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Method for eliminating dependence of methanol-induced promoter on single methanol carbon source

一种启动子、诱导型的技术，应用在生物工程领域，能够解决工业化生产麻烦、难满足氧气的需求、冷却能力要求越高等问题

Active Publication Date: 2012-10-31
CHINA RESOURCES BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the wider and wider scope of use, many problems are encountered in the actual fermentation amplification process: (1) the promoter used for expression needs to be induced by methanol, methanol is toxic and flammable, and special special measures are required in large-scale industrial fermentation. Anti-riot design; (2) Methanol fermentation consumes a lot of oxygen. Generally, it is difficult to meet the demand for oxygen by increasing the ventilation volume of air and increasing the rotation speed, so pure oxygen is needed. The amount of oxygen required for methanol metabolism is the amount of oxygen required for glucose as a carbon source three to four times of
The more methanol consumed, the more pure oxygen is needed, which brings great trouble to the actual industrial production
In addition, the more methanol consumed, the greater the heat produced, and the higher the cooling capacity requirements of the required equipment; (3) methanol, as a petrochemical product, is not suitable for the production of some food additives; (4) methanol metabolism will produce H 2 o 2 , leading to hydrolysis of the expressed polypeptide

Method used

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  • Method for eliminating dependence of methanol-induced promoter on single methanol carbon source
  • Method for eliminating dependence of methanol-induced promoter on single methanol carbon source
  • Method for eliminating dependence of methanol-induced promoter on single methanol carbon source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1, Pichia pastoris strain GS115-MIT1 that glycerol can induce the expression of AOX1 promoter

[0110] 1. Construction of PpMIT1 overexpression plasmid

[0111] Using PCR, the method of restriction restriction ligation, with pGAPZαA ( figure 1 ) is a vector that inserts the PpMIT1 gene (SEQ ID NO: 1, the full length of the gene is 2667bp) at the Asu II / Sal I restriction sites downstream of the GAP promoter, and the resulting recombinant plasmid is called pGM plasmid.

[0112] Using M1-GAP5 and M1-AOX1TT as primers (Table 1), the expression system of pGAP-PpMIT1-AOX1TT (full length 3615bp) was amplified from the pGM plasmid by PCR method, and pAG32( figure 2 ) is the vector inserted into the expression system at the Sac I / Spe I place. The obtained recombinant plasmid was pGMhph.

[0113] Table 1. Primer List

[0114]

[0115]

[0116] 2. Screening of electroporated Pichia pastoris and GS115-MIT1 strain

[0117] The PpMIT1 overexpression plasmid (pGMhp...

Embodiment 2

[0134] Example 2, Glycerol or Glucose Inducible AOX1 Promoter Expression Pichia strain

[0135] 1. Construction of HXS1 gene knockout plasmid

[0136] The Sh ble fragment of the Zeocin resistance gene was excised from the plasmid pGAPZαA with BamH I and Sal I enzymes, and converted to the pUC18 plasmid ( Figure 10 ) as a vector, the Zeocin resistance gene Sh ble fragment was inserted into the BamH I and Sal I enzyme-cut ligation, named pUC18-ble plasmid. Using the GS115 genome as a template, primers HS1-5F / HS1-5R were first used to amplify the 5' peripheral promoter region of the PpHXS1 gene (upstream of the start codon ATG), and the size of the amplified product was 728bp. Primers HS1-3F / HS1-3R were used to amplify the 3' end region of the PpHXS1 gene (downstream of the stop codon). The size of the amplified product was 1011 bp, and the fragments of the target size were recovered by cutting the gel separately.

[0137] The peripheral promoter DNA fragment at the 5' end of ...

Embodiment 5

[0155] Example 5. Pichia pastoris strain Δmig1ΔmigΔnrg1-MIT1 in which expression of AOX1 promoter can be induced by glycerol or glucose

[0156] 1. Construction of NRG1 gene knockout plasmid

[0157] Using the GS115 genome as a template, the primer 5'NRG1-F / 5'NRG1-R was used to amplify the 5' end peripheral region of the PpNIG1 gene to obtain a 320bp fragment, and the amplified product was named 5'NRG1, and the gel recovered 5'NRG1 fragment. The 5'NRG1 fragment obtained above was double-digested with Sac I and Sma I, and ligated with the pUC18 plasmid that was also double-digested with Sac I and Sma I to form pUC18 (SacI-5'NRG1-SmaI). The vector pUC18 (SacI-5'NRG1-SmaI) was transformed into competent Escherichia coli, and positive clones were screened by colony PCR.

[0158] Then, using primers HYG-F and HYG-R, the plasmid pRDM054 was used as a template to amplify the hygromycin B resistance gene HPH with a size of 1648bp. The HPH fragment obtained above was double digested...

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Abstract

The invention relates to a method for eliminating dependence of a methanol-induced promoter on a single methanol carbon source. By means of increasing the expression index of an Mit1 polypeptide in a methylotrophic yeast cell and activating the expression of a promoter to be induced by methanol, the promoter which is originally dependent on the methanol for induction can be also used for expressing foreign polypeptides without depending on single methanol.

Description

technical field [0001] The invention belongs to the field of bioengineering; more specifically, the invention relates to a method for expressing exogenous polypeptides by eliminating the single methanol carbon source dependence of methanol-inducible promoters. Background technique [0002] The expression system of methanolotrophic yeast (including Pichia, Hansenula, Candida, Torulopsis, etc.) has been widely used in industrial production, pharmaceuticals and other fields due to its high-efficiency expression ability of exogenous polypeptides. Its characteristic is that they have promoters that can be efficiently induced by methanol (AOX1 promoter, DHAS promoter, FDH promoter, MOX promoter, AOX2 promoter, ZZA1, PEX5-, PEX8-, PEX14-promoter, etc.), And these promoters are strictly dependent on methanol, other carbon sources (such as glucose, glycerol, etc.) will inhibit the expression of these promoters. [0003] With the outbreak of the oil crisis, the cost of using methanol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/84C12R1/78C12R1/72C12R1/88
CPCC12R1/88C12N15/81C12R1/72C12R1/78C12R1/84C12N15/815C07K14/39C12N1/32C12P21/00
Inventor 周祥山张元兴王锦佳王小龙柏鹏张平
Owner CHINA RESOURCES BIOPHARMACEUTICAL CO LTD
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