CN-122012544-A - Gene for improving yield of Pichia pastoris S-adenosylmethionine (SAM) and application thereof

Abstract

The invention relates to the technical field of microbial metabolism engineering, in particular to a gene for improving the yield of Pichia pastoris S-adenosylmethionine (SAM) and application thereof. The invention screens and obtains candidate genes related to SAM synthesis and energy metabolism by carrying out transcriptome sequencing analysis on the fermentation process of pichia pastoris, and further determines PAS_chr3_0066, PAS_chr1-1_0233 and PAS_chr1-1_0389 as target genes. Experimental results show that compared with the control strain, SAM yield in the strain respectively overexpressing the three genes is obviously improved. The invention screens and obtains a novel key target spot capable of regulating and controlling the synthesis of the SAM of pichia pastoris, and provides a theoretical basis for constructing engineering strains with high SAM yield.

Inventors

• HUANG MINGZHI
• SHANG YUANYUAN
• WU JIAMING
• Tang Zhenqian
• ZHU BOHUA
• XU FENG

Assignees

• 华东理工大学

Dates

Publication Date

20260512

Application Date

20260410

Claims (6)

1. 1. A gene for improving the yield of Pichia pastoris S-adenosylmethionine (SAM) is characterized in that the gene is one or more of PAS_chr3_0066, PAS_chr1-1_0233 and PAS_chr1-1_0389, and the nucleotide sequences of the genes are shown as SEQ ID NO. 1-3 in sequence.
2. 2. The gene according to claim 1, wherein the gene is derived from Pichia pastoris (Pichia pastoris), and the gene encodes Methionine Sulfoxide Reductase (MSR), isocitrate Dehydrogenase (IDP), argininosuccinate lyase (ASL), respectively.
3. 3. Use of the gene according to claim 1 or 2 for the preparation of recombinant pichia pastoris for the production of S-adenosylmethionine.
4. 4. A recombinant expression vector for increasing production of pichia pastoris S-adenosylmethionine, wherein the vector comprises one or more of the genes of claim 1.
5. 5. An engineered strain, characterized in that the engineered strain is pichia pastoris transformed with the recombinant expression vector of claim 4.
6. 6. The engineering strain according to claim 5, wherein the pichia pastoris is obtained by further genetic engineering with pichia pastoris G12'/AOX-acs2 as an original strain.

Description

Gene for improving yield of Pichia pastoris S-adenosylmethionine (SAM) and application thereof Technical Field The invention belongs to the technical field of microbial metabolism engineering, and particularly relates to a gene for improving the yield of Pichia pastoris S-adenosylmethionine (SAM) and application thereof. Background S-adenosylmethionine (S-Adenosyl-L-methionine, SAM) is an important methyl donor in organisms, and widely participates in key metabolic processes such as methyl conversion, thio conversion, polyamine synthesis and the like, plays a positive role in protecting liver, resisting depression, bone joint health and the like, and has wide market prospect in the fields of medicines, health foods and the like. At present, SAM industrial production is mainly based on a microbial fermentation method, and Pichia pastoris (Pichia pastoris) is taken as an excellent eukaryotic expression host, has the advantages of high-density fermentation potential, strong and strict regulation and control containing promoters (such as pAOX 1), stable integration of exogenous genes, perfect post-translational modification system and the like, and is considered as one of the most potential industrial production hosts. However, the synthesis of SAM in Pichia pastoris relies on the condensation of L-Met with ATP catalyzed by SAM synthase, and under conditions where SAM synthase and L-Met are sufficiently supplied, energy supply becomes a key bottleneck limiting efficient synthesis of SAM. Therefore, how to alleviate the energy limitation in pichia pastoris to achieve efficient enhancement of SAM synthesis is a core problem to be solved currently. In order to break through the bottleneck, the existing research focuses on metabolic engineering modification of known energy metabolism related targets, but the genetic attention indirectly related to energy metabolism but having potential important regulation and control effects on SAM synthesis is insufficient, so that the possibility of systematically improving SAM synthesis capacity is limited to a certain extent. Therefore, by means of the methods of transcriptome and other systematic biology, the gene expression change in the fermentation process of pichia pastoris is systematically analyzed, and potential recessive regulatory genes related to SAM synthesis and energy metabolism are excavated, so that new gene targets and breakthrough directions are provided for metabolic engineering of SAM biosynthesis. Disclosure of Invention The invention aims to solve the problem of insufficient energy supply in the production process of S-adenosylmethionine by pichia pastoris, and provides a key gene capable of effectively improving the SAM yield of pichia pastoris and application thereof. In order to achieve the above object, the present invention provides genes of Pichia pastoris genes with accession numbers of any one or more of PAS_chr3_0066, PAS_chr1-1_0233 and PAS_chr1-1_0389, respectively, in improving the yield of Pichia pastoris S-adenosylmethionine. The invention also provides a gene over-expression vector which can be used for improving the yield of the Pichia pastoris S-adenosylmethionine, and the vector can over-express any one or more than two of Pichia pastoris genes PAS_chr3_0066, PAS_chr1-1_023 and PAS_chr1-1_0389 in Pichia pastoris respectively. The invention also provides a gene overexpression strain which can be used for improving the yield of the Pichia pastoris S-adenosylmethionine, and the overexpression strain can overexpress any one or more than two of genes PAS_chr3_0066, PAS_chr1-1_0233 and PAS_chr1-1_0389. Preferably, the above-mentioned over-expression strain is selected from Pichia pastoris, preferably from Pichia pastoris G12'/AOX-acs2. The over-expression strain provided by the invention can be used for producing S-adenosylmethionine. Preferably, the over-expression bacteria can be used for improving the yield of S-adenosylmethionine in the industrial production of pichia pastoris. The pichia pastoris gene provided by the invention has the following advantages that 1, a novel gene target point is provided, and the key regulation and control effects of three genes PAS_chr3_0066, PAS_chr1-1_0233 and PAS_chr1-1_0389 in the synthesis of pichia pastoris SAM are screened and verified for the first time through transcriptomic analysis, so that a novel strategy is provided for metabolic engineering modification. 2. And the SAM yield is obviously improved, namely, the Pichia pastoris engineering strain with obviously improved SAM yield is successfully constructed by respectively over-expressing the genes. Compared with a control strain, SAM accumulation level of the over-expression strain is effectively improved, SAM yield is improved by 39.60%,50.65% and 56.71% respectively by the over-expression PAS_chr3_0066 strain, the over-expression PAS_chr1-1_0233 strain and the over-expression PAS_chr1-1_0389 strain compared with the original Pichia pastoris st