CN-122012357-A - Formic acid bioconversion engineering strain and application thereof

Abstract

The invention relates to the technical field of biology, in particular to the technical field of formic acid biological utilization and efficient bioconversion. The invention provides a formic acid bioconversion engineering strain, which is obtained by introducing plasmid pED31-FFM and plasmid pAD31-gcvTHP into escherichia coli, wherein plasmid pED31-FF M is obtained by connecting ftfl-fchA-mtdA (FFM) genes to pED31 empty plasmid, and plasmid pAD31-gcvTHP is obtained by connecting gcvTHP genes to pAD31 empty plasmid. The strain provided by the invention has the advantages that under the initial concentration of 20-60mM formic acid, the multiplication time is less than 5 hours, the biomass yield can reach 6gCDW/mol formic acid, the single cell protein yield can reach 2.45-4.2g/mol, and the international leading level of formic acid conversion and biomass is reached.

Inventors

• ZHANG YANPING
• ZHAO LEI
• ZHAO TONGXIN
• LI YIN

Assignees

• 中国科学院微生物研究所

Dates

Publication Date

20260512

Application Date

20241110

Claims (4)

1. 1. The engineering strain is characterized in that the engineering strain is obtained by introducing plasmid pED31-FFM and plasmid pAD31-gcvTHP into escherichia coli; the plasmid pED31-FFM is obtained by connecting ftfl-fchA-mtdA (FFM) genes to pED31 empty plasmid; the plasmid pAD31-gcvTHP was obtained by ligating the gcvTHP gene to the empty plasmid pAD 31.
2. 2. The engineered strain of claim 1, wherein the escherichia coli is obtained by one or more of the following genetic engineering procedures: (1) Knocking out the sdaB gene of the wild escherichia coli; (2) Integrating the escherichia coli sdaA gene into the sdaB gene position of the strain Δsdab; (3) Integration of formate dehydrogenase PseFDH into the E.coli genome; (4) Carrying out gene mutation on escherichia coli engineering bacteria, wherein a mutation site contains RpoB (I854N), pc nB (V102E) and ProA (A383E); (5) Carrying out gene mutation on the escherichia coli engineering bacteria, wherein mutation sites comprise gcvR (P (-45) Ins T), acs (delta 1915-1918) and Pat (L206P); (6) The engineering bacterium of the escherichia coli is subjected to gene mutation, wherein mutation sites comprise ArcA (E94) and Ac tP (A350T).
3. 3. Use of an engineered strain according to any one of claims 1 to 2 for the use of formic acid.
4. 4. Use of an engineered strain according to any one of claims 1 to 2 for the production of single cell proteins.

Description

Formic acid bioconversion engineering strain and application thereof Technical Field The invention relates to the technical field of biology, in particular to the technical field of formic acid biological utilization and efficient bioconversion. Background The renewable electric power is utilized to reduce CO 2 into carbon compounds such as formic acid, formaldehyde, methanol and the like, and the coupling microorganism is utilized to convert the carbon compounds into chemicals, so that the method is one of important strategies for realizing sustainable recycling of carbon resources and development of green industry. Formic acid is the easiest water-soluble carbon compound to prepare, has high solubility, low toxicity, mature technology, high economic and technical feasibility, good bioavailability, high conversion rate, incombustibility, and better electronic economy compared with methanol by electrocatalytic production compared with methanol. The transportation and storage are safer, the safety is higher in the microbial cultivation process, the strain is developed for efficiently utilizing the formic acid, and the carbon source of the formic acid is used for replacing the traditional industrial microbial glycosyl raw materials, so that the method is an effective means for reducing carbon emission and improving the green biological preparation route. The natural formic acid utilizing strain mainly comprises acetogenic bacteria such as acetic acid bacillus wushuriensis (Acetobacterium woodii), clostridium immortalnii (Clostridium ljungdahlii) and heat vinegar mushroom bacterium (Moorella thermoacetica), methanogenic bacteria such as hydrogenotrophic methane coccus (Methanococcus maripaludis), sulfate reducing bacteria such as common vibrio (Desulfovibrio vulgaris), vibrio pastoris (Desulfovibrio baarsii), vibrio pastoris (Desulfovibrio desulfuricans) and vibrio pastoris (Desulfarculus baarsii), and other microorganisms which utilize formic acid in a mode of oxidizing the formic acid to provide reducing power for CO 2 by utilizing formate dehydrogenase. Natural marine vibrio natrii (Vibrio natriegens) has strong formic acid tolerance (60 g/L) and metabolic capacity, and has huge formic acid bioavailability potential. However, the strain can not grow and metabolize formic acid as the only carbon source at present, and the assimilation rate of the formic acid is still to be improved. Most of natural formic acid is not fully researched by utilizing bacterial strains, has few genetic transformation tools and is not suitable for metabolic engineering transformation and industrial production. In contrast, the research of microorganisms in E.coli, S.cerevisiae and other modes is more sufficient, and the construction of the efficient formic acid utilization strain is more suitable for metabolic engineering and industrial production, and is more beneficial to the further development of formic acid bioeconomical. The formate assimilation pathway found and optimized includes serine cycle, modified serine cycle, reduced acetyl-CoA pathway (WL pathway), reduced glycine pathway (rGlyP), formate-formaldehyde-RuMP pathway, formate-formyl-CoA-serine pathway, and the like. Among them, the WL pathway is a natural formic acid assimilation pathway with few reaction steps and low ATP consumption, but the heterologous expression of the pathway is difficult. The rGlyP-way short (5 enzymes) has low energy (2 ATP) and reducing power (3 NAD (P) H) requirements, little interference with central metabolic ways and good oxygen resistance, and is a carbon fixation way which is of great interest. Successful construction in E.coli and Saccharomyces cerevisiae has been achieved, showing high tolerance to formate (750 mM) in Saccharomyces cerevisiae. However, the utilization rate of formic acid of the reconstructed escherichia coli and saccharomycetes is still low, and the multiplication time of engineering strains taking formic acid as the only carbon source is more than 6 hours. Using the reductive glycine pathway, 1.2mM lactate was produced by coupling LDH in E.coli (maximum yield 10%), and PHB production was observed in E.coli by introducing the PHB pathway. The existing formate nutrition type strain still has the problems of long growth period, weak utilization capacity of formate, low assimilation rate and the like, so that development of the formate efficient transformation strain is urgently needed. Disclosure of Invention In view of the above, the invention provides an efficient formic acid bioconversion engineering strain. The strain is obtained by introducing plasmid pED31-FFM and plasmid pAD31-gcvTHP into escherichia coli, wherein the plasmid pED31-FFM is obtained by connecting ftfl-fchA-mtdA (FFM) genes to pED31 empty plasmid, and the plasmid pAD31-gcvTHP is obtained by connecting gcvTHP genes to pAD31 empty plasmid. Further, the E.coli is obtained by one or more of the following genetic engineering procedures: (1) Knocking out