31 results about "Ribosomal binding site" patented technology

The invention belongs to the field of synthetic biology and metabolic engineering, and particularly relates to high-yield sialyl acyl-N-tetrasaccharide a escherichia coli as well as a construction method and application thereof. According to the method, recombinant escherichia coli is taken as an initial strain, and a beta1, 3-galactosyl transferase gene wbgO from escherichia coli O55: H7 is integrated into a chromosome in a multi-copy form, so that a lactyl-N-tetrasaccharide efficient synthesis chassis strain without plasmid dependence is constructed. And overexpression neuC, neuB, neuA and Nm3ST are introduced through plasmids, so that the supply of glycosyl donors is improved. The expression intensity of uridine diphosphate-N-acetylglucosamine synthesis pathway genes glmM and glmUS is regulated through ribosome binding site engineering, and a cytidine triphosphate regeneration module is introduced, so that the yield of sialyloyl-N-tetrasaccharide a is increased. And finally, the maximum titers of the sialyloyl-N-tetrasaccharide a respectively reach 2.238 g / L and 8.178 g / L by shaking a flask and culturing in batches. The invention provides an efficient technical scheme for industrial production of complex breast milk oligosaccharide.

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 corynebacterium glutamicum for secretory expression of lysozyme and a construction method and application thereof.The construction method comprises the steps that expression conditions of lysozyme of different sources in corynebacterium glutamicum are analyzed, lysozyme with the good expression effect is selected as target protein, artificially predicted recombinant lysozyme is selected as the target protein, and the corynebacterium glutamicum is constructed; a vector pGX19 is used for carrying out secretory expression, then signal peptide screening, promoter and ribosome binding site combination optimization are carried out on an expression element, and secretion of recombinant lysozyme is improved from the transport level; according to the invention, the CgR0949 signal peptide of a tat pathway can obviously improve the expression quantity of recombinant lysozyme; through combination optimization of the promoter and the RBS, the expression quantity is increased by 2.3 times; the recombinant lysozyme has the advantages that the expression quantity of the recombinant lysozyme is increased by 1.5 times through overexpression of tat transfer systems from different sources, and after multi-level optimization, the shake flask fermentation level of the recombinant lysozyme is increased by 8.3 times compared with that of an initial strain and reaches 0.2 g.L <-1 >.

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.

The invention discloses a construction method and application of a bacterial sensor for sensing iron and magnesium ions. The invention belongs to the field of microorganisms, and particularly relates to a construction method and application of a bacterial sensor for sensing iron and magnesium ions. The recombinant escherichia coli contains a super-folded green fluorescent protein sfGFP coding gene, a sensing protein PmrB mutant coding gene, a response regulatory protein PmrA mutant coding gene, a signal transduction protein PmrD coding gene and a strong ribosome binding site RBS. The gene contains a T7 RNA polymerase coding gene driven by a promoter of phosphorylase PmrG activated by regulatory protein PmrA and an sfGFP coding gene driven by a T7 promoter, and meanwhile, overexpresses a phosphoethanolamine transferase EptA coding gene. The bacterial sensor for detecting the concentration of iron ions is constructed, modification of lipopolysaccharide of the bacterial sensor can be changed through induction of iron and magnesium ions, and drug resistance of bacteria is enhanced.

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.

A broadly protective Shigella-ETEC vaccine includes components to cover multiple Shigella species and serotypes as well as multiple different colonization factors of ETEC. ETEC colonization factors CS2 and CS3 are among the most prevalent CFs found on ETEC isolates associated with diarrhea. These two factors are important components of a vaccine that can confer broad protection against ETEC. High level expression of these heterologous antigens in Shigella live vectors results in stronger immune responses. This specification describes the genetic engineering of DNA sequences encoding upstream sequences, including promoter and ribosome binding sites, that results in increased expression of CS2 and CS3 in Shigella live vectors and that leads to stronger immune responses when used as a vaccine in animal models.

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 belongs to the technical field of biology, and relates to a modular plasmid vector for constructing an FRET (Fluorescence Resonance Energy Transfer) probe. The modular plasmid vector comprises a promoter, a ribosome binding site and a terminator, and a modular probe assembly region is connected between the ribosome binding site and the terminator; the modular probe assembly region comprises an SH3 structural domain gene, a restriction endonuclease recognition site sequence for insertion of a fluorescent protein 1 gene, a connecting chain, a restriction endonuclease recognition site sequence for insertion of a fluorescent protein 2 gene, and a restriction endonuclease recognition site sequence for insertion of an SH3 structural domain ligand gene; the connecting chain comprises a connecting region 1, a restriction endonuclease recognition site sequence 1 for antigen epitope gene insertion, a connecting region 2, a restriction endonuclease recognition site sequence for protease recognition sequence gene insertion, a connecting region 3, a restriction endonuclease recognition site sequence 2 for antigen epitope gene insertion and a connecting region 4. The modular plasmid vector can quickly change and replace partial regions according to the application of the FRET probe, and can be used as a universal vector for constructing the FRET probe.

The invention belongs to the technical field of biology, and discloses a screening method and application of an antibacterial peptide and the antibacterial peptide. According to the screening method of the antibacterial peptide provided by the invention, a to-be-selected antibacterial peptide expression vector constructed by taking a nucleotide fragment with a sequence as shown in SEQ ID NO: 2 as a ribosome binding site is adopted, and protein expression in a cell-free protein expression system can be realized under the condition of no other treatment (such as transformation, transfection and the like) or specific condition stimulation; according to the method, the to-be-selected antibacterial peptide fermentation liquor is used for preparing the antibacterial peptide rapidly, then the growth condition of pathogenic bacteria after the to-be-selected antibacterial peptide fermentation liquor and the pathogenic bacteria culture solution are mixed is observed, screening of the antibacterial activity of the antibacterial peptide can be achieved, and huge potential is shown in rapid and high-throughput screening of the antibacterial peptide.

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.

A broadly protective Shigella-ETEC vaccine includes components to cover multiple Shigella species and serotypes as well as multiple different colonization factors of ETEC. ETEC colonization factors CS2 and CS3 are among the most prevalent CFs found on ETEC isolates associated with diarrhea. These two factors are important components of a vaccine that can confer broad protection against ETEC. High level expression of these heterologous antigens in Shigella live vectors results in stronger immune responses. This specification describes the genetic engineering of DNA sequences encoding upstream sequences, including promoter and ribosome binding sites, that results in increased expression of CS2 and CS3 in Shigella live vectors and that leads to stronger immune responses when used as a vaccine in animal models.

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.

The invention discloses a cis-element library for thermophilic microorganism gene expression regulation and application. The cis-element library comprises a constitutive promoter, an inducible promoter and a ribosome binding site (RBS). The invention also discloses application of the element library in gene expression of thermophilic microorganisms. The toxin-antitoxin gene is used as a new selective marker to be applied to genetic manipulation of thermophilic microorganisms by rationally controlling the strength of the promoter. Promoters of different types and strengths are used for replacing natural promoters of genes related to hydrogen production pathways, and hydrogen metabolic pathways in thermophilic microorganisms are modified, so that the yield of hydrogen is improved. And a cis-element library for accurately regulating and controlling gene expression is provided for carrying out basic research on metabolic engineering and molecular biology in thermophilic microorganisms.

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 relates to the field of gene engineering, in particular to a plasmid elimination module based on multiple input cascade reaction, which comprises an antisense RNA expression region, an antisense RNA, a constitutive promoter of the antisense RNA and a terminator of the antisense RNA; the antisense RNA is the antisense RNA of mRNA (messenger ribonucleic acid) of a gene capable of influencing microbial growth; the first antisense neutralizing RNA expression region comprises an inducible promoter of first antisense neutralizing RNA or a fragment containing a constitutive promoter and repressor protein binding site, a first antisense neutralizing RNA sequence, a first linker sequence and a terminator; the second antisense neutralizing RNA expression region comprises an inducible promoter of second antisense neutralizing RNA or a fragment containing a constitutive promoter and repressor protein binding site, a second linker sequence, a second antisense neutralizing RNA sequence and a terminator; and the repressor protein gene / transcription factor expression region comprises a constitutive promoter, a ribosome binding site, a repressor protein translation region / transcription factor translation region and a terminator of the repressor protein gene. The method is high in elimination efficiency, elimination objects can be freely set according to needs, and selective or synchronous elimination of various plasmids can be realized.

A method for screening αβ-TCR mutants, the method comprising the construction of a phagemid. In the construction, a gene encoding an A1 polypeptide and a gene encoding B1 polypeptide are inserted into the 3′ ends of genes encoding the constant regions of the α chain and the β chain of TCR respectively, and a ribosome binding site and a molecular chaperone gene are integrated into a phage vector, wherein the amino acid sequence of the A1 polypeptide is as shown in SEQ ID NO: 1 or is represented by the variant of the A1 polypeptide; the amino acid sequence of the B1 polypeptide is as shown in SEQ ID NO: 2 or is represented by the variant of the B1 polypeptide; and the variants at least retain the function of the polypeptides before mutation. The A1 peptide and the B1 peptide are used for promoting the correct pairing and folding of the αβ heterodimer TCR, so that the efficiency of displaying the correctly folded TCR on the surface of a phage is improved. The method can efficiently complete the screening of αβ heterodimer TCR mutants.