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A method for the construction of isobutanol-synthesizing strains guided by the regulation of intracellular reducing power based on a genome-scale metabolic network model

A genome-scale, metabolic network technology, applied in the field of isobutanol synthesis strain construction based on genome-scale metabolic network model to guide the regulation of intracellular reducing force, can solve the problem of not being able to achieve optimal intracellular reducing force state, strain growth and product Problems such as decreased synthesis ability

Inactive Publication Date: 2020-04-07
TIANJIN UNIV
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Problems solved by technology

To further modify the predicted target, simple methods such as knockout, overexpression or replacement usually cannot achieve the best intracellular reducing power state, and may cause over-regulation to cause strain growth and product synthesis ability to decline

Method used

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  • A method for the construction of isobutanol-synthesizing strains guided by the regulation of intracellular reducing power based on a genome-scale metabolic network model
  • A method for the construction of isobutanol-synthesizing strains guided by the regulation of intracellular reducing power based on a genome-scale metabolic network model
  • A method for the construction of isobutanol-synthesizing strains guided by the regulation of intracellular reducing power based on a genome-scale metabolic network model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of vector plasmid pTRCLA and pACYCLA

[0059] (1) Using pTRC99a as a template, design primers PLO-F and PLO-R, the primer sequences are SEQ ID NO: 6 and SEQ ID NO: 7, and use PCR to obtain the fragment PLO; using pTRC99a as a template, design primers TB-F and TB -R, the primer sequence is SEQ ID NO: 8 and SEQ ID NO: 9, fragment TB is obtained by PCR; after fragment PLO and TB are digested with KpnI and PstI, they are connected to obtain plasmid pTRCLA ( Figure 6 ).

[0060] Double digestion system: fragment PLO or fragment TB 20 μL, KpnI 1 μL, PstI 1 μL, 10×buffer 1 μL, ddH 2 O 23μL, react at 37°C for 1h.

[0061] Ligation system: TB fragment 7 μL, PLO fragment 1 μL, T4 ligase 0.5 μL, ddH 2 O 1.5 μL, react at 22°C for 1h.

[0062] (2) Using pTRC99a as a template, design primers PT-F and PT-R, the primer sequences are SEQ ID NO: 10 and SEQ ID NO: 11, and use PCR to obtain fragment PT; using pACYC184 as a template, design primers OC-F and OC -R...

Embodiment 2

[0068] The recombinant plasmid pACYCLA09 and pTRCLA10 construction of embodiment 2 isobutanol synthesis pathway

[0069] (1) First extract the DNA template

[0070] Bacillus subtilis 168 was cultured in LB medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, pH7.0) at 200rpm at 37°C, and after reaching the logarithmic phase, the genome extraction kit (Tiangen ) to extract total DNA.

[0071] Lactococus lactis was cultured in MRS medium (10g / L peptone, 10g / L beef extract powder, 5g / L yeast powder, 20g / L glucose, 1.0mL Tween 80, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate , 2g / L K 2 HPO 4 ·3H 2 O, 0.58g / LMgSO 4 ·7H 2 O, 0.25g / LMnSO 4 ·H 2 0, pH 7.0) to the logarithmic phase after static culture, the genome extraction kit (Tiangen) was used to extract the total DNA.

[0072] E.coli MG1655 was cultured in LB medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, pH 7.0) at 37°C and 200rpm, and after reaching the logarithmic phase, the genome extraction kit (Tian...

Embodiment 3

[0088] Embodiment 3 initial isobutanol synthesis bacterial strain is constructed

[0089] (1) Escherichia coli E.coli LA01 electroporation competent preparation

[0090] 1) Inoculate the strains transferred by streaking in -80 refrigerator, and culture at 37°C.

[0091] 2) Pick a single clone, transfer it to a 5 mL LB test tube, culture at 200 rpm, 37° C. overnight.

[0092] 3) Inoculate 1% inoculum into 50ml LB medium, 37°C, 200rmp, OD 600 Stop culturing when it reaches 0.5-0.6.

[0093] 4) Under sterile conditions, transfer the bacteria to an ice-precooled 50mL centrifuge tube, place on ice for 20min, centrifuge at 4200rpm, 4°C for 10min.

[0094] 5) Discard the medium, invert the tube for 1 min, resuspend the pellet with 50 mL pre-cooled 10% glycerol, and centrifuge and wash 3 times.

[0095] 6) Add 500uL of 10% glycerol to resuspend, and distribute in sterilized precooled centrifuge tubes, about 100uL in each centrifuge tube.

[0096] 7) Rapidly cooled with liquid nit...

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Abstract

The invention provides an isobutanol synthetic strain construction method implemented by guiding the adjustment of intracellular reducing power based on a genomic scale metabolic network model. Based on the genomic scale metabolic network model, by adopting flow balance analysis and metabolic minimum adjustment analysis, the action law of different reconstruction modes of an intracellular reducing power metabolism to strain growth and isobutanol synthesis is simulated, and according to phenotypic coefficients, a conclusion that glyceraldehyde-3-phosphate dehydrogenase is a key target spot of the intracellular reducing power adjustment of an isobutanol synthetic strain is obtained. By using a synthetic biological artificially-regulated element, an NADP+ depended glycerin-3-phosphate dehydrogenase metabolic pathway is built and adjusted so as to match and balance the intracellular reducing power metabolism, thereby obtaining an efficient isobutanol synthetic strain. The intracellular NADPH / NADP ratio of the strain reaches 0.4-0.8, and when 20-50 g / L glucose as a substrate is adopted for carrying out batch fermentation, the yield of isobutanol can reach over 8 g / L in 36 h, which is increased by over 60%.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and bioenergy, and specifically relates to the construction of an initial isobutanol synthetic strain, and the simulation and prediction of the key target points of reductive metabolism regulation through the genome-scale metabolic network model, and guides the construction of heterologous glycerol-3- that depends on NADP Phosphate dehydrogenase gene (gapN), and the expression level of gapN is controlled by constitutive promoters of different strengths to regulate intracellular reducing power, and the method for constructing isobutanol synthetic strains. Background technique [0002] With the rise of bioenergy, the biosynthesis of isobutanol has recently gained significant attention. Isobutanol has the advantages of high energy density, low volatility and corrosion, and high octane number. It is an ideal gasoline substitute and one of the most basic chemical platform substances. At pres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/53G16B5/00C12R1/19
Inventor 闻建平刘蛟齐海山
Owner TIANJIN UNIV
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