153 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 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 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 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 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 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 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.

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.

The application discloses a polyphosphate kinase mutant, a preparation method and application thereof, and wild-type polyphosphate kinase from Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1, The active site is mutated by using a directed evolution method, and a polyphosphate kinase mutant with high activity in catalyzing synthesis of ATP is obtained, the relative activity of the mutant to a substrate ADP or AMP is much higher than that of a wild type, and the mutation is at least one of D307M, W408A and Y431P, or at least one of E304V, P331S and Y431P. The application realizes efficient synthesis from a cheap substrate to a high-value product, is helpful to industrial production of ATP, and has a good application prospect in the fields of medicine and the like.

The invention belongs to the technical field of directed evolution modification and biological catalysis application of enzymes, and relates to a Candida antarctica lipase B mutant protein and application thereof, the sequence of the mutant protein comprises one or more of the mutation of the 195 S and F, the mutation of the 195 S and Y and the mutation of the 283 A and Y from the N terminal to the C terminal of the amino acid sequence shown in SEQ ID NO.1 of the original CALB; on the basis of a wild type lipase gene sequence, through rational design and site-specific mutagenesis, mutants S195F, S195Y, A283Y, S195F-A283Y and S195Y-A283Y, of which the Tm value and the optimal reaction temperature are obviously improved and the catalytic activity is improved by 1-2 times, are finally obtained; when the mutant is used for catalyzing synthesis of docosahexaenoic acid (DHA) glyceride, higher catalytic efficiency is shown, and the yield of the DHA glyceride is effectively improved.

A continuous directed evolution platform in human B cell lines (CODE-HB) that recruits and repurposes the inherent B cell somatic hypermutation (SHM) mechanisms to rapidly evolve proteins is described. This approach uses stable, non-immunoglobulin loci within the genomes of human B cell lines for integration of a heterologous gene, such as a gene encoding an antibody or antibody fragment. A B-cell surface display platform for displaying fragment antigen binding domains (Fab) of antibodies is also described, which adapts the CODE-HB platform to rapidly evolve antibody fragments in a continuous manner. Plasmids and kits for carrying out these methods are also described.

The invention belongs to the field of biology, and particularly relates to a botulinum toxin A light chain mutant and application. The BoNT / A LC mutant is obtained by performing directed evolution optimization on the BoNT / A LC by adopting a phage-assisted evolution method, and compared with a wild type BoNT / A LC, the activity of the mutant is remarkably improved, the defects of an existing botulinum toxin product and the problems of drug resistance and the like can be overcome, and the BoNT / A LC mutant can be applied to the fields of medical beauty and medical treatment and has a wide application prospect.

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

The invention relates to the technical field of metabolic engineering bacteria. The invention provides an engineering bacterium for improving the yield of poly-beta-hydroxybutyric acid and a construction method of the engineering bacterium. According to the invention, methylotrophic bacteria MB200 is taken as an original strain, a mispairing repair system MutL gene knock-down strain of CRISPRi is constructed, so that the mispairing repair system MutL gene knock-down strain has a higher gene mutation rate, laboratory directed evolution is carried out by utilizing a screening mode of'rich-deficient carbon sources', chassis cells with stable genomes after directed evolution are obtained, and the engineering strain is named as MB-P1. Compared with the original strain methylotrophic bacteria MB200, the PHB production capacity of the MB-P1 is improved by 72.61%. The method provided by the invention is suitable for optimizing the methylotrophic bacteria strain for producing the PHB, provides a new normal form for realizing efficient accumulation of the PHB, and has wide industrial application potential.

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.

The invention discloses a tylosin sensing biosensor, a construction method thereof and MphR mutant protein based on the tylosin sensing biosensor, and belongs to the technical field of gene engineering. The technical problem to be solved by the invention is how to detect tylosin. In order to solve the technical problem, the invention provides the biosensor for sensing tylosin, the biosensor can express an MphR31D1 protein and / or a coding gene containing the MphR31D1 protein, and the MphR31D1 protein can be a protein with an amino acid sequence of SEQ ID NO: 1. The invention also provides a preparation method of the biosensor, and application of the MphR31D1 protein and related biological materials thereof in detection of tylosin. According to the invention, escherichia coli Top10 is taken as a chassis bacterium, a macrolide compound transcriptional regulation factor MphR is taken as a starting template, and the tylosin whole-cell biosensor is successfully constructed through protein directed evolution.

The application belongs to the field of biology and relates to a nanobody targeting calexcin and a preparation method and application thereof, the nanobody targeting calexcin is successfully screened and prepared, and the nanobody-HRP fusion protein used for affinity identification in an enzyme-linked immunosorbent assay is successfully expressed; in combination with a phage-assisted continuous and discontinuous directed evolution (PACE) system, the nanobody screened is subjected to directed evolution, and stable mutation sites are found through sequence alignment analysis; it is proved through an indirect enzyme-linked immunosorbent assay and a luciferase complementation assay that the affinity of the nanobody after evolution to calexcin is improved by about 2 times.

The invention relates to an adenovirus associated virus 9 (AAV9)-based viral particle, for use in a method of treatment of a heart disease, whereby said viral particle is administered to the heart. The invention further relates to a pharmaceutical composition comprising the AAV9-based viral particle, and to methods of producing the AAV9-based viral particle.

The invention belongs to the technical field of biological catalysis and enzyme engineering, and discloses a fluorinated enzyme mutant, a recombinant plasmid and a screening method and application thereof. The amino acid sequence of the fluorinated enzyme mutant is obtained by mutating an amino acid sequence shown as SEQ.ID.NO.1 as follows: threonine at the 84th site is mutated into alanine, or / and tryptophan at the 144th site is mutated into phenylalanine. The catalytic activity of the fluorinase mutant is obviously higher than that of a wild type, the synthesis efficiency of organic fluoride can be effectively improved, and the fluorinase mutant has great application potential in the field of organic fluoride biosynthesis. A high-throughput screening method is used for the fluorinase mutant, the method is suitable for rapid screening of the fluorinase mutant, the directed evolution efficiency of the fluorinase is improved, and a feasible scheme is provided for exploring the fluorinase with excellent characteristics.

This invention relates to a high-yield coenzyme Q method. 10 The establishment and application of *Rhodotorula globulinii* strains. Compared to rational metabolic engineering, the coenzyme Q of this invention... 10 High-yielding Rhodopseudomonas aeruginosa bacteria were used as a template by the global regulatory factor prrA gene. Through directed evolution, different mutant strains were constructed, and combined with high-throughput screening methods, coenzyme Q was achieved. 10 Increased yield. This invention starts from global regulatory factors and leverages complex intracellular regulatory mechanisms to achieve fine-grained global regulation under minute genomic perturbations, providing a new approach for the engineering modification of strains.

The application belongs to the technical field of biology and specifically relates to a Cas12a protein mutant with high cleavage activity and application thereof. The Cas12a protein mutant has high trans-cleavage activity and has a good application prospect in nucleic acid detection. The Cas12a protein mutant changes the binding of the protein and crRNA by directional evolution of the RECII domain, improves the cleavage efficiency of the target strand in double-stranded DNA, and improves the detection sensitivity, and can distinguish single nucleotide polymorphism (SNP). The Cas12a protein mutant can hardly depend on PAM sequence recognition for SNP site detection, can distinguish SNP located between positions 1 to 17, and realizes high-sensitivity and high-precision molecular detection.

A method for protein engineering includes generating a fitness library by performing protein language model (PLM)-guided site selection to identify favorable mutagenesis sites and creating protein variants, training a machine learning model to predict protein fitness from sequence data, wherein the machine learning model comprises a convolutional neural network (CNN); and optimizing protein sequences using a phase transition-based algorithm that dynamically updates a score matrix A of dimensions L×20 using cumulative statistics from sampled sequences and implements heating and cooling cycles to maintain system criticality. The CNN has a three-component sequence representation comprising a latent embedding matrix of shape L×1280, a probability matrix of shape L×20, and a feature vector containing 7 values including normalized zero-shot scores, where L represents the number of residues in the protein, wildtype subtraction normalization applied to the latent embedding matrix, dual parallel convolution processing paths, and percentile-based pooling.

Disclosed are adeno-associated virus (AAV) capsid protein variants that have improved infectivity and transduction efficiency for target cells, and uses thereof. Specifically, the disclosure relates to AAV2 variant capsid proteins selected by directed evolution for gene delivery to retinal cells, recombinant AAV vectors comprising the same, and uses thereof as gene delivery vehicles to retinal cells.