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Construction method for pichia pastoris bacterial strain of high-yield S-ademetionine

A technology for adenosylmethionine and construction methods, applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc.

Inactive Publication Date: 2014-09-10
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

my country is a big country where hepatitis is prevalent, but there is no biological drug that can really improve liver cell metabolism in my country

Method used

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  • Construction method for pichia pastoris bacterial strain of high-yield S-ademetionine
  • Construction method for pichia pastoris bacterial strain of high-yield S-ademetionine
  • Construction method for pichia pastoris bacterial strain of high-yield S-ademetionine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Construction of recombinant expression vector pPIC9K-sam2

[0029] The gene sam2 was amplified from the S. cerevisiae genome using primers sam2-F: 5'-CAGGATCCACCATGACCAAGAGCAAAACT-3'; EcoR I and Bam H I respectively double-digested the PCR amplification product and pPIC9K plasmid DNA, recovered the target band by agarose gel electrophoresis, and used T 4 DNA ligase ligated the recovered target bands to obtain the eukaryotic expression vector pPIC9K-sam2, and used CaCl 2 Transform it into Escherichia coli DH5α by heat shock at 42°C for 90 seconds. Single colonies were screened for Kana resistance. The selected transformed clones were identified by PCR and enzyme digestion to prove that the clones were correct and then sent to Dalian Bao Biological Company for sequencing. For specific operations, see figure 1 .

Embodiment 2

[0030] Example 2. Obtaining of cbs gene knockout plasmid T-C-Z and fragment C-Z

[0031] Using primers cbs-F: 5'-TTCTGGAGCACATTGGAA-3'; cbs-R: 5'-AGTGTATGCCTAG ATGG-3', the cbs gene was amplified from the Pichia pastoris genome, and then subcloned into the vector pMD19T to obtain a recombinant vector T-cbs. Using primers zeocin-F: 5'-GGACTAGTAGACCTTCGTTTGTGC-3'; zeocin-R: 5'-GGACTAGTCGGTTCCTGGCCTTTTG-3' to amplify the zeocin gene from pPICZα-A, use Speech I digest the PCR product zeocin gene and the recombinant plasmid T-cbs respectively, and use T 4 DNA ligase ligated the recovered fragments to obtain the cbs gene knockout plasmid T-C-Z. After successful identification by PCR and enzyme digestion, it was sent to Dalian Bao Biological Company for sequencing. After successful sequencing, use Sal I and Bgl II double-digested the recombinant plasmid T-C-Z, recovered a 2100bp fragment from rubber tapping, and named it C-Z. See the specific operation process figure...

Embodiment 3

[0032] Example 3. Transformation and screening of recombinant Pichia pastoris

[0033] The identified correct recombinant expression vector pPIC9K-sam2 plasmid DNA was subjected to endonuclease Bpu1102I linearization treatment, electroporation transformation of Pichia pastoris GS115. Immediately after the electric shock, 1ml of pre-cooled 1mol / l sorbitol was added, centrifuged at 3000rpm for 5min, the bacteria were resuspended in 400μl of pre-cooled 1mol / l sorbitol, and 200μl was spread on MD plates (1.34% YNB, 4×10 -5 % biotin, 2% glucose, 2% agarose) at 30°C until colonies appeared. Randomly pick colonies and inoculate them on YPD plates containing different concentrations of -418 (0, 0.50mg / ml, 1.00mg / ml, 2.00mg / ml, 3.00mg / ml, 4.00mg / ml), and culture them at 30°C for 2-5 days. , and check the colony growth every day. The positive clone transformant ZJGSU01 with high resistance to -418 was quickly screened out according to the growth conditions.

[0034] Transform the...

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Abstract

The invention discloses a method for constructing a high-yield S-adenosylmethionine Pichia pastoris strain, which is characterized in that it comprises the following steps: PCR amplifying the S-adenosylmethionine synthetase gene sam2 from the Saccharomyces cerevisiae genome , connected to the plasmid pPIC9K to obtain the eukaryotic expression vector pPIC9K-sam2, which was then transformed into Pichia pastoris; the cystathionine β synthase gene cbs was amplified from the Pichia pastoris genome by PCR, and its sub Cloned into the vector pMD19T to obtain the recombinant vector T-cbs; the zeocin resistance gene zeocin was introduced into the cystathionine β synthase gene of the recombinant vector T-cbs to obtain the cbs gene knockout plasmid T-C-Z, and cbs was obtained after digestion Gene knockout fragment C-Z; transform fragment C-Z into the Pichia pastoris containing pPIC9K-sam2 obtained in step 1 to obtain high-production S-adenosylmethionine Pichia pastoris that knocks out the cbs gene and strengthens the sam2 gene strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a method for constructing a high-yield S-adenosylmethionine Pichia pastoris strain, and optimizes the process of fermenting and producing S-adenosylmethionine from the strain. S-adenosylmethionine The output of methionine can reach 4.37 g / L, reaching the level of industrial production. Background technique [0002] The English name of S-adenosylmethionine is S-adenosylmethionine, referred to as SAM, and the chemical name is 5'-[[(3S)-3-amino-propyl]methyl-(S)-sulfone]-5'-deoxyadenosine , formula C 15 h 22 N 6 o 5 S, the molecular weight is 399. [0003] S-adenosylmethionine is an important physiologically active substance that widely exists in organisms. It has the functions of transmethylation, transthiol, transaminopropyl, etc., and participates in more than 40 biochemical reactions in the body. It interacts with proteins, nucleic acids, nerves, etc. The synthesis of tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/54C12N15/81C12N15/09C12P19/40C12R1/84
Inventor 于平
Owner ZHEJIANG GONGSHANG UNIVERSITY
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