CN114958877B

CN114958877B - Deacetylated oxo cephalosporin C synthetase mutant, encoding gene and application thereof

Info

Publication number: CN114958877B
Application number: CN202210671678.7A
Authority: CN
Inventors: 梁恒宇; 周鹏; 韩超; 陈民良; 幸志伟; 张皓; 赵振华; 张敏; 李雪亮
Original assignee: Henan Jiankangyuan Biomedical Research Institute Co ltd
Current assignee: Henan Jiankangyuan Biomedical Research Institute Co ltd
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2024-02-20
Anticipated expiration: 2042-06-14
Also published as: CN114958877A

Abstract

The invention discloses a desacetoxy cephalosporin C synthetase mutant, a coding gene and application thereof, belonging to the technical field of enzyme engineering. The amino acid sequence of the deacetyl-oxy cephalosporin C synthetase mutant takes SEQ ID NO.1 as a reference, and has at least one mutation in threonine T72 at position 72 and glycine G92 at position 92; threonine at position 72 is mutated to any natural amino acid other than itself, and glycine at position 92 is mutated to any natural amino acid other than itself. The invention improves the catalytic activity of the enzyme on penicillin G by carrying out site-directed mutagenesis and genetic engineering transformation on the desacetyloxy cephalosporin C synthetase of streptomyces clavuligerus, can improve the yield of the trichothecene C of the trichothecene flavum, and is suitable for commercial and industrial application.

Description

Deacetylated oxo cephalosporin C synthetase mutant, encoding gene and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a desacetoxycephalosporin C synthetase mutant, a coding gene and application thereof.

Background

The desacetoxycephalosporin C synthetase (Deacetoxycephalosporin C synthase, DAOCS) is a key enzyme in the biosynthesis process of beta-lactam antibiotics, and catalyzes five-membered thiazole ring expansion to generate six-membered thiazine ring, so that corresponding beta-lactam metabolites are obtained.

Cephalosporin C (CPC) is an important natural β -lactam antibiotic produced by the filamentous fungus Acremonium chrysogenum. The cefEF gene encoding a bifunctional enzyme, namely desacetoxycephalosporin C synthase (expandase)/desacetylcephalosporin C synthase (hydroxylase) (Deacetoxycephalosporin/deacetylcephalosporin C synthase, DAOC/DACS), exists on the genome of A. Chrysogenum and is the rate-limiting enzyme for synthesizing CPC by A. Chrysogenum, and catalyzes the ring-expanding reaction of penicillin N (penicillin N, peN) to form desethoxy cephalosporin C (DAOC) and then catalyzes the hydroxylation reaction of DAOC to form desacetyl cephalosporin C (DACS). CPC and Pen N are main natural products secreted by Acremonium chrysogenum, and a large amount of Pen N can be secreted simultaneously by the Acremonium chrysogenum in the CPC fermentation production process; secretion of these two compounds depends on their biosynthesis at the cellular level; therefore, the improvement of the activity of the Acremonium chrysogenum expandase is likely to be beneficial to the conversion of the byproduct Pen N to the product CPC.

In addition, 7-amino-3-deacetyl-cephalosporanic acid (7-ADCA) is an important intermediate raw material for producing oral cephalosporin medicines, and is mainly used for synthesizing antibiotics such as cefalexin, cefradine, cefadroxil and the like. The chemical synthesis of 7-ADCA using penicillin G potassium salt as raw material is a method commonly adopted in industrial production at present, however, the synthesis process requires multi-step chemical ring expansion, the reaction condition is high, the yield of the reaction products of each step is low, and the chemical pollution at the end of production is heavy, so people try to realize the synthesis of cephalosporin mother nucleus by using a pollution-free and more economical enzyme conversion method. The penicillin G has large yield and low price, and a relatively effective mode is to utilize the deacetylated oxo cephalosporin C synthetase, namely penicillin expandase to expand the penicillin G to obtain G-7-ADCA (7-phenylacetyl deacetylated oxo cephalosporin C), and then remove the 7-side chain to obtain 7-ADCA. However, the natural substrate of wild-type DAOCS is penicillin N (penicillin N, peN) which is currently not commercially synthesized on a large scale; the enzyme has low reactivity to a large amount of non-natural substrate penicillin G, so that the actual conversion rate is low, and the application of the deacetoxycephalosporin C synthetase in industrial production is limited. Therefore, the enzymatic modification of DAOCS enhances the catalytic activity of DAOCS on a large amount of low-cost penicillin G and enhances the reaction stability of DAOCS, which becomes the primary problem to be solved when the DAOCS is introduced into industrial production.

Disclosure of Invention

The invention discloses a desacetoxy cephalosporin C synthetase mutant, a coding gene and application thereof, wherein the catalytic activity of the enzyme can be improved by carrying out site-directed mutagenesis on the desacetoxy cephalosporin C synthetase in streptomyces clavuligerus, thereby being beneficial to the synthesis and conversion of beta-lactam antibiotics.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a mutant of desacetyloxy cephalosporin C synthetase, the amino acid sequence of which is referenced to SEQ ID No.1 (wild type desacetyloxy cephalosporin C synthetase): MDTTVPTFSLAELQQGLHQDEFRRCLRDKGLFYLTDCGLTDTELKSAKDLVIDFFEHGSEAEKRAVTSPVPTMRRGFTGLESESTAQITNTGSYSDYSMCYSMGTADNLFPSGDFERIWTQYFDRQYTASRAVAREVLRATGTEPDGGVEAFLDCEPLLRFRYFPQVPEHRSAEEQPLRMAPHYDLSMVTLIQQTPCANGFVSLQAEVGGAFTDLPYRPDAVLVFCGAIATLVTGGQVKAPRHHVAAPRRDQIAGSSRTSSVFFLRPNADFTFSVPLARECGFDVSLDGETATFQDWIGGNYVNIRRTSKA; has at least one mutation in threonine T72 at position 72, glycine G92 at position 92; threonine at position 72 is mutated to any natural amino acid other than itself, and glycine at position 92 is mutated to any natural amino acid other than itself.

Further, the nucleotide sequence of the wild-type desacetoxycephalosporin C synthetase is shown in SEQ ID NO. 2: atggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccaccatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccggcagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatag.

Further, the amino acid sequence of the single-site mutant of the desacetoxycephalosporin C synthetase is shown in SEQ ID NO. 3: MDTTVPTFSLAELQGLHQDEFRRCDKGLQYLTDLTDELKQDLVIDEQVEQVEGEVEGEVEGEARVPQVEGEARTMSEARASTAQVEGEVETYVEGESTASTAQTYVESRARGGVEULAARGEGEVEQVPQVEQVEQVEQVEGGAFLPLPYRPDAVLVFCGAIATTGGQGQKVKAHHVAVVAV VAV-KappaRRDQVQVQVVVVVULASSVULASSVVULASSVEVPLARECARECADSLANGGETTQUATQVEGFQVEGGVPGVPGVPLARENGTAQVEGGUGVGVGVIRTV-VVIRTV the process comprises, the process comprises, wherein X at position 72 represents a natural amino acid other than threonine.

Further, the nucleotide sequence of the single-site mutant of the desacetoxycephalosporin C synthetase is shown in SEQ ID NO. 4: the ccgccgaccgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcg g cs g cs, cs g cs g, g, g, the process comprises, the process comprises, the codon sequence nnn of amino acid 72 represents the other natural amino acid codon sequences except for the threonine codon acc in the wild type.

Further, the amino acid sequence of the single-site mutant of the desacetoxycephalosporin C synthetase is shown in SEQ ID NO. 5: MDTTVPTFSLAELQGLHQDEFRRCDKGLQYLTDLTDELKQDLQVEQVEQVEQVEGEVEGEVEGEVERAVPTATQVEGESTRUSSAGARQVERACTGEMETHVEGEARTMSTRACTGESEQVESSTAQTYVEDSSDYSTADNPSPSGDFERRUQQYRQRQRARAVAARAGGEVEPLCEPLLRFRYFPQVPQVPAEQQPLARSEQPLARQQQQQQQQQQQPCPCPCPCVQQQQQGGAFLPLPYLPYLPTQVEQCAATTGATTGQQQQGQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVQVPQVPQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ the process comprises, the process comprises, wherein X at position 92 represents a natural amino acid other than glycine.

Further, the nucleotide sequence of the single-site mutant of the desacetoxycephalosporin C synthetase is shown in SEQ ID NO. 6: the ccacgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcg g cs g, g, g, the process comprises, the process comprises, the codon sequence nnn of amino acid 92 represents the other natural amino acid codon sequence except for the glycine codon ggc in the wild type.

Further, the amino acid sequence of the double-site mutant of the desacetyloxy cephalosporin C synthetase is shown in SEQ ID NO. 7: MDTTVPTFSLAELQGLHQDEFRRCDKGLQYLTDLTDELKQDLVIDEQVEQVEGEVEGEVEGEARQVEGEARTMASTAQVEGERRGEVEGEARTQSTASTAQVEGETAQTYVETYVERQVERAVEGGVEPLCEPLUQVPQVEQVEQVEQVEQQVEGGAFLPLPYRPDAVLVFCGAIATTGGQVKAVKAVKAHVVVAVVVVVAARGGUGGPQVPQVPQVPQVPQVPQVPQVPQVPQVEUGLQVESSGEGGVEGGUGGVEGGUGGUGGSQVEGEVEGGVEGGUGQVEGGSQVEGGUGQVEGGUGQVEGGSQVEGGUGQVEGGUGQVESQQVEGEGQVEVEGGQVEQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ the process comprises, the process comprises, wherein X at position 72 represents a natural amino acid other than threonine and X at position 92 represents a natural amino acid other than glycine.

Further, the nucleotide sequence of the double-site mutant of the desacetyloxy cephalosporin C synthetase is shown in SEQ ID NO. 8: the ccgccgaccgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcg g cs g, cs g, g, g, the process comprises, the process comprises, the codon sequence nnn of amino acid 72 represents the other natural amino acid codon sequence except for the threonine codon acc in the wild type, and the codon sequence nnn of amino acid 92 represents the other natural amino acid codon sequence except for the glycine codon ggc in the wild type.

Preferably, the amino acid sequence of the mutant is referred to as SEQ ID NO.1, and threonine at position 72 is mutated to tryptophan, glycine, glutamine, asparagine, aspartic acid, leucine, histidine, isoleucine, lysine or methionine.

More preferably, threonine at position 72 is mutated to tryptophan or glycine.

Most preferably, threonine at position 72 is mutated to tryptophan.

Preferably, the amino acid sequence of the mutant is referenced to SEQ ID NO.1, glycine 92 is mutated to methionine, cysteine, serine, asparagine, glutamine or histidine.

More preferably, glycine 92 is mutated to methionine or cysteine.

Most preferably, glycine 92 is mutated to methionine.

Preferably, the amino acid sequence of the mutant is referred to as SEQ ID NO.1, threonine at position 72 is mutated to tryptophan, glycine, glutamine, asparagine, aspartic acid, isoleucine or histidine; glycine 92 is mutated to methionine, cysteine, serine, asparagine, glutamine, phenylalanine, threonine or alanine.

More preferably, the mutant is the double site mutant T72D/G92C, T D/G92H, T G/G92 5495G/G92 5497G/G92 6272I/G92M, T N/G92M, T Q/G92C, T Q/G92H, T Q/G92 3995Q/G92S, T W/G92C, T W/G92F, T W/G92 3772W/G92H, T W/G92M, T W/G92 3996W/G92Q, T W/G92S or T72W/G92T.

More preferably, the mutant is a double site mutant T72G/G92M, T N/G92M, T Q/G92M, T W/G92C, T W/G92F, T72W/G92H, T W/G92M, T W/G92N, T W/G92S or T72W/G92T.

Most preferably, the mutant is a double site mutant T72W/G92M.

Further, the preparation of the mutant can be carried out by constructing a vector plasmid containing a wild-type desacetyloxy cephalosporin C synthetase gene, selecting site of site-directed mutation and amino acid type after mutation, synthesizing corresponding primers, using the vector plasmid containing the wild-type desacetyloxy cephalosporin C synthetase gene as a template, PCR amplifying mutant DNA fragments, separating and purifying, amplifying the obtained fragments into full-length mutant genes by PCR, cloning the full-length mutant genes onto a proper vector and transforming proper host cells, culturing and screening positive clones with high enzyme activity, extracting plasmid DNA from the positive clones, and carrying out DNA sequence determination analysis to determine the introduced mutation. Alternatively, the whole plasmid vector containing the mutant enzyme molecule DNA sequence can be synthesized by means of gene synthesis, then the proper host cell is transformed, and positive clones with high enzyme activity are screened out by culture.

In preparing the desacetylcephalosporin C synthetase mutants, any suitable vector may be used, for example, prokaryotic expression vectors pET28, pRSET, pET-30a (+), pGEMT-Easy, etc.; eukaryotic expression vectors pYD1, pYES2/GS and the like; pUC18/19, pBluscript-SK may also be used.

A biological material is an expression vector containing the gene or an enzyme protein expression host cell containing the expression vector.

Further, the above genes may be expressed in prokaryotic, eukaryotic cells, or extracellular expression in prokaryotic, eukaryotic, or cell-free systems may be performed by any suitable method known in the art. The chassis system of the expression vector can be prokaryotic microorganism cells, eukaryotic microorganism cells or a cell-free system; wherein the prokaryotic microorganism can be Escherichia coli, bacillus, corynebacterium glutamicum, streptomyces, etc.; the eukaryotic microorganism can be Saccharomyces cerevisiae, pichia pastoris, filamentous fungi, etc.; cell-free systems may be derived from E.coli lysates, yeast cell lysates, wheat germ extracts, mammalian cell lysates, and the like.

The application of the deacetylated oxo-cephalosporin C synthetase mutant, gene or biological material in preparing 7-phenylacetyl deacetylated oxo-cephalosporin.

Preferably, 7-phenylacetyl-desacetylcephalosporin is prepared using penicillin G as substrate by using a desacetylcephalosporin C synthetase mutant.

The application of the gene for encoding the desacetoxycephalosporin C synthetase mutant in improving the cephalosporin C yield by heterologous expression in Acremonium chrysogenum after codon optimization.

Preferably, the mutant desacetyloxy cephalosporin C synthetase uses penicillin N as a substrate.

In conclusion, the invention improves the catalytic activity of the enzyme on penicillin G by carrying out site-directed mutagenesis and genetic engineering modification on the deacetoxycephalosporin C synthetase of streptomyces clavuligerus, can improve the yield of the cephalosporin C of the acremonium chrysogenum, and is suitable for commercial and industrial application.

Drawings

FIG. 1 shows a wild-type expression plasmid for Streptomyces clavuligerus desacetyloxy cephalosporin C synthase.

FIG. 2 is a diagram showing a wild-type protein electrophoresis gel of Streptomyces clavuligerus desacetyloxy cephalosporin C synthetase;

m: a marker;1: a whole cell sample; 2: centrifuging the supernatant after cell wall breaking; 3: samples were precipitated after cell wall breaking.

FIG. 3 shows the results of a test of the ability of Streptomyces clavuligerus to convert wild-type cephalosporin C synthase G13571-56 to G-7-ADCA using penicillin G as a substrate.

FIG. 4 shows recombinant expression vectors of the highly active Streptomyces clavuligerus deacetylated cephalosporin C synthetase mutant G13571-56.

FIG. 5 shows the recombinant plasmid pBARGPE 1-ScDAOCS-G13571-56.

FIG. 6 shows the peak of HPLC overlay comparison of fermentation CPC yield of negative control A.chrysogenum strain ATCC11550-pBARGPE1, high activity Streptomyces clavuligerus desacetylcephalosporin C synthase mutant overexpressing strain ATCC11550-pBARGPE 1-ScDAOCS-G13571-56.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: acquisition of wild-type desacetyloxy cephalosporin C synthetase gene and construction of mutant library

Designing mutants according to a wild streptomyces clavuligerus (Streptomyces clavuligerus) deacetoxycephalosporin C synthetase amino acid sequence SEQ ID NO.1 (protein ID: AAA 26715.1) and a coding gene nucleotide sequence SEQ ID NO.2 (GenBank ID: m32324, CDS sequences 1559 to 2494 bp) published by NCBI website; the amino acid sequences of the mutants are shown as SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, and the nucleotide sequences of the encoding genes are shown as SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8.

Libraries of wild-type and mutant desacetyloxy cephalosporin C synthetases were constructed by Changzhou-ary biotechnology Co., ltd, including single point saturation mutation of desacetyloxy cephalosporin C synthetase site 72 threonine (T72), single point saturation mutation of glycine 92 (G92), partial double point combination mutation of threonine 72 and glycine 92 (T72/G92); the cloning vector is selected from pET-30a (+) fusion protein expression vector (kanamycin resistance, empty vector size is 5422 bp), cloning site is NdeI-HindIII (restriction enzyme NdeI recognizes 5'-CA ≡TATG-3' restriction site, restriction enzyme HindIII recognizes 5'-A ≡AGCTT-3' restriction site), host cell is Escherichia coli TOP10 competent strain (purchased from Beijing Soy Bao technology Co., ltd., product number C1200).

The nucleotide sequence of the wild insert fragment of the streptomyces clavuligerus desacetyloxy cephalosporin C synthetase library is SEQ ID NO.9: catatggacacgacggtgcccaccttcagcctggccgaactccagcagggcctgcaccaggacgagttccgcaggtgtctgagggacaagggcctcttctatctgacggactgcggtctgaccgacaccgagctgaagtcggccaaggacctcgtcatcgacttcttcgagcacggcagcgaggcggagaagcgcgccgtcacctcgcccgtccccaccatgcgccgcggcttcaccgggctggagtcggagagcaccgcccagatcaccaataccggcagctactccgactactcgatgtgctactcgatgggcaccgcggacaacctcttcccgtccggtgacttcgagcggatctggacccagtacttcgaccgccagtacaccgcctcccgcgcggtcgcccgggaggtcctgcgggcgaccgggaccgagcccgacggcggggtcgaggccttcctcgactgcgagccgctgctgcggttccgctacttcccgcaggtccccgagcaccgcagcgccgaggagcagcccctgcggatggcgccgcactacgacctgtcgatggtcaccctcatccagcagacaccctgcgccaacggcttcgtcagcctccaggccgaggtcggcggcgcgttcacggacctgccctaccgtccggacgccgtcctcgtcttctgcggcgccatcgcgaccctggtgaccggcggccaggtcaaggccccccggcaccatgtcgcggccccccgcagggaccagatagcgggcagcagccgcacctccagtgtgttcttcctccgtcccaacgcggacttcaccttctccgtcccgctggcgcgcgagtgcggcttcgatgtcagcctggacggcgagaccgccacgttccaggattggatcgggggcaactacgtgaacatccgccgcacatccaaggcatagtaaaagctt; the nucleotide sequence of the mutant insert of the streptomyces clavuligerus desacetyloxy cephalosporin C synthetase library is SEQ ID NO.10: the invention relates to a method for preparing a composite material, which comprises the steps of dividing a composite material into a plurality of pieces, dividing the pieces into pieces, dividing the pieces into pieces, dividing pieces into pieces, and dividing pieces. A. The invention relates to a method for producing a fibre-reinforced plastic composite a. The invention relates to a method for producing a fibre-reinforced plastic composite. The codon sequence nnn of amino acid 72 represents the other natural amino acid codon sequence except for the threonine codon acc in the wild type, and the codon sequence nnn of amino acid 92 represents the other natural amino acid codon sequence except for the glycine codon ggc in the wild type. Wherein the wild type enzyme expression plasmid map is shown in figure 1, and the plasmid size is 6192bp.

Example 2: culture and induced expression of host strain of mutant of desacetoxycephalosporin C synthetase

The wild-type or mutant expression strain of the desacetylcephalosporin C synthetase constructed in example 1 was taken out from a-80℃low-temperature refrigerator, slowly and completely dissolved in an ice bath, then 0.5mL of the strain frozen stock solution was drawn from the strain frozen stock tube on a sterile console and cultured in a sterile cooling LB liquid medium (containing 50. Mu.g/mL kanamycin) at 37℃with shaking for 12 hours.

After the completion of the cultivation, 0.5mL of the culture medium was transferred to a 250mL triangular flask containing 50mL of sterile LB medium, and the culture was performed at 37℃for 1.5 hours with shaking at 220rpm, and isopropyl-beta-D-thiogalactoside (IPTG, available from Shanghai Ala Biotechnology Co., ltd., product No. I104812-25 g) was added to the culture medium, followed by shaking at 25℃for 10 hours with shaking at 220 rpm.

SDS-PAGE protein electrophoresis was performed at the end of the culture.

1) The experimental reagent is prepared according to the following operation:

(1) 30% stock gum solution: 29.0g of acrylamide (Acr), 1.0g of methylenebisacrylamide (Bis), and ddH was added after mixing ₂ O was dissolved, the volume was set to 100mL and the brown bottle was stored at room temperature.

(2) 10 x running buffer (ph=8.3): 3.02g Tris,18.8g glycine, 10mL 10% SDS, ddH ₂ 0 to dissolve and to volume to 100mL.

(3) 10% Ammonium Persulfate (AP): 100mgAP plus ddH ₂ 0 to 1mL was fully dissolved.

(4) 2 XSDS electrophoresis loading buffer: 1mol/L Tris-HCl (pH=6.8) 2.5mL, beta-mercaptoethanol 1.0mL, SDS0.6g, glycerol 2.0mL,0.1% bromophenol blue 1.0mL, ddH ₂ O 3.5mL。

2) Preparing polyacrylamide gel:

(1) preparation of 12mL of isolation gel (12%): ddH ₂ O,3.96mL;30% stock gum, 4.8mL;1.5mol/L Tris-HCl,3mL;10% SDS,0.12mL;10% AP,0.12mL. Mixing the above solutions, adding TEMED (N, N, N ', N' -tetramethyl ethylenediamine) 10 μl, mixing, and pouring into glassCapping with water saturated n-butanol between glass plates, and keeping the liquid level flat, wherein the gel is polymerized completely for 30-60min.

(2) Preparation of 6ml laminating adhesive (4%): ddH ₂ O,4.2mL;30% stock gum, 0.99mL;1mol/L Tris-HCl,0.75mL;10% SDS,0.06mL;10% AP,0.06mL; TEMED,6 μl. The liquid on the separating gel was poured off, rinsed with ultrapure water and sucked dry with filter paper. Adding the above mixed solution, and immediately inserting the comb between glass plates, wherein the polymerization time is 15-30min.

3) Sample treatment: the sample was added to an equal amount of 2 XSDS loading buffer, heated at 100℃for 5min, centrifuged for 12000 g.times.1 min, and the protein Marker was subjected to parallel treatment.

4) Loading: 10uL of the treated sample was added to the gel tank and 20 uL of protein Marker was added as a control.

5) Electrophoresis: and (3) adopting a vertical electrophoresis tank device, adding 1 Xelectrophoresis buffer solution into the electrophoresis tank, connecting a power supply, enabling a negative electrode to be on the upper side, enabling a positive electrode to be on the lower side, laminating glue voltage 150V, separating glue voltage 200V, and stopping electrophoresis until bromophenol blue goes to the lower end of the electrophoresis tank. After about 1 hour the protein gel was removed from the glass plate and stained and destained using a pyxis protein rapid processing system. The decolorized albumin glue is used for photographing the decolorized gel under an image processing system, and the gel can be stored in 7% acetic acid solution.

A diagram of the wild-type protein electrophoresis gel of the streptomyces clavuligerus deacetylase cephalosporin C synthetase is shown in figure 2.

Example 3: test of the reaction Capacity of the Deacetoxycephalosporin C synthetase mutant to produce G-7-ADCA Using penicillin G as substrate

1) Wall breaking of chassis host cells: the wild-type and mutant host strains of desacetoxycephalosporin C synthetase were induced and cultured as described in example 2, after the culture was completed, the shake flask was removed, 4mL was aspirated into a 15mL centrifuge tube, and centrifuged at 6000rpm for 5min. After centrifugation, the supernatant was discarded and the cells were collected and resuspended in 4ml of Tris-ammonium sulfate buffer (50 mmol/L each, pH=7.4, and 1mmol/L final concentration of DTT was added). After the bacteria were resuspended, the tube was placed on an ice bath and subjected to ultrasonic disruption (ultrasonic parameters: 45% power, ultrasonic treatment for 3s, stop for 2s, and ultrasonic treatment for 15min total).

2) Enzyme ability to react with substrate test: after the bacterial heavy suspension is crushed by ultrasonic, 2.4mL is absorbed and transferred into a 50mL centrifuge tube, feSO with the final concentration of 2mmol/L is added ₄ 2mmol/L sodium ascorbate, 5mmol/L alpha-ketoglutarate, 5mmol/L potassium penicillin G; standing at 30deg.C for 30min. After the completion of the reaction, 1.2mL of acetonitrile (analytically pure) solution was added to terminate the reaction, and the obtained reaction solution was subjected to HPLC detection.

HPLC detection instrument: analytical balance sartorius BSA224S (0.0001 g), siemens U3000 high performance liquid chromatograph, ultrasonic cleaner, pH meter sartorius PB-10, agilent C ₁₈ Chromatographic column (4.6X1250 mm, particle size 5 μm).

HPLC detection reagent:

(1) 20mmoL/L phosphate solution (ph=3.50): 4.5644g of dipotassium phosphate trihydrate is taken and dissolved in water to 1000mL, and the pH is adjusted to 3.50 with phosphoric acid.

(2) Mobile phase: 20mmoL/L phosphate solution (ph=3.5): methanol (50%: 50%).

(3) System adaptation solution: 10mg of the G-7-ADCA standard, 2mg of penicillin G standard, alpha-ketoglutaric acid and L-ascorbic acid are respectively placed in a 50ml volumetric flask, and dissolved by mobile phase ultrasound, the volume is fixed and the mixture is shaken uniformly. In the ultrasonic process, the water temperature in the ultrasonic wave is controlled to be 2-8 ℃.

(4) Control solution: 10mg of G-7-ADCA standard is taken and placed in a 50ml volumetric flask, dissolved by ultrasound with phosphate solution with pH=3.5, and the volume is fixed and shaken well. Alpha-ketoglutaric acid, ferrous sulphate and L-ascorbic acid were formulated as solutions, diluted with mobile phase to 500. Mu.M, 200. Mu.M and 400. Mu.M, respectively. In the ultrasonic process, the water temperature in the ultrasonic wave is controlled to be 2-8 ℃.

(5) Test solution: the reaction solution was diluted 4 times with methanol and mixed well.

HPLC chromatographic conditions: the column temperature is 30 ℃, the dissolution temperature is 2-8 ℃, the detection wavelength is 225nm, the flow rate is 0.65mL/min, and the sample injection amount is 5 mu L.

HPLC detection result calculation:

C _sample -concentration of G-7-ADCA in the test sample, mg/mL;

C _{label (C)} -concentration of G-7-ADCA in standard solution, mg/mL;

A _sample -G-7-ADCA peak area in test sample;

A _{label (C)} -G-7-ADCA standard peak area;

relative enzyme activity calculation:

relative enzyme activity= (C _Mutant /C _{Wild type} )×100％。

C _Mutant -concentration of G-7-ADCA in the test sample after enzymatic reaction of the desacetoxycephalosporin C synthetase mutant, mg/mL;

C _{wild type} -concentration of G-7-ADCA in the test sample after the deacetoxycephalosporin C synthase wild-type enzyme reaction, mg/mL;

the results are shown in Table 1 and FIG. 3.

TABLE 1 relative enzyme Activity of desacetoxycephalosporin C synthetase mutants

Example 4: codon optimization of high-activity streptomyces clavuligerus deacetylase cephalosporin C synthetase mutant

Genetic information is described by triplet codons. Because of the degeneracy of codons, most amino acids are encoded by 2-6 synonymous codons. Different species use different Codon types that encode the same amino acid and the frequency of use, a phenomenon known as Codon Usage Bias. There is a clear difference in codon preference between prokaryotic and eukaryotic microorganisms. Thus, in this example, the codon of the prokaryotic microorganism Streptomyces clavuligerus deacetylase highly active mutant G13571-56 gene was optimized to a codon sequence suitable for expression of the filamentous fungus Acremonium chrysogenum.

In this example, alanine codon sequence was optimized for gcc cysteine codon sequence was tgc, aspartic acid codon sequence was optimized for gac, glutamic acid codon sequence was optimized for gag, phenylalanine codon sequence was optimized for ttc, glycine codon sequence was optimized for ggc, histidine codon sequence was optimized for cac, isoleucine codon sequence was optimized for atc, lysine codon sequence was optimized for aag, leucine codon sequence was optimized for ctc, asparagine codon sequence was aac, proline codon sequence was optimized for ccc, glutamine codon sequence was optimized for cag, arginine codon sequence was cgc, serine codon sequence was optimized for agc, threonine codon sequence was optimized for acc, valine codon sequence was optimized for gtc, tyrosine codon sequence was optimized for taa.

The nucleotide sequence of the high activity mutant gene G13571-56 of the streptomyces clavuligerus deacetylase cephalosporin C synthetase after codon optimization is shown in SEQ ID NO.11 (changed due to codon sequence optimization):

example 5: construction, transformation and verification of recombinant expression vector of high-activity streptomyces clavuligerus deacetylated oxygen cephalosporin C synthetase mutant escherichia coli after optimization of codon preference

The total length of the pET-28a (+) plasmid vector is 5369bp, and contains kanamycin resistance. The pET-28a (+) plasmid obtained by purification was subjected to double cleavage with restriction enzymes (both purchased from Shanghai Biotechnology Co., ltd.) and HindIII (5 '-A ∈T-3', cat# B600184) using cleavage recognition site 5'-CA ∈TATG-3', shanghai Biotechnology Co., ltd., cat# B600120-0500), and a 63bp multiple cloning site sequence was cut out. The enzyme digestion reaction system is as follows: plasmid vector, 3 μg; buffer, 5. Mu.L; ndeI restriction enzyme, hindIII 0.5. Mu.L; restriction enzyme, 0.5. Mu.L; ddwater, to 50 μl; incubate at 37℃for 3-4 hours, shake at intervals and centrifuge to prevent evaporation of droplets onto the tube cap. And (3) performing gel cutting and purification on the linear plasmid vector subjected to enzyme cutting after verification for standby.

G13571-56 is synthesized by Changzhou Kogyo Biotechnology Co., ltd, restriction enzyme NdeI recognition site (5 '-CA ≡TATG-3') and HindIII recognition site (5 '-A ≡AGCTT-3') are added to upstream and downstream of the mutant gene of desacetoxycephalosporin C synthetase, and the nucleotide sequence of the synthesized insert is shown in SEQ ID NO. 12:

The synthesized insert is digested with restriction enzymes NdeI and HindIII, and then subjected to enzyme ligation reaction with a pET-28a (+) linear plasmid obtained by double digestion and purification of restriction enzymes NdeI and HindIII. And (3) connecting a reaction system: annealing product, 2 μl; linearizing 25ng of plasmid; t4 DNA ligase (Shanghai Biotechnology Co., ltd.; cat# B110041) 0.5. Mu.L; connecting buffer,5 mu L; ddwater was added to 10. Mu.L and the ligation was carried out overnight at 16 ℃.

The ligation products were purified and validated to transform E.coli competent cells: 1. Mu.L of the expression vector plasmid was added to 100. Mu.L of E.coli BL21 commercial competent cells, incubated in an ice bath for 30min, removed and placed in a water bath at 42℃for 2min, immediately placed on an ice bath for 3min, added to 650. Mu.L of LB liquid medium (37 ℃) and cultured with shaking at 200rpm for 1h at 37℃and centrifuged at 5000rpm for 3min at room temperature, leaving approximately 150. Mu.L of coated resistant plates (50. Mu.g/mL kanamycin) and incubated overnight in an incubator at 37 ℃. A well-grown monoclonal colony on the resistance plate was picked up, inoculated into 5mL of LB liquid medium containing 50. Mu.g/mL kanamycin resistance, and shaken at 37℃for 200rpm overnight. After culturing, plasmid vector (shown in FIG. 4) was extracted by using plasmid extraction kit (Tiangen plasmid extraction kit, cat# DP 103), and purified and then sent to sequencing company for sequencing. And if the sequencing result is correct, storing the obtained product in a low-temperature refrigerator at-80 ℃ for standby.

Example 6: application of high-activity streptomyces clavuligerus deacetylase C synthetase mutant in improving cephalosporin C yield in acremonium chrysogenum

1) Construction of Gene overexpression plasmid vector pBARGPE1-ScDAOCS-G13571-56

The heterologous overexpression of the streptomyces flavus strain with the streptomyces flavus deacetylatus C synthetase mutant G13571-56 is shown in figure 5, and a schematic diagram of the recombinant plasmid pBARGPE1-ScDAOCS-G13571-56 is constructed.

The method comprises the following specific steps:

(1) plasmid pET28-G13571-56 was extracted as template DNA using bacterial plasmid extraction kit.

(2) Using the cDNA obtained in the step (1) as a template, carrying out PCR amplification by using PrimeSTAR high-fidelity enzyme and a primer G13571-56-F/G13571-56-R, and carrying out sequencing verification on PCR amplification products, wherein the primer G13571-56-F has the sequence: 5' -GGTTCCATGGACACGACGGTGCCCACCTTC-3' (SEQ ID NO.13 underlined is the recognition site for the restriction enzyme BamHI) and primer G13571-56-R sequence: 5' -GGGCCCCTAGGCCTTGGAGGTGCGGCGGAT-3' (SEQ ID NO.14, underlined is the recognition site for the restriction enzyme ApaI) to obtain a PCR amplified cDNA product of about 948 bp.

(3) And (3) connecting the PCR amplification product obtained in the step (2) with a vector pEASY-Blunt to obtain a recombinant plasmid pEASY-ScDAOCS-G13571-56.

(4) The recombinant plasmid pEASY-ScDAOCS-G13571-56 was digested with the restriction enzymes BamHI and ApaI, and a DNA fragment ScDAOCS-G13571-56 of about 948bp was recovered.

(5) The plasmid pBARGPE1-Hygro was digested with the restriction enzymes BamHI and ApaI to recover a vector backbone of about 5936 bp.

(6) The DNA fragment ScDAOCS-G13571-56 was ligated to the vector backbone to obtain the recombinant plasmid pBARGPE 1-ScDAOCS-G13571-56. The recombinant plasmid pBARGPE1-ScDAOCS-G13571-56 was sequenced. Based on the sequencing results, the structure of recombinant plasmid pBARGPE1-ScDAOCS-G13571-56 is described as follows: the small fragment between restriction enzymes BamHI and ApaI of plasmid pBARGPE1-Hygro is replaced by a DNA molecule shown as a sequence SEQ ID No.11 of a sequence table, so as to construct a recombinant plasmid pBARGPE1-ScDAOCS-G13571-56 for over-expression of a Streptomyces clavuligerus desacetyloxy cephalosporin C synthetase mutant in Acremonium chrysogenum. The recombinant plasmid pBARGPE1-ScDAOCS-G13571-56 has gpdA promoter, codon optimized streptomyces clavuligerus deacetylated oxy cephalosporin C synthetase mutant G13571-56 gene, trpC terminator and hygromycin resistance gene.

2) Culture of Cephalosporium chrysogenum ATCC11550 mycelium and protoplast preparation

(1) Scraping appropriate amount of Cephalosporium chrysogenum ATCC11550 spores from the culture slant, respectively inoculating into 100mL YPS liquid medium (glucose 2%, yeast extract 0.5%, polypeptone 1%, mgSO) ₄ ·7H ₂ O 0.1％，K ₂ HPO ₄ ·3H ₂ O0.13%, ph=7.0), 28 ℃, 230rpm shaking culture for 4-5 d;

(2) centrifuging at 8000rpm for 15min, collecting mycelium, and washing with sterile water once;

(3) 50mL of dithiothreitol (DTT, 5 mmol/L) solution is filtered by a sterile filter membrane with the thickness of 0.22 mu m, bacterial cells are resuspended, and the bacterial cells are incubated for 40 to 60 minutes at 30 ℃ under 150rpm oscillation;

(4) centrifuging at 8000rpm for 5min, and adding P Buffer (KCl 44.7g/L, mgCl) ₂ ·6H ₂ O 2.03g/L，CaCl ₂ 2.78 g/L) for 2 times at normal temperature;

(5) 60mL of lying enzymolysis liquid (prepared by P Buffer, 10 mg/mL) is filtered by a sterile filter membrane with the thickness of 0.22 mu m, bacterial cells are resuspended, and the bacterial cells are incubated for 3 to 4 hours at 30 ℃ under shaking at 150 rpm;

(6) microscopic examination, adding 4 times of PBuffer after most mycelia release protoplast, filtering with sterilized cylinder filled with absorbent cotton to remove residual mycelia;

(7) centrifuging at 3000rpm for 5min, washing with PBufer for 2 times, suspending protoplast in appropriate amount of PBufer to make protoplast concentration>10 ⁸ CFU；

(8) Protoplasts of Cephalosporium chrysogenum ATCC11550 were dispensed into 1.5mL centrifuge tubes, 100. Mu.L per tube.

3) Cephalosporium chrysogenum protoplast transformation and identification

PEG-CaCl is adopted ₂ The constructed plasmid is transformed into Cephalosporium chrysosporium by a mediated protoplast transformation method, and the experimental steps are as follows:

(1) 10 μg of recombinant plasmid pBARGPE1-ScDAOCS-G13571-56 of DNA is added into the protoplast, and the mixture is gently mixed and ice-bathed for 30min;

(2) add 900. Mu.L 30% PEG4000/CaCl ₂ Solution (weighing CaCl) ₂ ·2H ₂ Dissolving 14.7g of O and 90mL of pure water in a 100mL volumetric flask, adding 30g of PEG4000, then fixing the volume to 100mL by using pure water, filtering, sterilizing, sub-packaging and storing to 4 ℃, and incubating for 15min at 25 ℃;

(3) 6000rpm×5min, sucking out PEG4000/CaCl as much as possible ₂ The solution was washed 1 time with PBuffer;

(4) using 100 μl of PBuffer to resuspend protoplast, adding into upper soft agar culture medium (peptone, 1%, naCl,0.5%, yeast extract, 0.3%, sucrose, 2%, agar, 0.75%, natural pH) kept at 45 ℃ and gently shaking and mixing on a vortex shaker, then pouring into regenerated agar plate (soluble starch 2.4%, glycine 0.12%, polypeptone 0.4%, ammonium sulfate 0.6%, monopotassium phosphate 0.012%, calcium sulfate 0.8%, magnesium sulfate 0.06%, yeast extract 0.03%, agar 2%, ph=8.5, subpackaging to 500mL conical flask according to 250mL volume, sterilizing for 15min at 121 ℃, cooling for standby), rapidly rotating the plate to make soft agar uniformly cover the surface of lower medium; culturing at 28deg.C for 36h, covering with soft NaCl agar (NaCl, 4%; agar, 0.75%) containing hygromycin to give final hygromycin concentration of 5.0 μg/mL in the plate, solidifying the soft NaCl agar, and culturing at 28deg.C; (5) after 7d of culture, the strains are respectively selected for carrying out bevel culture on bleomycin resistance transformants, and after 7d, genomic DNA is extracted for carrying out PCR verification, so that the recombinant plasmid is successfully transformed.

4) Verification of yield increase effect of CPC of A. Chrysogenum by overexpression of high-activity streptomyces clavuligerus deacetylase cephalosporin C synthetase mutant gene

The transformed Cephalosporium chrysogenum ATCC11550 protoplast cells are mixed with soft agar and then spread on a regenerated agar medium for growth. According to the growth conditions of the colonies (after 3-5 days of colony growth, a relatively sparse single colony can be seen), a soft agar of NaCl containing hygromycin (final concentration 5.0. Mu.g/ml) was spread on the plate. After antibiotic application, the transformants were incubated at 28℃with daily attention to the presence and growth of the transformants. If the transformant grows well after 10-15 days, it can be picked up on a separate medium containing 5.0. Mu.g/ml hygromycin and incubated at 28℃for 5-7 days. And (3) carrying out shake flask fermentation culture on all the obtained transformants, fermenting for 5 days, and detecting by HPLC to obtain a fermentation result, wherein the CPC fermentation unit is obviously improved, and the CPC yield is up-regulated by the overexpression of the high-activity streptomyces clavuligerus desacetoxycephalosporin C synthetase mutant gene.

5) Fermentation of Cephalosporium chrysogenum and detection of CPC (CPC) as fermentation product

The triangular flask in this example had a 500mL gauge with a straight baffle at the bottom and three shake flasks were repeated for each experimental strain. A proper amount of Cephalosporium chrysogenum spores are scraped from the inclined surface of the culture 10d, a wild-type original strain ATCC11550 is used as a control, a strain containing a transformant is used as an experimental group, wherein the transformant 1 is a Cephalosporium chrysogenum strain ATCC11550-pBRGPE1-Hygro transformed with an empty plasmid pBARGPE1-Hygro, the transformant 2 is a Cephalosporium chrysogenum strain ATCC11550-pBARGPE 1-Hygro transformed with a high-activity Streptomyces clavuligerus deacetylase C synthetase mutant gene overexpression plasmid pBARGPE1-SCDAOCS-G13571-56, the Cephalosporium chrysogenum strain ATCC11550-pBARGPE1-ScDAOCS G13571-56 is respectively inoculated with 30mL seed culture medium (glucose, 5G/L, sucrose, 35G/L, corn steep liquor 10mL/L, ammonium sulfate, 8G/L, DL-methionine, 0.5G/L, calcium carbonate, 5G/L, soybean oil, 5mL/L, pH=7.20+/-0.121 min, and shaking table 35G/L), and shaking and sterilizing at a shaking flask at a rotation speed of 230 m.121 min for further culturing at a rotation speed of about 230 m for a certain time. Transferring to 30mL fermentation medium (corn starch, 30g/L, maltodextrin, 60g/L, alpha-amylase, 0.2g/L, corn steep liquor) with 10% (v/v) inoculum size 10mL/L; DL-methionine, 6g/L; urea, 2g/L; ammonium sulfate, 11g/L; mgSO (MgSO) ₄ ·7H ₂ O，3g/L；K ₂ HPO ₄ 9g/L; calcium carbonate, 5g/L; soybean oil, 10mL/L; ph=7.20±0.05;121 ℃ for 15min sterilization and then cooling for later use), and culturing for 7d at 25 ℃ and 230 rpm. The fermentation broth was filtered through plain filter paper, 0.22 μm, the filtrate was collected, diluted 20-fold and subjected to HPLC detection.

HPLC detection uses an Agilent 1260HPLC detector, a C18 chromatographic column, a column temperature of 40 ℃, a mobile phase of methanol: 0.2% (w/v) sodium dihydrogen phosphate=5:95, a flow rate of 1.0mL/min, a detection wavelength of 254nm, a sample injection amount of 10 mu L and an analysis time of 9min. And calculating the CPC content in the sample according to the peak area and the effective content of the standard substance.

As shown in FIG. 6, the wild-type starting control strain ATCC11550 had CPC yield of 2679.45.+ -. 138.9mg/L, the mutant control strain ATCC11550-pBRGPE1-Hygro transformed with the empty plasmid pBARGPE1-Hygro transformant had CPC yield of 2591.+ -. 214.6mg/L, and the mutant strain ATCC11550-pBARGPE1-ScDAOCS-G13571-56 transformed with the high-activity Streptomyces clavuligerus desacetylcephalosporin C synthase mutant enzyme overexpressing plasmid pBARGPE1-ScDAOCS-G13571-56 had CPC yield of 4357.4.+ -. 231.9mg/L. After statistical analysis of the data, the wild-type control strain ATCC11550 showed no significant difference in CPC yield from the control strain ATCC11550-pBRGPE1-Hygro transformed with the empty plasmid pBRGPE1-Hygro, but showed significant difference (p=0.011) from the experimental mutant strain ATCC11550-pBARGPE1-ScDAOCS-G13571-56 transformed with the high-activity Streptomyces clavuligerus deacetylase mutant gene overexpression plasmid pBARGPE 1-ScDAOCS-G13571-56; the same significant difference (p=0.024) was seen between the control strain ATCC11550-pBRGPE1-Hygro transformed with the empty plasmid pBRGPE1-Hygro and the experimental mutant strain ATCC11550-pBARGPE1-ScDAOCS-G13571-56 transformed with the high activity Streptomyces clavuligerus deacetylase C synthetase mutant gene overexpression plasmid pBARGPE1-ScDAOCS-G13571-56, which improved CPC yield by 68.2%. The experimental results prove that the exogenous high-activity streptomyces clavuligerus deacetylase C synthetase mutant gene is over-expressed in the acremonium chrysogenum ATCC11550, and the improvement of CPC yield can be effectively promoted.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Henan province health Yuan biological medicine institute of Co., ltd

<120> desacetoxycephalosporin C synthetase mutant, encoding gene and application thereof

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<400> 6

atggacacga cggtgcccac cttcagcctg gccgaactcc agcagggcct gcaccaggac 60

gagttccgca ggtgtctgag ggacaagggc ctcttctatc tgacggactg cggtctgacc 120

gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180

gcggagaagc gcgccgtcac ctcgcccgtc cccaccatgc gccgcggctt caccgggctg 240

gagtcggaga gcaccgccca gatcaccaat accnnnagct actccgacta ctcgatgtgc 300

tactcgatgg gcaccgcgga caacctcttc ccgtccggtg acttcgagcg gatctggacc 360

cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgggaggt cctgcgggcg 420

accgggaccg agcccgacgg cggggtcgag gccttcctcg actgcgagcc gctgctgcgg 480

ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540

gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacaccctg cgccaacggc 600

ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgtccggac 660

gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720

ccccggcacc atgtcgcggc cccccgcagg gaccagatag cgggcagcag ccgcacctcc 780

agtgtgttct tcctccgtcc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840

tgcggcttcg atgtcagcct ggacggcgag accgccacgt tccaggattg gatcgggggc 900

aactacgtga acatccgccg cacatccaag gcatag 936

<210> 7

<211> 311

<212> PRT

<213> Artificial

<400> 7

Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly

1 5 10 15

Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe

20 25 30

Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys

35 40 45

Asp Leu Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg

50 55 60

Ala Val Thr Ser Pro Val Pro Xaa Met Arg Arg Gly Phe Thr Gly Leu

65 70 75 80

Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Xaa Ser Tyr Ser Asp

85 90 95

Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser

100 105 110

Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr

115 120 125

Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu

130 135 140

Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg

145 150 155 160

Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln

165 170 175

Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile

180 185 190

Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val

195 200 205

Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val

210 215 220

Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala

225 230 235 240

Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser

245 250 255

Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr

260 265 270

Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp

275 280 285

Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn

290 295 300

Ile Arg Arg Thr Ser Lys Ala

305 310

<210> 8

<211> 936

<212> DNA

<213> Artificial

<400> 8

atggacacga cggtgcccac cttcagcctg gccgaactcc agcagggcct gcaccaggac 60

gagttccgca ggtgtctgag ggacaagggc ctcttctatc tgacggactg cggtctgacc 120

gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180

gcggagaagc gcgccgtcac ctcgcccgtc cccnnnatgc gccgcggctt caccgggctg 240

gagtcggaga gcaccgccca gatcaccaat accnnnagct actccgacta ctcgatgtgc 300

tactcgatgg gcaccgcgga caacctcttc ccgtccggtg acttcgagcg gatctggacc 360

cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgggaggt cctgcgggcg 420

accgggaccg agcccgacgg cggggtcgag gccttcctcg actgcgagcc gctgctgcgg 480

ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540

gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacaccctg cgccaacggc 600

ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgtccggac 660

gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720

ccccggcacc atgtcgcggc cccccgcagg gaccagatag cgggcagcag ccgcacctcc 780

agtgtgttct tcctccgtcc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840

tgcggcttcg atgtcagcct ggacggcgag accgccacgt tccaggattg gatcgggggc 900

aactacgtga acatccgccg cacatccaag gcatag 936

<210> 9

<211> 948

<212> DNA

<213> Artificial

<400> 9

catatggaca cgacggtgcc caccttcagc ctggccgaac tccagcaggg cctgcaccag 60

gacgagttcc gcaggtgtct gagggacaag ggcctcttct atctgacgga ctgcggtctg 120

accgacaccg agctgaagtc ggccaaggac ctcgtcatcg acttcttcga gcacggcagc 180

gaggcggaga agcgcgccgt cacctcgccc gtccccacca tgcgccgcgg cttcaccggg 240

ctggagtcgg agagcaccgc ccagatcacc aataccggca gctactccga ctactcgatg 300

tgctactcga tgggcaccgc ggacaacctc ttcccgtccg gtgacttcga gcggatctgg 360

acccagtact tcgaccgcca gtacaccgcc tcccgcgcgg tcgcccggga ggtcctgcgg 420

gcgaccggga ccgagcccga cggcggggtc gaggccttcc tcgactgcga gccgctgctg 480

cggttccgct acttcccgca ggtccccgag caccgcagcg ccgaggagca gcccctgcgg 540

atggcgccgc actacgacct gtcgatggtc accctcatcc agcagacacc ctgcgccaac 600

ggcttcgtca gcctccaggc cgaggtcggc ggcgcgttca cggacctgcc ctaccgtccg 660

gacgccgtcc tcgtcttctg cggcgccatc gcgaccctgg tgaccggcgg ccaggtcaag 720

gccccccggc accatgtcgc ggccccccgc agggaccaga tagcgggcag cagccgcacc 780

tccagtgtgt tcttcctccg tcccaacgcg gacttcacct tctccgtccc gctggcgcgc 840

gagtgcggct tcgatgtcag cctggacggc gagaccgcca cgttccagga ttggatcggg 900

ggcaactacg tgaacatccg ccgcacatcc aaggcatagt aaaagctt 948

<210> 10

<211> 948

<212> DNA

<213> Artificial

<400> 10

catatggaca cgacggtgcc caccttcagc ctggccgaac tccagcaggg cctgcaccag 60

gacgagttcc gcaggtgtct gagggacaag ggcctcttct atctgacgga ctgcggtctg 120

accgacaccg agctgaagtc ggccaaggac ctcgtcatcg acttcttcga gcacggcagc 180

gaggcggaga agcgcgccgt cacctcgccc gtccccnnna tgcgccgcgg cttcaccggg 240

ctggagtcgg agagcaccgc ccagatcacc aataccnnna gctactccga ctactcgatg 300

tgctactcga tgggcaccgc ggacaacctc ttcccgtccg gtgacttcga gcggatctgg 360

acccagtact tcgaccgcca gtacaccgcc tcccgcgcgg tcgcccggga ggtcctgcgg 420

gcgaccggga ccgagcccga cggcggggtc gaggccttcc tcgactgcga gccgctgctg 480

cggttccgct acttcccgca ggtccccgag caccgcagcg ccgaggagca gcccctgcgg 540

atggcgccgc actacgacct gtcgatggtc accctcatcc agcagacacc ctgcgccaac 600

ggcttcgtca gcctccaggc cgaggtcggc ggcgcgttca cggacctgcc ctaccgtccg 660

gacgccgtcc tcgtcttctg cggcgccatc gcgaccctgg tgaccggcgg ccaggtcaag 720

gccccccggc accatgtcgc ggccccccgc agggaccaga tagcgggcag cagccgcacc 780

tccagtgtgt tcttcctccg tcccaacgcg gacttcacct tctccgtccc gctggcgcgc 840

gagtgcggct tcgatgtcag cctggacggc gagaccgcca cgttccagga ttggatcggg 900

ggcaactacg tgaacatccg ccgcacatcc aaggcatagt aaaagctt 948

<210> 11

<211> 936

<212> DNA

<213> Artificial

<400> 11

atggacacga cggtgcccac cttcagcctg gccgagctcc agcagggcct gcaccaggac 60

gagttccgca ggtgcctgag ggacaagggc ctcttctacc tgacggactg cggcctgacc 120

gacaccgagc tgaagtcggc caaggacctc gtcatcgact tcttcgagca cggcagcgag 180

gcggagaagc gcgccgtcac ctcgcccgtc ccctggatgc gccgcggctt caccggcctg 240

gagtcggaga gcaccgccca gatcaccaac accatgagct actccgacta ctcgatgtgc 300

tactcgatgg gcaccgcgga caacctcttc ccgtccggcg acttcgagcg catctggacc 360

cagtacttcg accgccagta caccgcctcc cgcgcggtcg cccgcgaggt cctgcgcgcg 420

accggcaccg agcccgacgg cggcgtcgag gccttcctcg actgcgagcc gctgctgcgg 480

ttccgctact tcccgcaggt ccccgagcac cgcagcgccg aggagcagcc cctgcggatg 540

gcgccgcact acgacctgtc gatggtcacc ctcatccagc agacgccctg cgccaacggc 600

ttcgtcagcc tccaggccga ggtcggcggc gcgttcacgg acctgcccta ccgcccggac 660

gccgtcctcg tcttctgcgg cgccatcgcg accctggtga ccggcggcca ggtcaaggcc 720

ccccgccacc acgtcgcggc cccccgcagg gaccagatcg cgggcagcag ccgcacctcc 780

agcgtgttct tcctccgccc caacgcggac ttcaccttct ccgtcccgct ggcgcgcgag 840

tgcggcttcg acgtcagcct ggacggcgag accgccacgt tccaggattg gatcggcggc 900

aactacgtga acatccgccg cacctccaag gcctag 936

<210> 12

<211> 948

<212> DNA

<213> Artificial

<400> 12

catatggaca cgacggtgcc caccttcagc ctggccgagc tccagcaggg cctgcaccag 60

gacgagttcc gcaggtgcct gagggacaag ggcctcttct acctgacgga ctgcggcctg 120

accgacaccg agctgaagtc ggccaaggac ctcgtcatcg acttcttcga gcacggcagc 180

gaggcggaga agcgcgccgt cacctcgccc gtcccctgga tgcgccgcgg cttcaccggc 240

ctggagtcgg agagcaccgc ccagatcacc aacaccatga gctactccga ctactcgatg 300

tgctactcga tgggcaccgc ggacaacctc ttcccgtccg gcgacttcga gcgcatctgg 360

acccagtact tcgaccgcca gtacaccgcc tcccgcgcgg tcgcccgcga ggtcctgcgc 420

gcgaccggca ccgagcccga cggcggcgtc gaggccttcc tcgactgcga gccgctgctg 480

cggttccgct acttcccgca ggtccccgag caccgcagcg ccgaggagca gcccctgcgg 540

atggcgccgc actacgacct gtcgatggtc accctcatcc agcagacgcc ctgcgccaac 600

ggcttcgtca gcctccaggc cgaggtcggc ggcgcgttca cggacctgcc ctaccgcccg 660

gacgccgtcc tcgtcttctg cggcgccatc gcgaccctgg tgaccggcgg ccaggtcaag 720

gccccccgcc accacgtcgc ggccccccgc agggaccaga tcgcgggcag cagccgcacc 780

tccagcgtgt tcttcctccg ccccaacgcg gacttcacct tctccgtccc gctggcgcgc 840

gagtgcggct tcgacgtcag cctggacggc gagaccgcca cgttccagga ttggatcggc 900

ggcaactacg tgaacatccg ccgcacctcc aaggcctagt aaaagctt 948

<210> 13

<211> 30

<212> DNA

<213> Artificial

<400> 13

ggttccatgg acacgacggt gcccaccttc 30

<210> 14

<211> 30

<212> DNA

<213> Artificial

<400> 14

gggcccctag gccttggagg tgcggcggat 30

Claims

1. The desacetoxycephalosporin C synthetase mutant is characterized in that the mutant is based on SEQ ID NO. 1, single mutation is carried out on glycine G92 at 92 th position, and the glycine at 92 th position is mutated into methionine, cysteine, serine, asparagine, glutamine or histidine;

Or, double mutation of threonine T72 at position 72 and glycine G92 at position 92, the double mutation selected from the group consisting of:

(1) Mutation of T at position 72 to D and G at position 92 to A;

(2) Mutation of T at position 72 to D and G at position 92 to C;

(3) Mutation of T at position 72 to D and G at position 92 to H;

(4) The T at position 72 is mutated to G, and the G at position 92 is mutated to C;

(5) The T at position 72 is mutated to G, and the G at position 92 is mutated to M;

(6) The T at position 72 is mutated to G, and the G at position 92 is mutated to S;

(7) T at position 72 is mutated to I and G at position 92 is mutated to M;

(8) Mutation of T at position 72 to N and G at position 92 to M;

(9) Mutation of T at position 72 to Q and G at position 92 to C;

(10) Mutation of T at position 72 to Q and G at position 92 to H;

(11) Mutation of T at position 72 to Q and G at position 92 to M;

(12) Mutation of T at position 72 to Q and G at position 92 to S;

(13) Mutation of T at position 72 to W and G at position 92 to C;

(14) Mutation of T at position 72 to W and G at position 92 to F;

(15) Mutation of T at position 72 to W and G at position 92 to H;

(16) Mutation of T at position 72 to W and G at position 92 to M;

(17) Mutation of T at position 72 to W and G at position 92 to N;

(18) Mutation of T at position 72 to W and G at position 92 to Q;

(19) Mutation of T at position 72 to W and mutation of G at position 92 to S;

or alternatively, the first and second heat exchangers may be,

(20) T at position 72 is mutated to W and G at position 92 is mutated to T.

2. A nucleic acid encoding the desacetylcephalosporin C synthetase mutant of claim 1.

3. A biological material, characterized in that it is an expression vector comprising the nucleic acid of claim 2.

4. A biological material, characterized in that it is a host cell comprising the expression vector of claim 3.

5. Use of a mutant desacetylcephalosporin C synthase according to claim 1, a nucleic acid according to claim 2, or a biomaterial according to claim 3 or 4 for the preparation of 7-phenylacetyl desacetylcephalosporin.

6. The use according to claim 5, wherein 7-phenylacetyl-desacetyloxy-cephalosporin is prepared using penicillin G as substrate by using a desacetyloxy-cephalosporin C synthetase mutant.

7. Use of a codon optimized gene encoding a mutant desacetyloxy cephalosporin C synthetase of claim 1 for heterologous expression in A.chrysogenum to increase cephalosporin C production.

8. The use according to claim 7, wherein the mutant desacetoxycephalosporin C synthetase uses penicillin G as substrate.