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Temperature switch system and application thereof to increment of yield of amino acids

A technology of temperature switch and threonine, applied in the fields of genetic engineering and microbial fermentation, can solve the problems of difficulty in accumulating oxaloacetic acid and low conversion rate of oxaloacetic acid derivatives, and achieve high-efficiency fermentation production, rapid biomass accumulation, The effect of shortening the fermentation cycle

Active Publication Date: 2020-07-03
JIANGNAN UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, low-level expression of PYC is difficult to accumulate more oxaloacetate, resulting in a lower conversion rate of oxaloacetate derivatives

Method used

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  • Temperature switch system and application thereof to increment of yield of amino acids
  • Temperature switch system and application thereof to increment of yield of amino acids
  • Temperature switch system and application thereof to increment of yield of amino acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 temperature switch carrier construction and characterization

[0074] (1) Construction of temperature switch carrier

[0075] Based on the plasmid pFW001 (containing triclosan resistance and high copy number pMB1 replicon), the medium copy number replicon p15A was used to replace pMB1, and the temperature switch loop was used to replace the PJ23101 promoter to obtain a temperature switch vector. The plasmid pFW001 is derived from a paper published in 2018: Zhao, H., Fang, Y., Wang, X., Zhao, L., Wang, J., Li, Y., 2018.Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway.

[0076] The temperature switch circuit includes the temperature sensitive circuit cI ts -p R -p L , the TetR repressor gene tetR and the tightly regulated promoter P LtetO-1 . These genes were obtained by chemical synthesis. In order to obtain independent genetic regulation, dual multiple cloning sites (MC...

Embodiment 2

[0087] Embodiment 2 Application of temperature switch system to improve the production capacity of threonine strains

[0088] (1) Optimization and transformation of threonine production strains:

[0089] Taking TWF001 as the starting strain, the genome of this strain has overexpressed pyridine nucleotide transhydrogenase (pntAB) and related genes involved in threonine production (ppc, aspC, lysC, asd, thrA G433R BC and rhtA) (see figure 1 ). To improve the threonine production performance of the strain and reduce waste of carbon sources, some unimportant genes related to organic acid synthesis (poxB, pflB, ldhA, adhE, and pta) and a gene encoding a threonine transporter (tdcC) were knocked out, respectively. except (see figure 2 ), thereby producing platform strains TWF102, TWF103, TWF104, TWF105, TWF106, TWF107, TWF108, as shown in Table 1. These platform strains were subjected to shake-flask fermentation to produce threonine at 37°C constant temperature and variable te...

Embodiment 3

[0097] Embodiment 3 fermentation produces threonine

[0098] STF seed medium is based on LB medium and obtained after optimization and improvement. After adjusting the content of sucrose and peptone in the LB medium, it was used to cultivate the TWF001 strain, and the growth status and threonine production of the strain in the seed medium with different components were detected (such as Figure 8 shown). The results showed that the threonine yield of TWF001 was 15.37g / L, which was 50.54% higher than that of LB as the seed medium, when STF seed medium was used as the seed medium and fermented in a constant temperature shake flask at 37°C for 18 hours.

[0099] Recombinant strains TWF106 / pFT24r, TWF106 / pFT24p, TWF106 / pFT24pm and TWF106 / pFT24rp were fermented in variable temperature shake flasks (such as Figure 10 shown), its threonine yields are 17.24g / L, 20.15g / L, 20.60g / L and 23.29g / L respectively, and the corresponding molar sugar-acid conversion rates are 72.44%, 81.50%, ...

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Abstract

The invention relates to a temperature switch system and an application thereof to increment of yield of amino acids, in particular to a method for adjusting and controlling intracellular metabolism flow distribution to increment yield of threonine by using the temperature switch system, and belongs to the technical field of genetic engineering and microbial fermentation. The temperature switch system is used, so that the entire fermentation course is divided into two stages of cell growth and fermentation and production to be adaptive to intracellular environmental changes of different stagesin a fermentation process of an engineering bacterial strain. According to the system, the heterologous expression of pyruvate carboxylase is controlled, and through combination of a chemical property that oxaloacetic acid is thermo-sensitive and easy for decarboxylation, metabolism flows are rebalanced between pyruvate and oxaloacetic acid, a central metabolic pathway is dynamically regulated, and supply of reduced cofactors is guaranteed to promote production of L-threonine. The threonine mol conversion rate of obtained temperature control threonine production bacterial strains namely TWF106 / pFT24rp is 111.78% and the threonine mol conversion rate of obtained temperature control threonine production bacterial strains namely TW113 / pFT24rpal is 124.03%.

Description

technical field [0001] The invention relates to a temperature switch system and its application in increasing amino acid production, in particular to a method for improving threonine production by using the temperature switch system to regulate intracellular metabolic flow distribution, and belongs to the technical fields of genetic engineering and microbial fermentation. Background technique [0002] Metabolic engineering combined with metabolic regulation has been applied to enhance the production of natural chemicals, especially bulk amino acid products. However, the unbalanced distribution of cellular metabolic fluxes between cell growth and desired products has limited the further improvement of product yield and productivity for a long time. Traditional techniques (such as inactivation of genes involved in alternative pathways and overexpression of genes involved in heterologous pathways) cannot handle the challenges of more complex carbon distribution (such as the nee...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/52C12N1/21C12P13/08C12R1/19
CPCC12N15/70C12N9/93C12P13/08C12Y604/01001
Inventor 王小元方宇王建莉张淑嫣胡晓清
Owner JIANGNAN UNIV
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