23 results about "Directed evolution" patented technology

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 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.

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.

The application discloses a kind of deep learning assisted structure remodeling design and double-substrate collaborative screening lipase directed evolution method, including the key candidate residue site of lipase, the conservation residue in the key candidate residue site is eliminated, and first core mutation site is obtained;With the first core mutation site as the designable region, the mutation sequence is generated by resampling design using the protein unfolding model;The conservation residue in the first core mutation site is eliminated, and the second core mutation site is obtained, and the enzyme liquid containing lipase mutant is obtained after heterologous expression;Two kinds of substances are used as substrates for activity determination, and the mutant with increased activity under two substrates is selected to complete the screening of mutant.The present application greatly reduces the screening burden of wet experiment by using deep learning tools to assist pre-screening, and solves the problem that substrate preference is difficult to consider in traditional directed evolution by combining double-substrate evaluation system.

This invention discloses an R-type α-fluoroimine reductase mutant, its preparation method, and its applications. The wild-type imine reductase is derived from *Xanthomonas aeruginosa*. Luteolibacter luteus By using directed evolution to modify its active site, an imine reductase mutant with high activity, stereoselectivity, and high yield of 4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline was obtained. The mutation is at least one of S237T, I241L, or R248L. This invention achieves efficient synthesis of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline from inexpensive (Z)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine, which is beneficial for the industrial synthesis and application of (R)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline.

The application discloses an alcohol dehydrogenase mutant and application thereof in preparation of cis-4-propylcyclohexanol. The mutant is obtained by substituting at least one amino acid in three positions of an amino acid sequence shown in SEQ ID No. 1, i.e. the 43th, 271th and 282th positions. The 43th phenylalanine (F) is substituted into valine (V) or serine (S), the 271th isoleucine (I) is substituted into valine (V) or phenylalanine (F), and the 282th phenylalanine (F) is substituted into valine (V). The wild-type alcohol dehydrogenase gene is subjected to directional evolution modification, and the alcohol dehydrogenase mutant has higher catalytic activity and higher stereoselectivity than the wild-type alcohol dehydrogenase, so that the cis-4-propylcyclohexanol is efficiently and highly stereoselectively generated.

Compositions and methods are described demonstrating the ability to 1) use a virus assisted directed evolution platform to significantly improve the activity of engineered nonsense-suppressor tRNAs in mammalian cells, 2) provide mutants of E. coli tyrosyl-tRNAs that show remarkably improved Uaa incorporation efficiency in mammalian cells, and 3) use these tRNAs to express recombinant proteins in mammalian cells incorporating Uaas at significantly improved yields.

This invention relates to the field of base editing technology, specifically disclosing an R-loop binding domain and its application in base editing. The R-loop binding domain is PoRHBD, and its amino acid sequence is shown in SEQ ID NO:4. This invention also discloses a fusion protein containing PoRHBD, which, from the N-terminus to the C-terminus, sequentially includes a nuclear localization signal NLS1, an adenine deaminase TadA8e, a flexible linker peptide Linker1, PoRHBD, a flexible linker peptide Linker2, a nuclease-inactivating Cas12C9, and a nuclear localization signal NLS2. This fusion protein can effectively achieve A-to-G base substitution at the target sites of the rice genes OsACC and OsARF4. The R-loop binding domain PoRHBD and the fusion protein provided by this invention significantly improve the efficiency of adenine base editing, providing an efficient tool for precise crop improvement and directed evolution of endogenous genes.

The present application relates to the construction and application of the EcORep-based continuous directed evolution reporter system of aminoacyl-tRNA synthetase, and specifically, the reporter system comprises: a green fluorescent protein mutant sfGFP containing amber stop codon TAG N150TAG ; a chloramphenicol acetyltransferase mutant CAT containing amber stop codon TAG D112TAG ; a linear replicon containing aminoacyl-tRNA synthetase; a reporter plasmid containing a plus-strand mismatch DNA polymerase to continuously mutate the linear replicon, thereby generating an in-vivo library of aminoacyl-tRNA synthetase mutants; and a tRNA, which is orthogonal to the aminoacyl-tRNA synthetase and enables the incorporation of unnatural amino acids and the complete expression of proteins. The present application proves that the evolution reporter system can realize the efficient directed evolution of aminoacyl-tRNA synthetase, and further expands the application range of genetic code expansion technology.

In one embodiment, a method includes obtaining sequencing time-series data from a first round of directed evolution, wherein the sequencing time-series data for the first round comprises a first biomolecule frequency of a first biomolecule, the first biomolecule frequency of the first biomolecule being a non-zero frequency, obtaining sequencing time-series data from a second round of directed evolution, wherein the sequencing time-series data for the second round comprises a second biomolecule frequency of the first biomolecule, and outputting an inferred fitness score for the first biomolecule using the first biomolecule frequency and the second biomolecule frequency.

This invention discloses an S-type α-fluoroimine reductase mutant, its preparation method, and its applications. The wild-type imine reductase is derived from actinomycetes. Actinomadura welshii By using directed evolution to modify its active site, S-type α-fluoroimine reductase mutants with high activity, stereoselectivity, and high yield of 4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline were obtained. These mutants were D98G, D98G / M122L, or D98G / M122L / W216V. This invention achieves efficient synthesis of (S)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline from inexpensive (trans)-2,2,2-trifluoro-N-(4-methoxyphenyl)-1-phenylethylimine, which is beneficial for the industrial synthesis and application of (S)-4-methoxy-N-(2,2,2-trifluoro-1-phenylethyl)aniline.

The application discloses an enzyme directed evolution method based on a protein language model and multi-dimensional structure feature weighting, and belongs to the cross field of enzyme engineering and bioinformatics. The method first acquires target enzyme sequence and three-dimensional structure data, fine-tunes an ESM-1B model to identify key function anchors; after structure preprocessing, a local interaction network is constructed through a KD-Tree and a hydrogen bond geometry algorithm, and candidate mutation sites are screened by adopting multi-factor collaborative scoring; then, a sequence-activity dataset is constructed through saturation mutation verification, closed-loop iterative optimization is performed through an ESM-2+ random forest, and site synergistic effects are mined in combination with combined mutations. The application solves the problems of complex traditional directed evolution process, high cost, and difficulty in multi-property optimization, candidate site screening is accurate, trial and error cost is significantly reduced, single mutant enzyme activity can be increased by 2.0 times at most, combined mutant enzyme activity can be increased by 2.16 times at most, the research and development cycle is greatly shortened, and the application is suitable for efficient directed evolution of enzymes.

This invention relates to a method and apparatus for processing directed evolution data of enzymes, and a computer-readable storage medium. The processing method includes the following steps: acquiring retrieval information about a target enzyme; determining a corresponding candidate set based on the retrieval information, wherein the candidate set includes at least one data structure, and the data structure includes at least a directed evolution data field and a comparison information field; determining the similarity score between the retrieval information and the comparison information field of each data structure in the candidate set; selecting at least one data structure similar to the target enzyme from the candidate set according to a preset similarity threshold; and outputting the directed evolution data field and / or comparison information field of each data structure similar to the target enzyme.

The application discloses a brazzein mutant and application thereof, and belongs to the technical field of protein modification. In view of the defects that wild-type brazzein is insufficient in sweetness, difficult to meet the application demand of industrialized sugar substitute, and the yield of a microbial heterologous expression system needs to be improved, the application adopts protein engineering directed evolution technology, screens single mutants P13E, D26K and I49G, and further constructs combination mutants P13E / D26K, P13E / I49G, D26K / I49G and P13E / D26K / I49G. The mutants are heterogeneously and efficiently expressed in a Pichia pastoris expression system, and the sweet taste performance of the mutants is systematically evaluated. The results show that, compared with the wild-type brazzein, the sweetness of each mutant is increased by 60% to 340%, and the mutants have excellent stability and pure sweet taste, and can be used as a new type of sweetener and applied to the field of food processing, and have important industrial application value.

The present application relates to the technical field of base editing, and specifically provides a fusion protein based on Cas12C9 and its application in adenine base editing. The R loop binding domain is RHBD1, and the amino acid sequence is shown as SEQ ID NO: 4. The present application also discloses a fusion protein containing RHBD1, which sequentially contains a nuclear localization signal NLS1, an adenine deaminase TadA8e, a flexible linker peptide Linker1, RHBD1, a flexible linker peptide Linker2, a nuclease-inactivated Cas12C9 and a nuclear localization signal NLS2 from N-terminal to C-terminal. The fusion protein can effectively realize A to G base substitution at the target sites of rice genes OsACC and OsARF4. The R loop binding domain RHBD1 and the fusion protein provided by the present application significantly improve the adenine base editing efficiency, and provide an efficient tool for crop precision improvement and endogenous gene directed evolution.

The application discloses a kind of heme oxygenase mutant and its multienzyme coupling preparation of application of bilirubin, belong to biological catalysis engineering field.The application obtains the positive mutant of heme oxygenase by directed evolution technology, can efficiently catalyze heme to synthesize chlorophyllin;Further introduce chlorophyllin reductase, coupling cytochrome P450 reductase, formic acid dehydrogenase, and two kinds of core enzyme are coupled with exogenous auxiliary enzyme, constructs recombinant escherichia coli, catalyzes heme to synthesize bilirubin.In the system, without exogenous addition NADPH, reaction time is about 4h, and bilirubin yield is as high as 2.10g / L, and no intermediate accumulation is observed in catalytic process, reaches the highest yield of the synthesis of bilirubin reported at home and abroad, and the shortest reaction time level.Compared with traditional pig bile extraction, the method of the application breaks through the resource limit, and the operation is simple, the yield is high, realizes the efficient, green synthesis of bilirubin.

This invention discloses a high-yield S-type amino acid ester imine reductase mutant, its preparation method, and its applications. The wild-type imine reductase is derived from actinomycetes. Actinomadura welshii By using directed evolution, the active site was modified to obtain imine reductase mutants with high activity, stereoselectivity, and (S)-2-((4-methoxyphenyl)amino)-2-phenylacetic acid methyl ester yield. The mutants are D98G / M122L / W216V, D98G / M122L / W216V / L15Y, D98G / M122L / W216V / L15Y / A120V, and D98G / M122L / W216V / L15Y / A120V / V124L / T238A. This invention enables the efficient synthesis of (S)-2-((4-methoxyphenyl)imino)-2-phenylacetic acid methyl ester from inexpensive (trans)-2-((4-methoxyphenyl)amino)-2-phenylacetic acid methyl ester, which is beneficial for the industrial synthesis and application of (S)-2-((4-methoxyphenyl)amino)-2-phenylacetic acid methyl ester.

The present disclosure belongs to the field of computational biology and protein engineering technology. The present disclosure provides a protein engineering and directed evolution method based on graph deep learning and applications thereof, the method includes the following steps: S1, construction of a protein structural dataset; S2, protein graph representation; S3, graph neural network model architecture; S4, model training and performance evaluation; S5, model inference, and finally identification of potential mutations that can improve the fitness. The present disclosure can realize zero-shot, low-cost, high-efficiency, and accurate prediction of protein variants with improved properties; meanwhile, TadA8ePro with improved A-to-G base editing efficiency, Cas9Plus with higher gene knockout efficiency, and OsPHR2 transcription factor with improved binding activity are also provided. The present disclosure realizes the rapid, low-cost, and efficient engineering of genome editing proteins and transcription factors, and provides a powerful tool for accelerating crop breeding and synthetic biology.

The application belongs to the technical field of genetic engineering, and specifically discloses a glutamine transaminase and application thereof in food. The application obtains a glutamine transaminase mutant with significantly improved enzyme activity and heat resistance by in vitro directional evolution of wild-type glutamine transaminase through error-prone PCR technology. The enzyme activity of the mutant is about 2.4 times that of the wild-type glutamine transaminase, the mutant has strong temperature / pH stability and iron ion tolerance, and can tolerate a salt concentration of 18%. The mutant is used in the preparation of food rich in protein, such as meat products, dairy products, and bean products. The mutant can not only maintain sufficient cross-linking activity under high-temperature heat processing conditions, but also effectively resist the inhibitory effect of Fe 2+ and Fe 3+ in food systems, and still has high catalytic efficiency under conventional or high-iron ion content raw material conditions. With lower enzyme addition amount, the mutant can achieve better protein cross-linking effect, and significantly improve the gel strength, elasticity, water retention and other properties of food.

The application discloses a sorbitol dehydrogenase mutant capable of efficiently degrading vomitoxin without relying on exogenous mediators, and belongs to the field of agricultural biotechnology. The mutant is obtained by rational design and directional evolution of wild-type sorbitol dehydrogenase (SDH), and contains three mutant sites of F103A, S454E and T492E, and the amino acid sequence and the nucleotide sequence are respectively SEQ ID NO. 1 and SEQ ID NO. 2. F103A / S454E / T492E The mutant SDH can efficiently degrade vomitoxin (DON) without relying on electron transfer replacement phenazine methosulfate (PMS), and the degradation efficiency can reach 82% in the absence of PMS. The application can be applied to the preparation of vomitoxin detoxification enzyme, and has a good application prospect in the biological detoxification of DON in feed, grain and food.

The present application belongs to the field of bioengineering technology, and particularly relates to a xylanase mutant with improved heat resistance, an engineering bacterium and application thereof. The present application takes the wild-type xylanase shown in SEQ ID NO. 1 as a template, and obtains a series of xylanase mutants with improved heat resistance, such as G38P, D160R, E263R single mutant, G38P / D160R, G38P / E263R, D160R / E263R double mutant and G38P / D160R / E263R triple mutant, and a recombinant engineering bacterium containing the mutant gene, especially the triple mutant G38P / D160R / E263R, which shows the best heat resistance, and the enzyme activity residual rate is greater than 90% after 2 h of treatment at 90℃, far more than that of the wild type (less than 30%), and can completely adapt to the high-temperature industrial process of 80-100℃, effectively solving the problem of high-temperature inactivation of traditional xylanase, so that it can be widely applied in the fields of feed, cellulose bioconversion, food processing and the like.

The present application provides a thermostable reductive aminase, its preparation method and application. Specifically, the present application relates to a thermostable reductive aminase and its functional identification and in vitro directed evolution. The reductive aminase of the present application is a reductive aminase from bacteria (BaRedAm) and its mutants (including a reductive aminase mutant with significantly improved enantiomeric selectivity of product rasagiline). The present application also provides an in vitro reductive amination method, comprising the steps of: (i) in the presence of a thermostable reductive aminase BaRedAm, subjecting (S1) a ketone substrate or an aldehyde substrate to a reductive amination reaction with (S2) an amine substrate. The enzyme and mutant of the present application can be used for in vitro enzymatic synthesis of different products such as enantiomerically pure rasagiline.