83 results about "Operon" patented technology

The invention provides a valine production strain and a construction method and application thereof, a designed acetohydroxy acid synthase mutant is that the 88th basic group of an ilvB gene is changed from a to c, the 382nd basic group is changed from a to g, the 413th basic group is changed from c to t, the gene sequence of a designed artificial operon comprises a promoter, an ilvB (A138V) gene or ilvB (Q30K, S128G, A138V) gene of coding mutated acetohydroxy acid synthase, and an ilvN (G20D, I21D, I21D, I21D, I21D, I21D, I21D, I21D) gene. I22F) gene, a pyk gene for coding pyruvate kinase, and a terminator; by designing a specific acetohydroxyacid synthase mutant and related biological materials and artificial operon, the strain constructed by directional modification of the strain by using a pK18mobsacB system gene editing technology based on allele exchange has the advantages of good genetic stability, high fermentation yield and the like, and valine can be stably produced.

The invention provides a valine production strain as well as a construction method and application thereof, the strain is constructed by utilizing a CRIPSR / Cas9 system gene editing technology based on allele exchange, an artificial operon is designed and constructed, the artificial operon is constructed at a ygaY site, a strong promoter Ptrc is used for controlling ilvIH (G14N S17F)-pykF to carry out synchronous multi-copy expression, and the valine production strain can be used for producing valine. On the basis of the strain, an artificial operon is constructed at an ycgH site, a strong promoter Ptrc is used for controlling ilvC-ilvD to carry out synchronous multi-copy expression, meanwhile, feedback inhibition of valine on ilvIH is relieved, and an ilvIH natural promoter is replaced by the strong promoter Ptrc, so that more pyruvic acid flows to valine; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for efficiently synthesizing valine from the beginning by taking glucose as a substrate.

The invention provides an L-lysine production strain as well as a strain construction method and application. A hom (V59A) is introduced on a genome by utilizing a homologous recombination gene editing technology to weaken a competitive approach of lysine; the lysC (T311I) which is not inhibited by L-lysine is overexpressed; the method comprises the following steps of: overexpressing lysE, lysA, ddh, dapB and asd, knocking out a lysI gene and multi-copying the lysE, carrying out synchronous multi-copying on the dapA and EcdapB genes through a manual operon P1676-EcdapB-Pa12-dapA, and carrying out synchronous multi-copying on the dapB, ddh, lysA and lysO genes through a manual operon Psod-dapB-ddh-Ptuf-lysO-lysA; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for stably producing L-lysine.

The present invention relates to nucleic acids and modified immune cells or precursors thereof, comprising a first polynucleotide encoding an NF-κB p65-rTetO fusion protein comprising one or more transactivation domains (TAD) of human NF-κB p65 fused to a reverse Tet operator (rTetO) binding domain; a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereoto; and a third polynucleotide comprising a promoter directing transcription of p65-rTetO from a first strand of DNA, where the p65-rTetO is configured to induce transcription of a transgene of interest operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA.

The invention belongs to the technical field of biological gene engineering, and particularly relates to escherichia coli with high RNA content and a construction method and application thereof.A strong promoter is used for replacing a promoter of escherichia coli rDNA, an expression element of strong promoter + rDNA + terminator is constructed, plasmid is used as a carrier, the expression element is transferred into escherichia coli, and an escherichia coli engineering strain with high RNA yield is constructed; wherein the rDNA is derived from one or more of rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH of rDNA operons of escherichia coli, and comprises 16S rDNA, 23S rDNA and 5S rDNA in the corresponding operons. By adopting the construction method, the RNA content of the escherichia coli can be obviously improved, and when the method is used for producing RNA, the RNA yield is obviously higher than that of an initial escherichia coli strain and common saccharomycetes for RNA production at present.

The invention relates to the technical field of microorganisms, and particularly discloses a construction method and application of a recombinant microorganism. According to the construction method of the recombinant microorganism, compared with an original strain, the expression quantity of threonine operon leader peptide of the recombinant microorganism is reduced, and the expression quantities of glyceraldehyde-3-phosphate dehydrogenase mutant, aspartate kinase / homoserine dehydrogenase I mutant, homoserine kinase and threonine synthase are increased; the glyceraldehyde-3-phosphate dehydrogenase mutant is characterized in that glycine at the 188th site in a glyceraldehyde-3-phosphate dehydrogenase protein sequence is mutated into serine; the aspartate kinase / homoserine dehydrogenase I mutant is characterized in that glycine at the 433rd site in an aspartate kinase / homoserine dehydrogenase I protein sequence is mutated into arginine; the starting strain is escherichia coli. According to the construction method disclosed by the invention, a novel recombinant microorganism with improved threonine yield can be obtained, and a novel method is provided for fermentation production of threonine.

The invention discloses an artificially synthesized corrinic acid operon, and the sequence of the artificially synthesized operon is obtained by integrating corrinic acid synthesis related genes and connecting and constructing the corrinic acid synthesis related genes in a reordering and end-to-end manner. According to the operon, a recombinant strain is synthesized on the basis of various chassis strains such as escherichia coli and rhodobacter sphaeroides, the corrinic acid and the derivatives thereof are synthesized through fermentation, the corrinic acid and the derivatives thereof can also be synthesized through a cell-free catalytic system, the problem that overlapping genes of a natural operon are difficult to modify is solved, and the synthesis efficiency and controllability of the corrinic acid are improved.

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.

Disclosed herein are programmable bacteria for tumor-targeted immunotherapeutic delivery. In certain embodiments, the programmable bacteria comprise at least one synchronized lysis circuit contained in a single operon which are capable of being further engineered to cyclically produce anti-cancer therapeutic agents including but not limited to nanobodies against immune checkpoint inhibitors and over-expressed markers in cancers, toxins, tumor antigens, cytokines, and chemokines. In some embodiments, the programmable bacteria comprise at least one synchronized lysis circuit contained in a single operon and at least one plasmid producing a therapeutic agent, i.e., at least one plasmid comprising a nucleic acid sequence which encodes a therapeutic agent. The disclosure also provides methods of curing and treating cancer using the programmable bacteria.

The invention belongs to the technical field of vitamin B2 production, and particularly relates to a preparation method of a safe high-yield strain for vitamin B2 production, which comprises the following steps: S1, strain source and screening: analyzing the current situation and development trend of vitamin B2, and screening the strain; s2, genetic engineering modification: evaluating the production performance and genetic stability of the strain, and carrying out mutagenesis treatment on the screened strain by using a chemical mutagenic agent to obtain a genetically mutated strain; s3, screening and identifying high-yield mutant strains: integrating the fragments containing the spore cereus dry riboflavin operon into a vitamin B2 genome to generate the mutant strains, and performing fine adjustment on the riboflavin operon in the mutant strains by utilizing a multiple genome editing system. And through technical popularization, the manufacturing cost of the product can be reduced, and the social benefit is obvious.

The invention discloses a high-induction-activity promoter derived from a tagatose operon of bacillus licheniformis, and belongs to the field of gene engineering. The promoter PfruK2 capable of being induced by a carbon source in a PTS (Pleurotropin Tyrosine Sequence) way is screened, the induced carbon substrate spectrum capable of being responded by the promoter is wide, the promoter is not affected by a glucose-mediated CCR effect, the induction intensity is high, and the induction peak value is improved by more than 50% compared with that of a quorum sensing strong promoter PLan.

The present disclosure relates to an L-arginine-producing microorganism in which the activity of a regulator of nitrate reductase operon expression is attenuated and the activity of glutamine synthetase is enhanced, and to a method for producing L-arginine using same, wherein the L-arginine-producing microorganism, in which the activity of the regulator of nitrate reductase operon expression is attenuated and the activity of glutamine synthetase is enhanced, has been confirmed to exhibit increased L-arginine production capability compared to a parent strain, and thus can be widely used for L-arginine production.

The application discloses a method for identifying Klebsiella pneumoniae gene circadian rhythm expression by using a bioluminescence reporting system. The application constructs a recombinant bioluminescence Klebsiella pneumoniae driven by a metabolic gene malate:quinone oxidoreductase mqo promoter (Pmqo), and directly observes the change rule of bacterial gene expression under entrainment and free-running conditions of temperature cycle by monitoring the luminescence characteristics. The luxCDABE gene operon comprises genes for coding luciferase and substrate synthesis enzyme, wherein luxA and luxB code for heterodimer luciferase, luxC, luxD and luxE synthesize aldehyde as a substrate for the luciferase luminescence reaction. The luciferase catalyzes the oxidation of aldehyde to generate visible blue-green light with a wavelength of 450-490 nm. The Lux bioluminescence reporting system has the advantages of not needing to add an exogenous substrate and not affecting the normal physiological functions of the host, and can be applied to the identification of Klebsiella pneumoniae gene circadian rhythm expression.

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.

This invention provides an L-lysine-producing strain, its construction method, and its application. The strain utilizes homologous recombination gene editing technology to first overexpress... dapA , aspB Introducing mutations ppc (A482V) Knockout amn Multiple copies are synchronized by manually manipulating Pa12-dapB(T1)-Psod-aspB(T1)-Pa16-Ec_lysA. dapB , aspB Ec. from E. coli W3110 lysA Multiple copies are synchronized via the artificial manipulator Pa12-ddh(RBS)-lysE(T1)-Pa16-gapN(RBS)-amtB. ddh , lysE Streptococcus equi subsp. porphyria gapN , amtB The strain is plasmid-free, defect-free, requires no induction, and has advantages such as good genetic stability and high fermentation yield, enabling stable production of L-lysine.

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) transposon gene integration system. The CRISPR transposon gene integration system comprises a vector pQCasTns containing transposase and Cas protein, and a vector pDoner containing a functional sequence, the pQCasTns comprises an operon and a T7 terminator which are connected in sequence; the pDoner comprises a functional sequence recognized by transposase and a crRNA sequence expression module of a targeted genome. The CRISPR transposon system provided by the invention solves a series of problems existing in genome editing of halomonas, and can realize multi-target knockout, high-efficiency deletion of long fragments and high-efficiency integration of exogenous genes.

The present application relates to a kind of genetically engineered bacteria of high-yield L-cysteine, construction method, and its application in the microbial fermentation preparation L-cysteine in L-cysteine. The present application is integrated by Esa quorum sensing system and lactose operon system, constructs a multifunctional QSI dynamic regulation system, realizes the coupling of target gene expression and cell growth, and is applied to the biosynthesis of L-cysteine, and obtains a high-yield L-cysteine of escherichia coli gene engineering strain. In addition, the QSI dynamic control system constructed in the present application dynamically controls lactose operon system using quorum sensing system, replaces the addition of traditional inducer IPTG, effectively saves the cost of industrial production and reduces the toxicity influence of inducer IPTG to cell, and has good production application value.

Disclosed herein are polynucleotides comprising sequences encoding coenzyme M synthase (ComF) linked to a heterologous regulatory element and methods of using the same. The polynucleotides may comprise synthetic operons comprising additional sequences encoding enzymes, e.g., a taurine-pyruvate aminotransferase, a sulfoacetaldehyde acetyl transferase, or a sulfopyruvate decarboxylase. Also disclosed herein are recombinant prokaryotic cells, e.g., recombinant bacterial, e.g., E. coli, or archaeal cells, e.g., Methanosarcina acetivorans with improved tolerance to oxidative stress.

The present invention relates to a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is a strong constitutive promoter or a strong vacuole-induced promoter, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding a lysin upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

The invention provides a salmonella paratyphi A strain for recombining and expressing Vi polysaccharide, which is characterized in that a viaB operon for expressing the Vi polysaccharide from salmonella paratyphi is cloned into a salmonella paratyphi A genome by a genetic engineering technology, so that the expression of the Vi polysaccharide in the salmonella paratyphi A is realized; furthermore, a constitutive strong promoter (Tac promoter) is used for replacing a viaB natural promoter to realize high expression of the Vi polysaccharide under an in-vitro culture condition; further, the msbB gene of the strain is knocked out to reduce the acylation level of the LPS and reduce the toxicity of the LPS; the vesicle vaccine prepared from the strain can induce immune protection effects on two serotypes.

The application discloses an Aeromonas phage recombinant system, an expression vector, a recombinant strain and application thereof, and the recombinant system comprises a recombinase operon 109TES derived from a salmonella Aeromonas phage vB_AsaM-56; the operon comprises at least one gene with exonuclease function and one single-stranded DNA annealing protein gene. Through function verification in Escherichia coli, 109TES can efficiently mediate linear circle recombination and linear linear recombination by using a short homologous arm (50 bp), and has stable and reliable recombination function in a heterologous host, indicating that the recombinase operon from a non-Escherichia coli phage can also exert efficient homologous recombination function in Escherichia coli, and can be used as a powerful candidate for constructing a new type of recombination tool, and provides a new choice for developing a multi-host available gene editing system.

The invention belongs to the field of biology, and discloses a proteus mirabilis homologous recombination system expression plasmid which comprises a pBR322 replication starting point, a resistance gene, an arabinose inducible promoter and a proteus mirabilis-derived homologous recombination operon. The homologous recombination operon is used for coding a TPM66325 protein, an EPM66325 protein, a single-chain binding protein SSB and a fake release protein. The expression plasmid has relatively high recombination efficiency in proteus mirabilis GDMCC 66325 and can also play a recombination role in a proteus mirabilis standard strain ATCC35659, and the expression plasmid is obtained by knocking out a related gene bcsB formed by a hemolysin related gene hpmA and a biological membrane of the proteus mirabilis GDMCC 66325 and directly knocking in a heterologous gene after a related gene cluster is adjusted by flagellum. The effectiveness of the expression plasmid in recombinant operation of proteus mirabilis is verified; meanwhile, the invention further discloses a proteus mirabilis homologous recombination system, application and a gene knockout / knockin method.

The invention provides a DNA marker plasmid as well as a combination system and application thereof, and relates to the field of molecular biology. The DNA marker plasmid is formed by connecting a linearized skeleton sequence and an insertion fragment sequence; the linearized skeleton sequence is derived from a reference plasmid from which a lactose operon sequence is deleted, and a selectable marker gene of the linearized skeleton sequence has a preset recognition site of restriction enzyme; the insertion fragment sequence is formed by connecting a plurality of DNA fragments in series, and recognition sites of restriction endonuclease are arranged between the adjacent DNA fragments. According to the plasmid, a restriction enzyme cutting site is introduced into a selective marker, so that a skeleton is directly used as a characteristic band, and the complete utilization of a sequence is realized. Waste and purification are avoided after enzyme digestion, the cost is remarkably reduced, and the preparation efficiency is improved.

The invention provides a strain for producing L-isoleucine and a construction method and application of the strain. Pck genes of the strain are knocked out, ilvBN (G20D, I21D and I22F), ilvA (V140M, F383A), lysC (T311I), hom (G378I), thrB (A20G), ppc, aspB, thrC, asd and brnFE are over-expressed, and the strain is prepared through operons Psod-ilvBN (G20D, I21D and I22F)-ilvA (V140M, F383A)-Ptuf-thrA-thrB-thrC and Psod-ppc-aspB-Ptuf

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 application belongs to the technical field of genetic engineering, and particularly relates to an engineered bacillus subtilis, a preparation method and application thereof. In the present application, in order to improve the supply of precursor cytidine triphosphate (CTP) in the process of fermentation of bacillus subtilis for producing cytidine, first, the feedback-inhibited CTP synthetase PyrG E156K is overexpressed to promote the conversion of uridine triphosphate to CTP; then, the repressor gene of the pyrimidine operon pyrR is knocked out to promote the synthesis of uridine monophosphate, and further promote the synthesis of CTP; finally, the pyrimidine nucleotide degradation genes pdp and cdd are knocked out to block the conversion of uridine / cytidine to uracil / cytosine and the conversion of cytidine to uridine, respectively, reduce the consumption of pyrimidine nucleotides, and further facilitate the intracellular accumulation of CTP. The yield of cytidine of the finally constructed engineering bacteria is about 12.6 times that of the original starting bacteria, and the results of the present application further prove the importance of CTP for cytidine synthesis.

The invention discloses an HlyA secretion system for increasing the secretion amount of substrate protein and a construction method and application thereof.The HlyA secretion system comprises a first component, a second component and a third component, the first component comprises an inner membrane transporter HlyB and a membrane interstitial adaptor protein HlyD, and the inner membrane transporter HlyB and the membrane interstitial adaptor protein HlyD are obtained by expressing modified hlyBD operons; according to the modified hlyBD operon, a nucleotide sequence for coding a hexa-polyhistidine tag is inserted behind an hlyD gene initiation codon in the hlyBD operon; and a second component: an outer membrane channel protein TolC derived from a host cell. According to the invention, the HlyD protein is specifically modified, so that the secretion amount of the HlyA secretion system to the substrate protein is obviously increased.

The present invention relates to a bio-attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment operably linked to an independently controlled promoter, the first segment comprising a heterologous polynucleotide encoding a cargo molecule upstream of an hlyAs translocation sequence, the heterologous polynucleotide encoding the cargo molecule replacing the hlyA gene, and the second segment comprising an hly gene involved in secretion. Thus, the present invention relates to a bacterial delivery system and various uses and methods thereof.

Disclosed herein are methods and systems to generate novel nucleic acid sequences that bind to an allosteric transcription factor and novel nucleic acid sequences generated by said methods.

Escherichia coli is a widely studied model organism and an integral component of the human gut microbiome, offering significant potential for bacteria-based therapeutic applications. However, engineering native E. coli strains poses persistent challenges. In this study, the chassis- independent recombinase-assisted genome engineering technique was leveraged to engineer the native gut strain E. coli EcAZ-1 and the probiotic strain E. coli Nissle 1917 (EcN). The bioluminescent lux operon, green fluorescent protein (GFP), and the oxygen-independent fluorescent protein IFP 2.0 were successfully introduced into both strains. To further enhance IFP 2.0 fluorescence, a heme oxygenase was co-expressed, and the chromophore biliverdin was supplemented, achieving robust IFP 2.0 expression under both anaerobic and aerobic conditions. Also, both strains were engineered to biosynthesize bioactive compounds, including the plant- derived flavonoid naringenin and mycosporine-like amino acids. The results underscore the potential of native E. coli strains as flexible and robust platforms for synthetic biology, enabling novel applications in biomedical research and therapeutics. Based on these results, this application provides recombinant bacteria generating IFP2.0 fluorescence under anaerobic condition, bacterial composition for gastrointestinal cancer detection and use method thereof, and naringenin or MAA overproducing recombinant bacteria.