Oxymethyltransferase mutant and application thereof in production of prodigiosin

An oxymethyltransferase, enzyme mutant technology, applied in transferase, microorganism-based methods, applications, etc., can solve the problems of hindering the industrialization process of microbial fermentation method and low yield.

Active Publication Date: 2020-09-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there are still certain defects in the existing microbial fermentation method, among which, the low yield is the most important defect hindering the industrialization process of the microbial fermentation method

Method used

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  • Oxymethyltransferase mutant and application thereof in production of prodigiosin
  • Oxymethyltransferase mutant and application thereof in production of prodigiosin
  • Oxymethyltransferase mutant and application thereof in production of prodigiosin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of Oxymethyltransferase Mutants

[0049] Specific steps are as follows:

[0050] 1. Construction of recombinant Escherichia coli

[0051] With the genome of Serratia marcescens JNB5-1 as a template, the gene pigF encoding oxymethyltransferase PigF was amplified by PCR (the amino acid sequence of oxymethyltransferase PigF is shown in SEQ ID No.1, encoding oxygen The nucleotide sequence of the gene pigF of the methyltransferase PigF is shown in SEQ ID No.2); The gene pigF of the obtained coding oxygen methyltransferase PigF and pET-28a after EcoR I / HindIII double digestion The plasmids were ligated to obtain the ligated product; the ligated product was transformed into Escherichia coli BL21 to obtain the transformed product; the transformed product was spread on LB solid medium (containing 50 μg·mL -1 Kanamycin), cultured upside down in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants; pick transformants and inoculat...

Embodiment 2

[0091] Embodiment 2: the production of prodigiosin

[0092] Specific steps are as follows:

[0093] 1. Construction of recombinant Serratia marcescens

[0094] Using the genome of Serratia marcescens JNB5-1 as a template, the upstream fragment pigFU (SEQ ID NO: 13) of the gene pigF encoding oxymethyltransferase and the gene encoding oxymethyltransferase were respectively obtained by PCR amplification The downstream fragment of pigF, pigFD (SEQ ID NO: 14); with the recombinant plasmid pET28a-S53C PigF As a template, the mutant 53C encoding oxymethyltransferase was obtained by PCR amplification PigF Apramycin resistance gene fragment Apm (pigF) (SEQ ID NO: 15) was chemically synthesized; the upstream fragment pigFU and the mutant 53C encoding oxygen methyltransferase were sequentially synthesized by overlap extension PCR PigF The gene, the apramycin resistance gene fragment Apm (pigF) and the downstream fragment pigFD were connected to obtain the gene fragment; the gene fragm...

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Abstract

The invention discloses an oxymethyltransferase mutant and application thereof in production of prodigiosin, and belongs to the technical field of biology. The invention provides serratia marcescens engineering bacteria JNB5-1 / S53CPigF and JNB5-1 / G176C / M245C / S53CPigF capable of realizing high yield of prodigiosin. The serratia marcescens engineering bacteria S53CPigF and JNB5-1 / G176C / M245C / S53CPigF are used for expressing the S53CPigF and the G176C / M245C / S53CPigF respectively; and the serratia marcescens engineering bacteria JNB5-1 / S53CPigF and JNB5-1 / G176C / M245C / S53CPigF are respectively inoculated into a fermentation culture medium to be fermented for 96 hours, and the yield of prodigiosin in the fermentation liquor can be respectively up to 7.24 g / L and 7.02 g / L.

Description

technical field [0001] The invention relates to an oxygen methyltransferase mutant and its application in the production of prodigiosin, belonging to the field of biotechnology. Background technique [0002] Prodigiosin is a kind of natural red pigment containing tripyrrole skeleton structure, which is mainly produced by some microorganisms. It has various biological activities such as anti-cancer, immunosuppression, and anti-insect. extensive attention of researchers. [0003] At present, the methods for producing prodigiosin mainly include chemical synthesis and microbial fermentation. Among them, the chemical synthesis method mainly obtains prodigiosin through tandem conjugate addition and high-temperature dehydrogenation. However, due to complex and difficult pathways and low yields, chemical synthesis is difficult to achieve large-scale industrial production. The principle of microbial fermentation is mainly to obtain prodigiosin through microbial fermentation. Comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/74C12N1/21C12P17/16C12R1/425
CPCC12N9/1007C12P17/165
Inventor 饶志明孙杨杨套伟徐美娟张显邵明龙付维来易敢峰
Owner JIANGNAN UNIV
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