus cinerea, the wild type beta-mannase is modified by using a directed evolution means to obtain the high temperature resistant beta-mannase mutant, and the mutation of the mutant comprises at least one of N381K, R123Y and M439L. The beta-mannase mutant has good thermal stability under the alkaline condition and longer heating time, the enzyme activity of the mutant is improved by 107.0% compared with that of an original enzyme when the beta-mannase mutant is treated for 2 h at the high temperature of 90 DEG C in a buffer solution with the pH value of 10, the capacity of catalyzing beta-mannase hydrolysis by the enzyme mutant is obviously improved, and the beta-mannase mutant has wide application prospects.

The invention discloses a heme oxygenase mutant and application of the heme oxygenase mutant in preparation of bilirubin through multienzyme coupling, and belongs to the field of biological catalysis engineering. The forward mutant of the heme oxygenase is obtained through a directed evolution technology, and heme can be efficiently catalyzed to synthesize biliverdin; the bilirubin is synthesized by further introducing biliverdin reductase, coupling cytochrome P450 reductase and formate dehydrogenase, and coupling the two core enzymes with foreign assistant enzymes to construct recombinant escherichia coli to catalyze heme to synthesize bilirubin. In the system, NADPH does not need to be exogenously added, the reaction time is about 4 h, the yield of bilirubin reaches 2.10 g / L, accumulation of intermediate products is not observed in the catalysis process, and the highest yield and the shortest reaction time in bilirubin synthesis reported at home and abroad at present are achieved. Compared with the traditional pig bile extraction, the method disclosed by the invention breaks through the resource limitation, is simple to operate and high in yield, and realizes efficient and green synthesis of bilirubin.

The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to an alkaline protease mutant and application thereof in leather unhairing. According to the invention, alkaline protease derived from Virgibacillus byunsanensis and alkaline protease derived from Ordinitinibacillus caprae are subjected to directed evolution in vitro, so that an alkaline protease mutant of which the enzyme activity and the stability are remarkably improved is obtained, and active extracellular expression of a coding gene of the mutant is realized in an engineering bacterium bacillus subtilis WB600. The optimal reaction temperature of the alkaline protease mutant is 45 DEG C, the optimal pH value of the alkaline protease mutant is 8, the residual enzyme activity of the alkaline protease mutant can be kept at 90% or above after the alkaline protease mutant is incubated for 12 h under the conditions that the pH is 8 and the temperature is 50 DEG C or below or the temperature is 45 DEG C and the pH is 7-9, and the alkaline protease mutant has good thermal stability and pH stability, has high tolerance to NaCl, is applied to unhairing of salt wet sheepskin subjected to preservative treatment through NaCl with the concentration smaller than or equal to 100 g / L, and has good application prospects. Under the conditions that the pH is 7-9 and the temperature is 40-50 DEG C, the unhairing time is shortened to 1.5-2 hours.

The invention belongs to the field of biotechnology and environmental governance, and particularly relates to a manganese peroxidase mutant based on high-throughput screening modification and application of the manganese peroxidase mutant to straw degradation. Aiming at the problems of low natural secretion amount, insufficient specific activity, complex preparation process and the like of the existing manganese peroxidase, the method comprises the following steps: firstly, constructing engineering bacteria based on an escherichia coli exocrine system, constructing a mutant library through error-prone PCR in combination with a directed evolution technology, and performing high-throughput screening by utilizing an ABTS colorimetric method; the recombinant manganese peroxidase mutant with high catalytic activity is obtained, and the enzyme activity is improved by 1.29 times compared with that of a wild type. Furthermore, recombinant engineering bacteria are constructed on the basis of a bacillus subtilis exocrine system, and efficient secretion of the mutant enzyme is achieved. The mutant enzyme preparation is used for wheat straw degradation, and the lignin degradation rate reaches 41.37% (improved by 30.09% compared with that of a wild type). The method is simple and convenient in process, low in cost and remarkable in effect, and has a wide industrial application prospect.

The present invention belongs to the field of enzyme engineering and food engineering technology, and specifically relates to the directed evolution and site-directed mutagenesis of a lactase enzyme molecule and its application in fresh milk. The amino acid sequence of the lactase mutant obtained by the present invention is shown in SEQ ID NO.4. Through the directed artificial evolution of the lactase molecule, a new lactase molecule is obtained that can efficiently convert lactose in fresh milk and synthesize oligosaccharides at a relatively low temperature, and is rapidly inactivated at a subsequent elevated temperature (such as pasteurization). This facilitates the highly nutritious in situ bioconversion of lactose in fresh milk into prebiotic oligosaccharides, further improving the nutritional value of fresh milk and expanding the fresh milk consumer group.

The invention discloses a beef cattle straw feed preparation method based on compound strain synergistic fermentation, and relates to the technical field of feed processing, and the beef cattle straw feed preparation method comprises the following steps: screening bacillus, lactic acid bacteria and the like to construct an initial compound strain, and screening a high-degradation-ability strain combination through a directed evolution system (30-45 DEG C, pH 4.0-7.0, and lignocellulose 5-15g / L); the straw lignocellulose structure is destroyed through microwave-assisted pretreatment (the power is 200-800 W, and the time is 1-5 min); high-efficiency fermentation is realized through double-stage fermentation (the pH is reduced at 30-35 DEG C for 12-24 hours in the first stage, and stubborn components are degraded at 35-40 DEG C for 36-60 hours in the second stage) and intelligent regulation and control. According to the method, the content of crude protein in the straw is increased to 15.2%, the content of crude fibers is reduced to 19.8%, the degradation rate of lignocellulose reaches 62%, the quality of the feed is remarkably improved, and a new path is provided for recycling of agricultural wastes.

The invention relates to the technical field of gene engineering, in particular to a glycosyl transferase mutant and application thereof in synthesis of rebaudioside M. Wild type glycosyl transferase is modified through a directed evolution theory to obtain the glycosyl transferase mutant, so that the efficiency of synthesizing rebaudioside D through reaction of rebaudioside A is effectively improved. Besides, recombinant bacteria capable of simultaneously expressing the glycosyl transferase mutant, a second glycosyl transferase gene and a sucrose synthase gene are constructed, a wet cell obtained by induced culture of the recombinant bacteria or a crude enzyme liquid extracted by crushing the wet cell is used as a catalyst, rebaudioside A is used as a substrate, sucrose is used as an auxiliary substrate, and the rebaudioside M is catalytically synthesized by a one-pot method. According to the present invention, the complete catalysis of 100 g / L of rebaudioside A to produce rebaudioside M only needs 24 h, the conversion rate reaches 97.5%, the yield of rebaudioside M reaches 130 g / L, the raw material conversion rate and the yield of rebaudioside M are effectively improved, and the synthesis time is shortened.

The invention discloses a germacene A synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. Aiming at the bottleneck of the catalytic efficiency of the germacene A synthase, the optimal germacene A synthase mutant DlGASS32H-I97V-Y315F is obtained through semi-rational design based on sequence conservative analysis by taking the germacene A synthase from fungi with a separation wheel layer carbon shell as an object, and the optimal germacene A synthase mutant DlGASS32H-I97V-Y315F is verified through shake flask fermentation in engineering bacteria saccharomyces cerevisiae, so that the yield of beta-elemene is increased by 24%. The combined mutant obtained through a key gene conserved sequence mutation prediction strategy shows remarkably improved catalytic performance, the effectiveness of guiding an enzyme directed evolution strategy based on multi-species sequence conservative analysis is verified, and the yield of germacene A or beta-elemene is remarkably improved. Meanwhile, the method has the advantages of simplicity and convenience in operation, environmental friendliness, mild reaction conditions and the like, and has a very good application prospect in the field of production of germacene A.

The present disclosure belongs to the field of synthetic biology and relates to a valencene synthase mutant and a valencene high-yield strain. An enzyme for synthesizing valencene is derived from Eryngium glaciale, and upon enzyme directed evolution of the enzyme, a valencene synthase mutant with improved enzyme performance is obtained, and the yield of a strain containing the mutant is 3.15 times the yield of a strain containing a wild-type synthase. The valencene synthase mutant of the present disclosure enhances the capability of synthesizing valencene by a strain, and a powerful foundation is laid for the industrial production thereof. A high-yield strain for synthesizing valencene is constructed by using the valencene synthetase mutant, and the yield of a fermentation tank reaches 12.4 g / L, which is the highest level reported to date.

The present disclosure generally relates to an evolved cytidine deaminase derived from a cytidine deaminase, and a method of editing DNA using the same. In some aspects, the present disclosure describes directed evolution of a TadA-derived adenosine deaminase (TadA-CD) for cytidine deamination. In some embodiments, the TadA-CD comprises a plurality of mutations as compared to the parent TadA variant. In some embodiments, the TadA-CD is fused to a programmable DNA binding protein. Other aspects of the present disclosure generally relate to a cytosine base editor (CBE) comprising a programmable DNA binding protein and a TadA-CD. In some embodiments, the disclosed cytosine base editor has an improved conversion efficiency and a reduced off-target editing frequency compared to a naturally occurring CBE. Polynucleotides, vectors, and kits for generating and delivering CBE are also provided. Also provided are cells containing such vectors and CBE. In addition, methods of treatment comprising administration of CBE are also provided.

The invention discloses a high-throughput enzyme directed evolution detection method and a magnetic micro-droplet sorting system, and belongs to the field of biological detection. Aiming at the technical defects of low separation flux, high cost of a separation device and insufficient magnetic separation precision in the existing enzyme directed evolution screening technology, a magnetic micro-droplet technology and a micro-fluidic chip technology are combined, and a micro-fluidic magnetic separation chip system integrating a detection module and a magnetic field separation module is constructed. The system has the technical advantages that the sorting flux and accuracy are remarkably improved, the structure of the sorting device is simplified, and the operation cost is reduced. A high-throughput, high-selectivity, high-universality, economical and efficient screening platform is provided for directed evolution of enzyme molecules.

The invention discloses a 4-propyl guaiacol oxidase mutant as well as a preparation method and application thereof, wild type 4-propyl guaiacol oxidase comes from Penicillium simplicissimum, an active center of the wild type 4-propyl guaiacol oxidase is modified by using a directed evolution means, and the mutant is obtained by modifying the active center of the wild type 4-propyl guaiacol oxidase. 4-propyl guaiacol oxidase mutants D170E, S426T, S50Q / D170E, D170E / L411V and D170E / L411V / S106C with relatively high activity, cis-trans selectivity and trans-isoeugenol yield are obtained, efficient synthesis from cheap 4-propyl guaiacol to high-value trans-isoeugenol is realized, and industrial synthesis and application of the trans-isoeugenol are facilitated.

The invention discloses a directed evolution strategy of type II sesquiterpene cyclase (SsDMS) derived from Streptomyces shedoensis based on alanine scanning, and an application of the type II sesquiterpene cyclase (SsDMS) derived from Streptomyces shedoensis based on alanine scanning. The invention relates to a plurality of SsDMS key mutant sites capable of catalytically generating a novel drive type sesquiterpenoid compound, which comprise substitution of sites 208, 248, 249, 497 and 505, and the SsDMS key mutant sites comprise substitution of sites 208, 248, 249, 497 and 505. The invention discloses an efficient escherichia coli cell factory applied to heterologous expression of an SsDMS mutant and efficient production of a novel drive type sesquiterpene compound, and further obtains the drive type sesquiterpene compound with a novel structure. According to the present invention, the efficient production of the drive type sesquiterpene compound by using the SsDMS and the mutant thereof is achieved, the modification strategy has wide applicability, the important enzymology resource is provided for the biosynthesis of the novel terpene compound, and the more choices are provided for the discovery of the drug lead compound.