An alditol oxidase and application thereof. The method uses D-glucose as a substrate to dock the alditol oxidase derived from Streptomyces coelicolor A3, and selects amino acid residues around an active center for saturation mutagenesis, and screens for alditol oxidase with D-glucose oxidizing activity by plate color development. An amino acid sequence of the alditol oxidase is shown in SEQ ID NO: 2 or SEQ ID NO: 4. The alditol oxidase has the activity of converting D-glucose to D-gluconic acid and D-glyceraldehyde to D-glyceric acid. By using the alditol oxidase, the conversion of D-glucose to pyruvic acid is realized by only three enzymes for the first time and does not depend on any coenzyme.

Various aspects relate to a cell-free protein expression method. The method includes exposing a microorganism to substantially anaerobic growth conditions to produce a conditioned microorganism. The method further includes lysing the conditioned microorganism to produce a lysate. The method further includes combining the lysate with a nucleic acid and producing a protein of interest a metabolic pathway, a molecule, or a mixture thereof from the lysate.

The present disclosure discloses an Escherichia coli-based genetically-modified recombinant strain, a construction method therefor and use thereof. A mutant gene obtained by subjecting a wild-type deoB gene (ORF sequence is shown in a sequence 3902352-3903575 in GenBank accession No. CP032667.1) and a wild-type rhtA gene promoter sequence PrhtA (shown in a sequence 850520-850871 in GenBank accession No. AP009048.1) of an E. coli K12 strain and a derivative strain thereof (such as MG1655 and W3110) to site-directed mutagenesis, and a recombinant strain obtained therefrom can be used for the production of L-threonine, and compared with an unmutated wild-type strain, the obtained strain can produce L-threonine with a higher concentration and has good strain stability, and also has lower production cost as an L-threonine production strain.

Disclosed is recombinant Escherichia coli for producing L-tyrosine and application thereof, and belongs to the technical fields of genetic engineering and bioengineering. According to the present disclosure, genes aroP and tyrP are knocked out, expresses the endogenous gene yddG of E. coli, then heterologously expresses fpk from Bifidobacterium adolescentis, expresses the endogenous genes ppsA and tktA of E. coli, and then expresses aroG.sup.fbr and tyrA.sup.fbr. Knocking out tyrR, trpE and pheA, so that the synthesis flux of L-tyrosine is increased. Finally, an endogenous gene poxB is knocked out to realize stable fermentation performance at high glucose concentration.

Recombinant E. coli strains and synthetic protein sequence designs are leveraged for production of disordered polypeptides such as spidroins and elastin-like peptides (ELPs). These disordered polypeptides, the high-titer production of which has proven difficult, include repeating structural motifs from a small selection of amino acid residues, resulting in lack of well-defined tertiary and quaternary structure. The recombinant E. coli include expression vectors with genes encoding for the disordered polypeptide product. Expression of these genes is controlled by a promoter that downregulates and substantially inhibits basal expression in the recombinant bacteria. Further, the recombinant bacteria include mutations to one or more stress-response genes from wild-type E. coli, such as yggw, yedv, yedw, yedy, spec, speb, uspc, hcha, loip, mltc, envz, ompr, yhgf, or hupb. The recombinant E. coli enable production of high titers of disordered protein product while minimizing the toxic effects thereof on the host.

The present invention relates to a recombinant strain for producing shikimic acid, in which a target gene that regulates the asymmetric cell division and target genes that regulate the shikimic acid production are expressed The target gene that regulates the asymmetric cell division includes cytoskeletal protein PopZ coding gene popZ, and the target genes that regulate the shikimic acid production include DAHP synthase coding gene aroG, 3-dehydroquinate synthase coding gene aroB, and transketolase coding gene tktA. The recombinant strain of the present invention realizes the de novo synthesis of shikimic acid using glucose as a substrate, with a low cost. After fermentation with the strain in a 7.5 L fermentor, the highest production of shikimic acid is 88.1 g/L, the yield is 0.33 g/g, and the production intensity of shikimic acid is 1.1 g/L/h.

Non-naturally occurring vesicles derived from bacteria, e.g., pathogenic bacteria, methods for making the vesicles, and methods for using compositions of these vesicles are disclosed. Methods of using the vesicles include prevention and/or treatment of bacterial infections. Also provided herein are compositions that include vesicles derived front bacteria and tumor vesicles, methods for making the tumor vesicles, and methods for using the compositions of bacterial vesicles and tumor vesicles. Methods of using the compositions of bacterial vesicles and tumor vesicles include treatment of cancer in a subject. Tumor vesicles may be derived from cancer cells present in the subject to be treated or from a cancer cell line expressing at least one neoantigen. The neoantigen may be specific to the subject and may have been identified by sequencing of the cancer cells from the subject. The neoantigen may be a neoantigen known to be commonly expressed in a particular type of cancer.

The present invention provides a nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid, the nitrilase mutant is obtained by mutating one or two of the amino acids at position 180 and 205 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by semi-rational design and protein molecular modification, the specific enzyme activity of the nitrilase double mutant AcN-G180D/A205C was increased by up to 1.6 folds, and the conversion rate>99%. And the reaction time was shortened to a quarter of the original using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50 C.). Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.

An amino acid sequence of the transaminase mutant is an amino acid sequence obtained by a mutation of an amino acid sequence is shown in SEQ ID NO: 1. The mutation occurred at least one of the following mutation sites: G17V, L36P, Q40H, G69Y, H70T, L73A, V77G, V77S, V77T, A78I, Y130M, Y130V, Y130T, N132I, N132T, K141S, K142S, K142T, R143P, G144F, G144W, G144Y, E145D, E145S, E145G, K146R, L148A, L148I and the like.

The present disclosure discloses the identification and introduction of a specific heterologous gene (denoted as smob), which encodes an -1,2-fucosyltransferase, into an LNT production strain to produce LNFP-I in particular. The smob gene originates from the organism Sulfuriflexus mobilis (https://www.dsmz.de/collection/catalogue/details/culture/DSM-102939), which is a sulfur-oxidizing bacterium isolated from a brackish lake sediment.