An engineering strain and method for preparing farnesene from cellulose

A technology for engineering strains and cellulose, applied in the field of bioengineering, can solve problems such as low yield, and achieve the effects of increasing yield and improving stress resistance

Active Publication Date: 2022-06-28
CHINA PETROLEUM & CHEM CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, during the research process, it was found that if the products containing monosaccharides produced by hydrolysis of cellulose are used for the fermentative production of farnesene, there is a defect of low yield

Method used

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  • An engineering strain and method for preparing farnesene from cellulose
  • An engineering strain and method for preparing farnesene from cellulose
  • An engineering strain and method for preparing farnesene from cellulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This example is used to illustrate the preparation of cellulose enzymatic hydrolysis solution.

[0041] Weigh 100 g of corn stover with a particle size of 20-80 mesh, add 1.5 L of formic acid solution (containing 88 wt % formic acid and 1 wt % HCl), and place it in a treatment tank. After reacting in a constant temperature water bath at 65°C for 3 h, the reaction slurry was separated into solid and liquid. The solid components were then treated with ammonia water, and the treatment conditions were: ammonia concentration 15wt%, volume of ammonia water used per kilogram of solid components was 8 liters, treatment temperature was 60°C and reaction time was 16h. After the reaction, the material is separated from solid and liquid, and the solid component is washed with distilled water to pH 6-7, and dried to obtain a pretreated solid material.

[0042] The pretreated solid material is subjected to enzymatic hydrolysis, and the enzymatic hydrolysis conditions include: the te...

Embodiment 2

[0044] This example is used to illustrate the sequence structure of the plasmid. Commercially available vectors used in this example include the pet-22b vector, pBAD33 vector, pACYDCDuet-1 vector, pBBR1MCS-4 vector, and pet-28a vector for knock-in of the acetate kinase ack (gene) into the E. coli genome .

[0045] According to the instructions for use of the above-mentioned vectors, molecular biology operations such as PCR cloning, whole gene synthesis, restriction endonuclease digestion and ligation were used to construct a set of plasmids for the MVA metabolic pathway, specifically including pMevT plasmid, pMBIS plasmid and pFII plasmid, and then One plasmid was constructed to replace the pFII plasmid, that is, the pFG plasmid, and then two plasmids were constructed to replace the pMevT plasmid, namely pMevT-1 and pMevT-2. The sequence information of the above plasmids is specifically shown in Table 2.

[0046] Table 2

[0047]

[0048]

[0049]

Embodiment 3

[0051] This example is used to illustrate the construction process of the engineered strain.

[0052] Construction of engineering strain F1 in the experimental group: Escherichia coli BL21 was selected as the host cell, and acetate kinase (ack) was knocked in according to the following method to obtain a strain overexpressing acetate kinase (ack) after knock-in.

[0053] The first step to prepare competent cells: after the plasmid pREDKI (shown in SEQ ID NO. 10) was transferred into Escherichia coli BL21, it was inoculated into LB liquid medium (kana resistance), and placed on a shaker at 30°C for overnight cultivation. The next day, it was transferred to fresh LB liquid medium (kana resistant) at a volume ratio of 1:100, cultured at 30°C to an OD600 of 0.2 to 0.3, and induced by adding L-arabinose with a final concentration of 10 mmol / L. After induction for 1-1.5 hours, take out the ice bath from the shaker at 30 °C for more than 10 min, centrifuge at 3500 rpm / min and 4 °C fo...

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Abstract

The present disclosure provides an engineering strain, which is Escherichia coli with a preservation number of CGMCC NO.14821. The present disclosure also provides a method for preparing an engineering bacterium, the method comprising: knocking in acetate kinase in the genome of the engineering bacterium host bacterium and then transferring the recombinant plasmid, so that the engineered bacterium host bacterium after the transfer can pass through expresses acetate kinase and can express the gene inserted in the recombinant plasmid; the recombinant plasmid includes the plasmid shown in SEQ ID NO.1, the plasmid shown in SEQ ID NO.2 and the plasmid shown in SEQ ID NO.7 plasmids indicated. The present disclosure also provides the engineering bacteria prepared by the method, the engineering strain and the application of the engineering bacteria, and a method for preparing farnesene from cellulose. The present disclosure significantly improves the yield of farnesene produced by fermenting cellulose hydrolyzate through the above technical scheme.

Description

technical field [0001] The present disclosure relates to the field of bioengineering, in particular, to an engineering strain, a method for preparing an engineering bacterium, an engineering bacterium prepared by the method, uses of the above-mentioned engineering strain and engineering bacterium, and a method for preparing farnesene by utilizing cellulose Methods. Background technique [0002] Farnesene can be hydroconverted to farnesane, a renewable fuel that can be blended with petrodiesel and aerospace fuels. With the advantages of no sulfur, high cetane number, low temperature performance and low GHG emission, farnesene is a bio-based chemical with wide application and ideal effect. For example, CN101553558A describes the production of farnesene by the MEV pathway in E. coli using a medium containing glucose. [0003] An important problem to be solved in the production of farnesene by microorganisms is the source of glucose. Cellulose is the most widely distributed a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/12C12P5/02C12R1/19
CPCC12N9/1217C12Y207/02001C12N15/70C12P5/026C12P2203/00
Inventor 高岚齐崴尤生萍陶志平李妍苏荣欣李宝石蔺建民
Owner CHINA PETROLEUM & CHEM CORP
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