241 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 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 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 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 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 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 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 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 relates to the technical field of biochemistry, in particular to an enzyme mutant and application thereof in preparation of conopeptide. According to the preparation method, a strategy of combining green chemistry and an enzyme method is adopted, seven tripeptide fragments are taken as raw materials, a conopeptide main chain is prepared through a liquid-phase synthesis method, and then enzymatic precise modification and synthesis are realized by utilizing a multiple directed evolution enzyme concerted catalysis system. Based on the efficient connection characteristic of a liquid-phase synthesis system and the high substrate specificity of an enzymatic system, the complex protection group operation in traditional solid-phase synthesis is effectively avoided, generation of by-products is remarkably reduced, meanwhile, fixed-point modification and precise assembly of a conopeptide main chain are achieved, and the application prospect is wide. And an efficient and green new path is provided for the continuous production of the cosmetic-grade high-purity conopeptide.

The invention discloses a methanol dehydrogenase mutant with improved catalytic activity, and belongs to the technical field of enzyme directed evolution and protein engineering. The methanol dehydrogenase is subjected to random mutation by means of directed evolution. After the construction of the plasmid library is completed, the methanol dehydrogenase with higher activity is screened by using a high-throughput and simple in-vivo screening system. Two methanol dehydrogenase mutants S1 (V37A) and S2 (I58T / M292T) with significantly improved methanol catalytic activity are obtained, V37 is subjected to saturation mutation on the basis, and mutants with improved formaldehyde generation rate are further screened. The methanol dehydrogenase mutant obtained by the invention can provide a good catalytic element for bioconversion of methanol, and has a wide application prospect.

In one embodiment, a method includes accessing a biomolecule representation of a first biomolecule and processing the biomolecule representation by a machine-learning model trained using sequencing time-series data. The sequencing time-series data was obtained from directed evolution of a population of biomolecules over multiple enrichment rounds where the population of biomolecules in each enrichment round was a unique set of biomolecules with respect to each other enrichment round. The sequencing time-series data for each enrichment round comprises a biomolecule frequency of each biomolecule of the population of biomolecules in the respective enrichment round. The training comprises learning inferred fitness scores of the population of biomolecules for each enrichment round by predicting biomolecule frequencies of the population of biomolecules in the respective enrichment round given biomolecule frequencies of the population of biomolecules in prior enrichment rounds. The method further includes outputting an inferred fitness score for the first biomolecule.

The invention relates to the technical field of biology, in particular to a pichia pastoris recombinant strain for expressing non-specific peroxygenase CciUPO and application of the pichia pastoris recombinant strain. The pichia pastoris recombinant strain is obtained by the following steps: S1, synthesizing a sequence of a CciUPO original gene sequence optimized based on a yeast codon; s2, carrying out recombination with the EV signal peptide after the directed evolution; s3, connecting the recombined target gene with an expression vector to construct a recombinant plasmid; and S4, transforming the recombinant plasmid into pichia pastoris competent cells, and screening out a recombinant strain containing the CciUPO recombinant gene. The expression quantity of the CciUPO obtained through the operation is increased by 10 times compared with that of a wild type CciUPO, the CciUPO has strict catalytic regioselectivity, can catalyze vitamin D3 to synthesize calcifediol, the selectivity is as high as 95%, the catalytic conversion rate of toluene is 95%, the selectivity is 78%, and the CciUPO has a relatively high application prospect in industrial production.

The invention discloses a new enzyme with acylsulfonate transferase activity or a mutant thereof and application thereof, and belongs to the field of enzyme engineering. On the basis of an NCBI (National Center of Biotechnology Information) database, a new enzyme sequence is mined through a bioinformatics means, a new enzyme is obtained through codon optimization, gene synthesis and cell expression, the activity of acylsulfonate transferase is measured, and a corresponding mutant is obtained by using a directed evolution method. Compared with reported mouse acyl sulfonate transferase (ASTIV), the enzyme sequence obtained in the invention has better catalytic activity in a regeneration system of PAPS, and provides a new choice for enzymatic synthesis of PAPS.

The invention relates to the technical field of agricultural biology, in particular to a rational design method of a rice growth-promoting synthetic microbial community based on collaborative optimization of directed evolution and a metabolism model. The method comprises the following steps: continuously subculturing a multi-source soil microbial community to be stable, co-culturing the multi-source soil microbial community and rice seeds, and selecting an optimal community based on a growth phenotype; the method comprises the following steps: selecting an optimal community, applying a bottleneck to the optimal community, externally adding flora disturbance, constructing a filial generation community, repeating'screening-disturbance-subculture 'for five rounds to obtain a growth promoting function enhanced directed evolution synthesis microbial community, dividing the microbial community into a plurality of modules, and constructing a genome scale metabolism model to simulate the influence of interaction between different modules on rice growth. And combining the directed evolution synthesized microbial communities in pairs according to different proportions so as to obtain rational design synthesized microbial communities with better growth promoting effects. According to the method, the problem of model prediction deviation caused by lack of a host-microorganism interaction mechanism in an existing method is solved.

HPV16 is a main cause of malignant tumors such as cervical cancer, and E6 protein of HPV16 becomes a key treatment and diagnosis target. In the prior art, an HPV16 detection antibody has the problems of poor stability, dependence on cold chain transportation, low preparation efficiency and the like. According to the invention, a nano antibody sequence which is high in affinity (dissociation constant KD is less than or equal to 1 * 10 <-7 > M) and stable at room temperature is obtained through total synthesis library construction and a phage-assisted directed evolution (PACE) screening technology. After the antibody is stored at 25 DEG C for 30 days, the activity of the antibody is kept to be greater than or equal to 95%, the antibody can be directly used for household test paper, the accuracy rate reaches 98%, and meanwhile, the antibody can be expanded to medical applications such as CAR-T cell therapy. Compared with a traditional antibody, the antibody has the advantages of being short in preparation period, low in cost, wide in application scene and the like, and a breakthrough solution is provided for early screening of cervical cancer.

This invention discloses a method for directed evolution of antibiotic-resistant Escherichia coli strains based on cytidine deaminase, belonging to the field of microbial directed evolution and genetic engineering technology. This method uses E. coli as the host, introducing a recombinant plasmid expressing an optimized double-stranded cytidine deaminase mutant. Utilizing the low toxicity and high mutagenicity of this mutant, continuous passage evolution is carried out under gradient concentrations of aminoglycoside antibiotics (kanamycin and streptomycin). Combined with whole-genome sequencing, molecular docking, and reverse genetics verification, the A145T missense mutation in the wcaE gene is identified as the core functional site. This invention overcomes the shortcomings of traditional spontaneous and chemical mutagenesis, which suffer from low efficiency and significant strain damage. The mutation type is controllable, the evolutionary cycle is short, and the obtained engineered strains can tolerate up to 300 mg / L kanamycin while exhibiting streptomycin cross-resistance, and the genetic stability of the tolerance trait is strong. This method is simple, highly reproducible, and suitable for industrial breeding of stress-resistant E. coli, and can be widely applied in antibiotic fermentation, industrial microbial culture, and other scenarios.

The invention relates to the fields of enzyme engineering and microbial fermentation production, in particular to an N-acetylglutamate synthetase mutant for producing N-acetyl blue and application of the N-acetylglutamate synthetase mutant. According to the invention, the N-acetylglutamate synthetase mutant EcargAV323A / S435A is constructed by researching the structure of N-acetylglutamate synthetase ArgA (EcargA) from Escherichia coli and mutating the 323rd site and the 435th site of EcargA protein in combination with a directed evolution technology, so that the N-acetylglutamate synthetase mutant EcargAV323A / S435A is constructed. When the engineering strain of the N-acetylglutamate synthetase mutant is used for producing the N-acetylobservation blue through fermentation, the yield of the N-acetylobservation blue can be remarkably improved, the generation of a by-product observation blue is reduced, the fermentation process is simplified, and the production cost is reduced. The N-acetylglutamate synthetase mutant and the engineering strain provided by the invention have good industrial application value.

The invention discloses a urate oxidase mutant and application thereof in improving the thermal stability of urate oxidase, and belongs to the technical field of biology. The urate oxidase mutant disclosed by the invention is T68L / T75S / E222D / K299E, T68L / T75S / K299E or T75S / K299E, and the urate oxidase mutant disclosed by the invention is T68L / T75S / K299E. According to the method, the key sites influencing the thermal stability are mined in the modes of directed evolution, high-throughput screening, multiple virtual screening and the like, and the stability is improved through mutation. And finally, a plurality of key sites and mutants influencing the stability are excavated, and the thermal stability is improved.

The invention belongs to the technical field of directed evolution modification of protein, and particularly relates to an engineered bacillus subtillis chassis cell for inserting unnatural amino acid, which is formed by uniformly replacing termination codons UAG or UGA of 57 essential genes of bacillus subtillis with UAA. On the basis, two sets of mutually orthogonal translation systems are introduced to decode the UAG and the UGA respectively, and finally site-specific insertion of the two non-natural amino acids at the preset site of the target protein is realized; a stable and reliable cell platform is provided for obtaining protein molecules carrying two different chemical functional groups and carrying out multi-site refined modification research.

The application discloses 4-propyl guaiacol oxidase mutants, a preparation method and application thereof, wherein wild-type 4-propyl guaiacol oxidase is from Penicillium simplicissimum Penicillium simplicissimum ​ The active center of the wild-type 4-propyl guaiacol oxidase is reformed by using a directed evolution method, and 4-propyl guaiacol oxidase mutants D170E, S426T, S50Q / D170E, D170E / L411V and D170E / L411V / S106C with high activity, cis-trans selectivity and trans-isoeugenol yield are obtained, efficient synthesis of high-value trans-isoeugenol from cheap 4-propyl guaiacol is realized, and the industrialized synthesis and application of the trans-isoeugenol are facilitated.

A thermostable DNA polymerase mutant with high amplification activity. Using protein directed evolution technology, a random mutation library was constructed for the polymerase active domain targeting Taq enzyme. By gradually adding screening pressure, unsuitable mutations were naturally eliminated, and mutations with advantageous traits gradually accumulated. Finally, a series of amino acid sites and their mutations that play a key role in the amplification performance and polymerization performance of Taq enzyme were screened out, and a Taq enzyme mutant with high amplification performance was obtained.

The invention, in part, includes systems for conducting continuous directed evolution in a plurality of sample and methods of using the systems. The invention, in part also provides systems for evaluating the suitability of diverse engineered cells to accomplish directed evolution and methods of use of such systems.

The present invention provides engineered lysine decarboxylases that can be used to synthesize 1,5-diaminopentane under industrially relevant conditions. The present invention also provides polynucleotides encoding engineered lysine decarboxylases, host cells capable of expressing the engineered lysine decarboxylases, and methods for preparing 1,5-diaminopentane using the engineered lysine decarboxylases. The engineered lysine decarboxylase of the present invention was developed from a wild-type lysine decarboxylase through a creative process of directed evolution, and the engineered lysine decarboxylase of the present invention has a better activity and / or stability and tolerates a high substrate concentration compared to other lysine decarboxylases for the preparation of 1,5-diaminopentane, and thus has a good prospect for industrial application.