21 results about "Ribosomal binding site" patented technology

Provided are a method for constructing a soluble expression plasmid mutant library, and the use thereof. The method for constructing the soluble expression plasmid mutant library comprises: introducing mutations into a ribosome binding site on a soluble expression plasmid, a sequence composition and length of a region between the ribosome binding site and an initiation codon of a fusion protein to be expressed, and a translation initiation region, so as to obtain a mutant library. By means of the method, plasmid mutants with increased expression levels are obtained, thereby increasing the expression level of a target fusion protein and the final yield of a target polypeptide. The present invention solves the problem of low soluble expression levels of certain polypeptides in Escherichia coli.

The invention belongs to the technical field of biological medicines, and particularly relates to a granzyme B expression system as well as a preparation method and application thereof. According to the invention, granzyme B with bactericidal activity is screened out, a corresponding expression vector is designed by adopting circular RNA, a bacterium-specific ribosome binding site (RBS) is taken as a protein translation regulation and control element, and a Lipid nano-particle (LNP) is taken as a delivery vector, so that efficient delivery of the circular RNA for coding GZMB and specific expression of GZMB in bacteria are realized. According to the strategy, efficient delivery and specific expression of circRNA for coding GZMB in bacteria are achieved, and a safe and accurate scheme is provided for GZMB antibacterial treatment.

The invention discloses an Escherichia coli strain capable of bidirectionally regulating the copy number of CoIE1 type plasmids and application of the Escherichia coli strain. According to the method, escherichia coli Mach1-T1 is taken as an initial strain, a CRISPR / CAS9 gene editing technology is utilized, RNase E Q36R variant genes, rssB genes and pcnb genes are constructed into a polycistron expression cassette, and the polycistron expression cassette is placed under the control of an arabinose operon to replace endogenous pcnb genes of a genome. Each gene in the polycistron expression cassette is driven to translate by an independent ribosome binding site (RBS). By adjusting the concentration of the arabinose, continuous and bidirectional regulation and control of the copy number of the CoIE1 type plasmids from'inhibition 'to'overproduction' can be realized, and the method has important significance on industrial mass production of the CoIE1 type plasmids and diversified application of the CoIE1 type plasmids.

Described herein are compositions and methods for generating a chassis for high-capacity production of different classes of high-value terpenoids. The chassis is a microorganism host that expresses a first operon comprising a modified ribosomal binding site, a Hmg-CoA reductase (HmgR) polypeptide, a 3-hydroxy-3-methylglutaryl CoA synthase (HmgS) polypeptide, and a β-ketothiolase (PhaA) polypeptide; and a second operon comprising a modified ribosomal binding site, a mevalonate kinase (MvK1) polypeptide, a phospho-mevalonate kinase (MvK2) polypeptide, and a diphosphomevalonate decarboxylase (MvD) polypeptide.

The invention discloses a BioB protein mutant and application of the BioB protein mutant in improving the conversion rate of desulfurized biotin to biotin, and relates to the technical field of genetic engineering. According to the method disclosed by the invention, the BioB is analyzed by using paver software, key amino acid sites influencing reactants / products to enter and exit from a protein binding cavity in the BioB are optimized, and amino acid residues with relatively large steric hindrance are replaced by amino acid residues with relatively small steric hindrance; according to the present invention, the BioB protein activity is changed through site-specific mutagenesis, and ribosome binding site optimization is performed on the BioB protein, such that the conversion rate of the desulfurization biotin to the biotin is substantially improved so as to obtain the mutant strain with the high biotin yield, and the high application value is provided.

The application provides a ribosome binding site, a promoter and application thereof. The ribosome binding site is based on an RBS sequence of a trc promoter, a translation initiation region is rationally designed and optimized by using a Salis RBS Calculator, an optimized RBS is obtained, a nucleotide sequence of the RBS is shown in a sequence table SEQ ID NO. 1 or shown in a sequence table SEQ ID NO. 2; a promoter composed of a fixed element upstream of the trc promoter and the optimized RBS is integrated into a valine synthesis pathway gene ygaZH, ilvC, ilvD, ilvE of an Escherichia coli W3110 genome by gene editing technology upstream of an initiation codon ATG, and a valine production strain constructed does not contain a plasmid, has no growth defect, does not need to be induced and has good genetic stability.

Provided are efficient heterologous expression of a nicotinamide ribokinase mutant in Escherichia coli and a use thereof. Provided are nicotinamide ribokinase capable of being heterologously expressed in Escherichia coli and a mutant thereof. The activity of the nicotinamide ribokinase of the mutant is significantly improved as compared with that of a parent. On the basis of the nicotinamide ribokinase and the mutant thereof, further provided is a method for the efficient heterologous expression of the nicotinamide ribokinase, that is, during the construction of a recombinant plasmid, by introducing a ribosome binding site into the plasmid, inserting a specific promoter sequence, etc., the enzyme activity of the expressed nicotinamide ribokinase is made higher than that of a control. In addition, further provided is a method for culturing a recombinant bacterium expressing the nicotinamide ribokinase, such that the highest enzyme activity of the expressed nicotinamide ribokinase is 208.3 U / mL. The present invention is conducive to efficient biosynthesis of β-nicotinamide mononucleotide and is suitable for industrial production and use.

The invention provides a method for producing beta-hydroxy-beta-methylbutyric acid through double-enzyme co-expression, and relates to the technical field of enzyme engineering. According to the invention, L-amino acid deaminase and 4-hydroxyphenylpyruvate dioxygenase are expressed by using the same promoter in a host cell, and a key RBS combination is determined by optimizing and screening ribosome binding site RBS sequences, so that expression balance and efficient synergy of the two enzymes are realized; on the basis, biosynthesis of beta-hydroxy-beta-methylbutyric acid is carried out by taking L-leucine as a substrate, the yield of beta-hydroxy-beta-methylbutyric acid reaches 14.12 mM, and the molar conversion rate is 56.5%. Through series co-expression design of double enzymes, efficient biosynthesis of beta-hydroxy-beta-methylbutyric acid is achieved, and the method has the advantages of being mild in reaction condition, high in product purity, environmentally friendly and the like and is suitable for industrial popularization.

Described herein are compositions and methods for generating a chassis for high-capacity production of different classes of high-value terpenoids. The chassis is a microorganism host that expresses a first operon comprising a modified ribosomal binding site, a Hmg-CoA reductase (HmgR) polypeptide, a 3-hydroxy-3-methylglutaryl CoA synthase (HmgS) polypeptide, and a β-ketothiolase (PhaA) polypeptide; and a second operon comprising a modified ribosomal binding site, a mevalonate kinase (MvK1) polypeptide, a phospho-mevalonate kinase (MvK2) polypeptide, and a diphosphomevalonate decarboxylase (MvD) polypeptide.

The present invention provides an AP plasmid, characterized in that the plasmid expresses the gIII-neg protein and has one or more RyhB binding sequences shown in SEQ ID No. 1 inserted into the rear of its ribosome binding site. Also disclosed are its application in the PACE directed evolution of the PAP I enzyme and a PACE directed evolution system for the PAP I enzyme. Also disclosed are screened PAP I enzyme mutants, encoding genes, prokaryotic expression vectors, and prokaryotic expression systems. The present invention introduces Escherichia coli non-coding RNA RyhB sRNA into the PACE directed evolution method to inhibit the translation of the gIII-neg protein and eliminate the adverse effects of the gIII-neg protein on the packaging of progeny phages. The stronger the polyadenylation activity of the PAP I, the longer the half-life of RyhB can be extended, promoting the latter's translational inhibition of the gIII-neg protein. After multiple passages, the phage forms a population advantage and is isolated and sequenced, ultimately obtaining a PAP I with significantly improved activity.

This invention discloses a PLA-degrading lipase mutant and its application. First, the lipase PaPlaA gene with the amino acid sequence shown in SEQ ID NO.1 was selected. Highly efficient heterologous expression was achieved by constructing a PgapA-T7 dual promoter system and optimizing the RBS sequence through ribosome binding site design, resulting in a recombinant enzyme activity yield of 6.25 U / mL, approximately 2.5 times higher than the original strain. Then, combining molecular dynamics simulations and rational design, the substrate pocket and flexible region were structurally modified to obtain the PLA-degrading lipase mutant. The obtained mutant exhibited excellent catalytic performance when applied to PLA degradation. This invention also provides a composite enzyme preparation obtained by combining the PLA-degrading lipase mutant with the protease nprT612. This composite enzyme preparation can achieve a degradation rate of up to 78.2% for PLA nonwoven fabrics. This provides a stable enzyme catalyst and a feasible synergistic degradation process for the efficient biodegradation of PLA at high temperatures.

The application discloses an engineered bacillus subtilis strain for producing tagatose, a construction method and application thereof, and belongs to the technical field of bioengineering. The engineered strain takes bacillus subtilis as a host, co-expresses L-arabinose isomerase from Erysipelothrix larvae and beta-galactosidase from escherichia coli, and constructs the bacillus subtilis for co-expressing double enzymes through ribosome binding sites or promoters in series. The double-enzyme bacillus subtilis whole cell catalyzes lactose to produce D-tagatose, and the yield and conversion efficiency of D-tagatose are significantly improved after optimization of the enzymatic reaction. The application adopts single-strain double-enzyme co-expression and one-step whole-cell catalysis, uses low-cost lactose as a substrate, and has the advantages of mild reaction, less by-product, simplified process, no endotoxin safety hazard, and the like. The application solves the problems of low conversion rate, high substrate cost and complicated strain compounding in the prior art, and is suitable for industrialized production of food-grade D-tagatose.

The application discloses a gene expression stabilization method and system based on sRNA feedback and non-periodic sampling regulation, relates to the technical field of gene expression optimization and regulation, and comprises the following steps: firstly, an sRNA-mediated feedback control gene circuit is designed and constructed; secondly, a state space model describing system dynamics is established, and minimization of the steady-state expression deviation of a target protein before and after ribosome disturbance is taken as an optimization target; through real-time calculation of a stability evaluation index, a non-periodic sampling strategy is used to dynamically judge a regulation triggering time; finally, when the triggering condition is met, the ribosome disturbance level is estimated in real time, and the optimal ribosome binding site strength adjustment value is calculated through solving an optimization problem and is implemented. The application can adaptively maintain the stability of target protein expression in a culture environment with dynamic competition of ribosome resources at a low intervention frequency, and significantly improves the robustness and output consistency of a synthetic biology system.

The invention relates to the technical field of construction of recombinant bacteria, in particular to recombinant corynebacterium glutamicum for producing hydroxytyrosol as well as a construction method and application of the recombinant corynebacterium glutamicum. On one hand, by knocking out the SigM gene, the non-productive metabolism consumption of the strain is reduced, and a carbon source and energy are accurately guided to hydroxytyrosol synthesis; on the other hand, the optimal combination of a Psod strong promoter and an RBS1 ribosome binding site is screened, the hpaBC gene expression efficiency is enhanced, and then fermentation condition optimization of tyrosol separate addition (6 g / L initial addition + 4 g / L supplementation) is matched, so that key data that 10.99 g / L of hydroxytyrosol is produced through shake flask fermentation and 15.81 g / L of hydroxytyrosol is produced in a 5L fermentation tank is finally achieved. The yield is obviously higher than that of existing engineering bacteria such as escherichia coli and yeast, and the technical bottlenecks of low yield and difficulty in large-scale production of a microbiological method are broken through.

The invention discloses a gene expression cassette and application thereof in preparation of recombinant collagen, and belongs to the field of molecular biology. Corynebacterium glutamicum is used as an expression system, and through promoter screening, ribosome binding site and spacer sequence optimization and serial combination of gene expression cassettes, on the basis of improving the collagen yield of the Corynebacterium glutamicum, the structure and stability of the protein are ensured, and the yield of the collagen is improved. When a plurality of target gene combinations of the gene expression cassette are connected in series, the highest collagen yield of the constructed recombinant bacteria reaches 697mg / L.

The present application relates to a variant gene encoding a phosphoenolpyruvate carboxylase comprising a variant 5 '-untranslated region (5' UTR), the variant gene comprising a ribosome binding site (RBS) variant sequence, a polyhydroxyalkanoate (PHA)-producing microorganism comprising the gene, and a PHA production method using the same.

The application discloses a NADH pyrophosphatase mutant with improved catalytic performance and application thereof. The application provides an optimized NADH pyrophosphatase, which has improved catalytic performance compared with that before optimization. On this basis, the application further provides a NADH pyrophosphatase mutant with improved catalytic performance through site-directed mutagenesis. In addition, the application further provides a method for further improving the catalytic performance of the provided optimized NADH pyrophosphatase and the mutant thereof, and when a recombinant plasmid is constructed, specific promoter sequences and ribosome binding sites are inserted into the plasmid, so that the catalytic performance of the obtained NADH pyrophosphatase induced and expressed is higher than that of the parent. The provided optimized NADH pyrophosphatase and the mutant thereof not only have high catalytic performance, but also have good thermal stability, and can be used for biosynthesis of reduced nicotinamide mononucleotide, and are suitable for industrial production and application.

The invention relates to an application of glyA-coded serine hydroxymethyltransferase in preparation of L-serine, in particular to an application of glyA-coded serine hydroxymethyltransferase in preparation of L-serine. Specifically, the invention provides a genetically engineered bacterium, and the sequence of a ribosome binding site of a glyA gene in the bacterium is shown as any one of SEQ ID NO: 53-58. The invention also provides a construction method of the genetically engineered bacterium for producing the L-serine or the L-serine derivative, and the ribosome binding site of the glyA gene of the bacterium is replaced by the ribosome binding site as shown in SEQ ID NO: 57 or 58.

This invention discloses a method for constructing a bacterial sensor that senses iron and magnesium ions and its application. This invention belongs to the field of microbiology, specifically relating to a method for constructing a bacterial sensor that senses iron and magnesium ions and its application. Recombinant *Escherichia coli* contains a gene encoding a superfolded green fluorescent protein (sfGFP), a mutant gene encoding a sensor protein (PmrB), a mutant gene encoding a response regulatory protein (PmrA), a gene encoding a signal transduction protein (PmrD), and a strong ribosome binding site (RBS). It also contains a T7 RNA polymerase gene driven by the promoter of the phosphorylase PmrG activated by the regulatory protein PmrA, and a T7 promoter-driven sfGFP gene, while simultaneously overexpressing the gene encoding the phosphorylethanolamine transferase (EptA). This invention constructs a bacterial sensor for detecting iron ion concentration, and the modification of the lipopolysaccharide in the bacterial sensor can be altered by the induction of iron and magnesium ions, thereby enhancing bacterial drug resistance.

This invention discloses a bicistronic translation-coupled expression vector. The expression vector is constructed by inserting a molecular chaperone, a novel ribosome binding site, and the target protein gene into the pET-28a(+) plasmid. Furthermore, this invention constructs a genetically engineered bacterium using *E. coli* BL21(DE3) as the expression host. Utilizing the bicistronic translation-coupled expression vector, the co-expression of mussel protein Mgfp-3B with the molecular chaperones SUMO and / or TrxA is achieved, promoting the correct folding and translation of Mgfp-3B for expression in a soluble form. This invention enables a simple and rapid production of bioactive soluble mussel protein in *E. coli*, and the bicistronic expression vector avoids the enzymatic chaperone manipulation required for fusion expression. In addition, this invention, through optimized fermentation medium and fermentation process, achieves a soluble mussel protein yield of 200-300 mg / L, simplifying downstream purification processes.

The invention discloses a genetic engineering strain for efficiently expressing nitrile hydratase as well as a construction method and application of the genetic engineering strain. On the basis of a nitrile hydratase (PtNHase) gene of pseudonocardia thermophila, the following optimization and transformation are carried out: (1) carrying out complete sequence optimization on a gene sequence according to codon preference of escherichia coli; (2) replacing a ribosome binding site (SD sequence) at the upstream of the alpha subunit; and (3) carrying out site-specific mutagenesis on tryptophan (Trp) at the 72nd site of the beta subunit to obtain phenylalanine (Phe). The nitrile hydratase produced by the engineering strain constructed by the invention has significantly improved enzyme activity, thermal stability and tolerance to a substrate / product, can be used for industrial biosynthesis of acrylamide and nicotinamide and degradation of nitrile-containing pollutants, is suitable for the fields of industrial large-scale production and environmental governance, and has broad application prospects.