Genome-scale metabolic network model of fk506-producing strain Streptomyces tsukuba to guide next-level pathway transformation

Abstract

The invention discloses a secondary approach transformation method based on an instruction of an FK506 production bacterial strain wave chain streptomycete genome scale metabolic network model. The model is based on annotation genes and physiology and biochemistry information. By comparing and analyzing the model with a streptomyces coelicolor genome, metabolic genes are found being highly conservative. Metabolic flux analysis is performed on a genome scale metabolic network, and therefore the model predicts a mutation bacterium secondary approach gene cluster transformation strategy for improving a production level. According to the secondary approach transformation method based on the instruction of the FK506 production bacterial strain wave chain streptomycete genome scale metabolic network model, the transformation method utilizes the genome scale metabolic network model to predict special structural genes in an FK506 bacterial strain secondary approach gene cluster, the production level of bacterial strains after transformation is improved by 20 percent to 90 percent, the special structural genes in the gene cluster are augmented to improve production capacity, and large application value is achieved in secondary approach rational transformation of microorganism immunosuppressor production bacterial strains. The high-efficiency and systematic method is provided for optimizing of the bacterial strains.

Description

technical field

[0001] The invention belongs to the technical field of metabolic engineering molecular transformation of microbial strains, and particularly relates to a method for transforming secondary pathways under the guidance of a genome-scale metabolic network model of FK506 producing strain Streptomyces tsukuba. Background technique

[0002] FK506, also known as tacrolimus, is a 23-membered polyketide macrolide compound with high immunosuppressive effect synthesized by Streptomyces tsukuba. FK506 is widely used in the treatment of autoimmune diseases such as atopic dermatitis, allergic contact dermatitis, psoriasis, lupus erythematosus, lichen planus, vitiligo, Nethenton syndrome, and host suppressive diseases. It is currently clinically used to prevent graft rejection after liver or kidney transplantation, and to treat graft rejection that cannot be controlled by other immunosuppressive drugs after liver, pancreas, kidney, heart, lung and other solid organ transplan...

Examples

Embodiment 1

[0046] The model is based on annotated genes and physiological and biochemical information, including 865 biochemical reactions and 621 metabolites. 735 reactions of this network are unique, and the rest are identical reactions encoded by isozymes. Through comparative analysis with the genome of Streptomycescoelicolor, it was found that the metabolic genes were highly conserved. The model mainly includes glycolysis pathway, pentose phosphate pathway, tricarboxylic acid cycle, pyruvate metabolism, glyoxylate cycle metabolism, biomass precursor synthesis, coenzyme synthesis, nitrogen-sulfur metabolism and porphyrin and other related reactions. Including the synthesis pathway reaction of FK506 and its by-products FK520 and FK506D, the bacterial synthesis reaction ( figure 1 ).

Embodiment 2

[0048] According to the prediction of the secondary pathway gene cluster special pathway gene in Example 1, the initial intracellular flux distribution of Streptomyces tsukuba obtained by the flux balance analysis calculation, each non-zero flux reaction was doubled, and the MOMA algorithm was used to obtain the expansion Intracellular flux distribution of each mock strain after multiplication, according to f PH The calculation formula for screening secondary pathway amplified target genes of FK506 biosynthesis ( figure 1 ).

[0049] f PH ≡ ( f biomass ) ( f FK 506 ) = ( v biomass , overexpression ...

Embodiment 3

[0051] According to Example 2, predict secondary pathway gene cluster special pathway genes—cyclohexanedicarboxylic acid synthesis pathway gene fkbO, percolic acid synthesis pathway gene fkbL and fkbP, FK506 rear modification pathway structural gene fkbM and fkbD, to Streptomyces tsukuba carry out molecular modification. Using the S.tsukubaensis genome as a template, fkbO-F / fkbO-R, fkbL-F / fkbL-R, fkbP-F / fkbP-R, fkbM-F / fkbM-R, fkbD-F / fkbD-R were used as Primers (Table 1) amplify the fkbO gene, fkbL gene, fkbP gene, fkbM gene and fkbD gene, and the PCR products include the ribosome binding sites of each gene itself. Respectively, the fkbO gene, fkbL gene, fkbP gene, fkbM gene and fkbD gene PCR products were digested with NdeI-XbaI and then ligated into the same digested pIB139, and the ligated products were transferred into the prepared Escherichia coli competent cells JM109 or DH5α , after transformation, spread onto a screening plate containing 50 μg / mL apramycin and culture ...

technical field

[0001] The invention belongs to the technical field of metabolic engineering molecular transformation of microbial strains, and particularly relates to a method for transforming secondary pathways under the guidance of a genome-scale metabolic network model of FK506 producing strain Streptomyces tsukuba. Background technique

[0002] FK506, also known as tacrolimus, is a 23-membered polyketide macrolide compound with high immunosuppressive effect synthesized by Streptomyces tsukuba. FK506 is widely used in the treatment of autoimmune diseases such as atopic dermatitis, allergic contact dermatitis, psoriasis, lupus erythematosus, lichen planus, vitiligo, Nethenton syndrome, and host suppressive diseases. It is currently clinically used to prevent graft rejection after liver or kidney transplantation, and to treat graft rejection that cannot be controlled by other immunosuppressive drugs after liver, pancreas, kidney, heart, lung and other solid organ transplan...

Examples

Embodiment 1

[0046] The model is based on annotated genes and physiological and biochemical information, including 865 biochemical reactions and 621 metabolites. 735 reactions of this network are unique, and the rest are identical reactions encoded by isozymes. Through comparative analysis with the genome of Streptomycescoelicolor, it was found that the metabolic genes were highly conserved. The model mainly includes glycolysis pathway, pentose phosphate pathway, tricarboxylic acid cycle, pyruvate metabolism, glyoxylate cycle metabolism, biomass precursor synthesis, coenzyme synthesis, nitrogen-sulfur metabolism and porphyrin and other related reactions. Including the synthesis pathway reaction of FK506 and its by-products FK520 and FK506D, the bacterial synthesis reaction ( figure 1 ).

Embodiment 2

[0048] According to the prediction of the secondary pathway gene cluster special pathway gene in Example 1, the initial intracellular flux distribution of Streptomyces tsukuba obtained by the flux balance analysis calculation, each non-zero flux reaction was doubled, and the MOMA algorithm was used to obtain the expansion Intracellular flux distribution of each mock strain after multiplication, according to f PH The calculation formula for screening secondary pathway amplified target genes of FK506 biosynthesis ( figure 1 ).

[0049] f PH ≡ ( f biomass ) ( f FK 506 ) = ( v biomass , overexpression ...

Embodiment 3

[0051] According to Example 2, predict secondary pathway gene cluster special pathway genes—cyclohexanedicarboxylic acid synthesis pathway gene fkbO, percolic acid synthesis pathway gene fkbL and fkbP, FK506 rear modification pathway structural gene fkbM and fkbD, to Streptomyces tsukuba carry out molecular modification. Using the S.tsukubaensis genome as a template, fkbO-F / fkbO-R, fkbL-F / fkbL-R, fkbP-F / fkbP-R, fkbM-F / fkbM-R, fkbD-F / fkbD-R were used as Primers (Table 1) amplify the fkbO gene, fkbL gene, fkbP gene, fkbM gene and fkbD gene, and the PCR products include the ribosome binding sites of each gene itself. Respectively, the fkbO gene, fkbL gene, fkbP gene, fkbM gene and fkbD gene PCR products were digested with NdeI-XbaI and then ligated into the same digested pIB139, and the ligated products were transferred into the prepared Escherichia coli competent cells JM109 or DH5α , after transformation, spread onto a screening plate containing 50 μg / mL apramycin and culture ...

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