68 results about "Genomic engineering" patented technology

The present disclosure provides compositions and methods for genomic engineering.

The invention discloses a method for synthesizing L-theanine through an enzyme process, and belongs to the biotechnical field. The method is characterized in that a gamma-glutamyltranspeptidase gene is obtained through chemical synthesis, a gene engineering bacterium over-expressing gamma-glutamyltranspeptidase is constructed by treating Escherichia coli as a host bacterium, glutamine and ethylamine hydrochloride having different concentrations are acted by a recombinase, and theanine is efficiently produced at a temperature of 37-50DEG C under a pH value of 9.5-10.5. The enzyme source preparation process has the advantages of simplicity, low cost, and large enzyme amount, and the theanine production method has the advantages of simplicity, high conversion rate, high output, short time and the like, and is in favor of the industrialized amplification production.

Provided are methods and compositions for obtaining genome-engineered iPSCs, and derivative cells with stable and functional genome editing at selected sites. Also provided are cell populations or clonal cell lines derived from genome-engineered iPSCs, which comprise targeted integration of one or more exogenous polynucleotides, and/or in/dels in one or more selected endogenous genes.

Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with improved targeting range and enhanced on-target activity, and their use in genomic engineering, epigenomic engineering, base editing, genome targeting, genome editing, and in vitro diagnostics.

The invention discloses an application of heat-resistant beta-glucosidase in preparation of gentiooligosaccharide and belongs to the field of genetic engineering and enzyme engineering. A beta-glucosidase TSBGl gene from Thermotoga sp.KOL6 takes Bacillus subtilis WSH11 as an expression host, and high-efficiency expression of the tsbgl gene in bacillus subtilis is achieved. The optimum temperatureof the beta-glucosidase TSBGl is 90 DEG C, the optimum pH is 6.0, and the beta-glucosidase TSBGl has relatively high thermostability at 90 DEG C. The beta-glucosidase is added to a reaction system employing 1200 g/L of glucose as a substrate, enzyme reaction is carried out under the conditions of pH 6.0 and 90 DEG C, and the yield of the gentiooligosaccharide reaches 178.2 g/L. The beta-glucosidase meets the requirements for foods and other industrial applications and can be applied to industrial production of the gentiooligosaccharide.

The invention discloses a keratinase mutant with improved catalytic performance and an application, belongs to the technical field of genetic engineering and provides recombinant keratinase constructed on the basis of leading peptide engineering and multi-site saturated mutation and a leading peptide engineering method. A recombination strain M7 shows high keratinase catalytic potential, enzyme activity is remarkably improved from 179 U/mL to 1114 U/mL, the optimal reaction temperature is 40 DEG C, the optimal reaction pH is 10.0, over 80% enzyme activity is retained after water bath processing is performed for 90 min at 40 DEG C, heat stability is better, and the keratinase mutant is suitable for industrial large-scale production. Besides, the method for modifying protease with the leading peptide engineering can be a universal way for modifying industrial enzyme with the self-shearing characteristic.

The present disclosure provides a HTP genomic engineering platform for improving escherichia coli. that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. The integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition.

The invention relates to the field of gene engineering, in particular to acid amylase AMYA4 and gene and application thereof. The amino acid sequence of the acid amylase AMYA4 is expressed as SEQ ID NO.1. The optimal pH value of the AmyA4 is 4.2, the optimal temperature is 75 DEG C, and the AmyA4 has high activity at the temperature of between 55 and 75 DEG C and has strong raw starch degrading capability at the same time. The zymology properties meet the process requirement for preparing sugar by a double-enzyme method, so the acid amylase has great application potential. In the process for preparing sugar by simulating the double-enzyme method, no matter paste starch or raw starch is used as a substrate, the starch can reach good hydrolysis rate under the action of matching commercial saccharifying enzyme, so the AmyA4 has huge application potential.

The invention relates to an nsdBmgh gene of Streptomyces roseoflavus Men-myco-93-63 as well as a preparation method and application thereof. In the invention, primers Ns8F and Ns8R are designed by utilizing an important negative regulator gene nsdB sequence in a Streptomyces coelicolor A3(2) antibiotic metabolic pathway, and genomic DNA (deoxyribonucleic acid) of Streptomyces roseoflavus Men-myco-93-63 is amplified to obtain a 1121bp segment; and two ends of a target sequence are extended by utilizing a primer anchoring polymerase chain reaction) (NPA-PCR ) method by a non-specific primer, thus obtaining the nsdBmgh gene of Streptomyces roseoflavus Men-myco-93-63. The preparation of the nsdBmgh gene provides an important basis for further researching and verifying the Streptomyces roseoflavus antibiotic metabolic pathway and regulatory mechanism, and establishes an important base for obtaining high-yield antibiotic gene engineering strains.

The present disclosure provides a HTP microbial genomic engineering platform that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. The HTP genomic engineering platform described herein is microbial strain host agnostic and therefore can be implemented across taxa. Furthermore, the disclosed platform can be implemented to modulate or improve any microbial host parameter of interest.

The invention discloses a 1,3-1,4-beta-glucanase mutant and belongs to the field of gene engineering and enzyme engineering.40-bit serine and 43-bit serine, 46-bit glutamic acid and 205-bit histidine of 1,3-1,4-beta-glucanase from bacillus terquilensis CGX 5-1 are mutated into glutamic acid, glutamic acid, praline and praline respectively through an iterative saturation mutation method, and finally four strains of single mutants and three strains of compound mutants are obtained.Seven strains of mutate enzyme all represent better heat stability, and particularly S40E/S43E/E46P/H205P mutate enzyme has extremely good heat stability.Compared with wild enzyme, the mutate enzyme can be used in the industry more easily.

The invention discloses an expansion protein and xylanase fusion protein, a coding gene and applications thereof, wherein the expansion protein and xylanase fusion protein comprises an expansion protein EXCL and xylanase XYN, the EXCL is located at the N terminal of the fusion protein, XYN is located at the C terminal of the fusion protein, the amino acid sequence of the expansion protein EXCL isrepresented by SEQ ID NO:2, and the amino acid sequence of the xylanase XYN is represented by SEQ ID NO:4. The invention further provides a preferred method for inserting a linker peptide into the fusion protein. According to the present invention, the expansion protein and xylanase fusion enzyme is constructed by using the gene engineering technology, wherein the enzyme activity is increased by 1.3 times compared to the natural xylanase, and the obtained product can adsorb lignocellulose.

The present disclosure provides a HTP microbial genomic engineering platform that is computationally driven and integrates molecular biology automation and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries which are derived from inter alia scientific insight and iterative pattern recognition. The HTP genomic engineering platform described herein is microbial strain host agnostic and therefore can be implemented across taxa. Furthermore the disclosed platform can be implemented to modulate or improveany microbial host parameter of interest.

The present disclosure is directed to a method of high-throughput (HTP) microbial genomic engineering, which utilizes in vivo transposon mutagenesis to develop strain libraries for the perturbation of microbial phenotypes.

The invention relates to a method for preparing gentiooligosaccharide by using beta-1,6-glucanase and an application of the method and belongs to the technical field of genetic engineering and fermentation engineering. The invention firstly provides beta-1,6-glucanase TcBgn with transglycosylation activity on gentiobiose, the beta-1,6-glucanase TcBgn can transform glucose into gentiooligosaccharide under the condition of relatively small additive amount of enzyme, and the transformation rate is high, so that the production cost can be significantly reduced. The gentiooligosaccharide is prepared by double-enzyme compounding of the beta-1,6-glucanase and beta-glucosidase, and the glucose is transformed into the gentiobiose and gentianose by using a double-enzyme compounded system, so that the transformation rate is high, and the proportion of the gentianose in a product is significantly improved. Therefore, the method for preparing the gentiooligosaccharide by a single enzyme of the beta-1,6-glucanase or double-enzyme compounding has good industrial application value.

The invention discloses a Cas9-scForkI fusion protein and application thereof, and belongs to the field of molecular biology. According to the Cas9-scForkI fusion protein and application, an inactivate Cas9 protein is transformed through a genetic engineering method; (G4S)10, namely (Gly-Gly-Gly-Gly-Ser)10, serves as a linker to be used for connecting two ForkIs; the ForkIs and the inactivate Cas9 are fused into Cas9-scForkI; and according to the Cas9-scForkI fusion protein, genomic editing can be carried out through sequence specificity. When the Cas9-scForkI fusion protein is used for drug screening or gene modification, more safety is obtained; and the fusion protein can further be used for gene treatment. Compared with the prior art, the design is more flexible; the cutting activation is higher; the input efficiency is higher when the fusion protein serves as a gene; and the fusion protein can be used for gene modification or gene treatment.

The invention discloses a phenylpyruvic acid reductase sourced from lactobacillus plantarum and an application thereof and belongs to the technical field of bioengineering. In the invention, for the first time, the phenylpyruvic acid reductase, which has the activity of catalyzing phenylpyruvic acid to generate phenyllactic acid, is screened and produced. The invention belongs to the fields of gene engineering and protein expression. The enzyme is successfully cloned and expressed in Escherichia coli BL21 and is applied to production of the phenyllactic acid through whole-cell catalysis of phenylpyruvic acid. After the catalytic reaction is carried out for 5 h, conversion of the substrate reaches 98.38% and ee value reaches 99.9%.

The present disclosure provides a HTP microbial genomic engineering platform for Saccharopolyspora spp. that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterativepattern recognition.

The invention discloses saccharomyces cerevisiae engineering bacteria for producing campesterol and a construction method, and belongs to the field of genetic engineering and bioengineering. An expression cassette element of 7-dehydrocholesterol reductase is introduced into a yeast cell body, and is integrated into a yeast genome by utilizing a CRISPR/Cas9 gene operating system, C-22 sterol desaturase is knocked out, competition of an ergosterol branch path is relieved, and yeast synthesis from glucose to campesterol is realized. The method is simple in process, green and environment-friendly, and can be used for producing campesterol through fermentation. The yield of campesterol prepared by adopting the saccharomyces cerevisiae engineering strain BY4742-delta erg5-TEF1p-dhcr7-ADH1t provided BY the invention can reach 253.35mg/L in a shake flask stage, and the horizontal yield of a fermentation tank can reach 916.88mg/L, which is increased by 2.6 times compared with the yield of campesterol in the shake flask stage of the strain.

The invention relates to a method for realizing the engineering expression of glycosyl endoglycosidase EGCase I. The method can realize the effective soluble expression of the EGCase I and maintain excellent hydrolysis and transglycosylation activity; a novel EGCase I mutant enzyme developed on the basis of the method has a broad substrate spectrum, and also has the activity of glycosidase synthetase; and the obtained glycosphingolipid endoglycosidase is very suitable for being applied to the analysis and the synthesis of industrial glycosphingolipids.

The invention discloses a method for preparing a porcine kidney epithelial cell line for stably expressing BE4 protein, which comprises the following steps: by using porcine kidney epithelial cells (PK15) with good passage as host cells, co-transfecting a piggyBac transposon (PB-BE4max) vector containing a BE4 fusion protein sequence and a transposase expression vector plasmid, screening a positive cell population by puromycin, and selecting a monoclonal cell line; and verifying the editing activity amplifying the characteristic fragment of BE4 and transfecting active sgRNA, so as to obtain the transformant for stably expressing BE4 protein. The porcine kidney epithelial cell line prepared by the method can stably express BE4 protein, and has important application value in the porcine genetic engineering fields of porcine genome function research, transgenic pig preparation, drug-resistant target screening and the like.