The technique of extragnus Clostridium DNA dna insertion based on phage-specific integration system.

On January 8, 2019, the international academic journal Blycoma Engineering published an online paper entitled Phage Serine Integrase-Mediated Engineering for the e'ion serium-biosynthetic in gas-fermenting Crods
the study established a new technique for the integrated expression of large fragmentgene cluster chromosomes based on the specific recombination mechanism of phage site-based carbon gas-based carbon gas-using bacteria-gas clostridium bacteria, which provides a useful genetic operation tool for the creation and research of a new type of carbon gasification cell plant.
carbon gas (e.g. CO2, CO) is a huge amount of free carbon resources.
in recent years, in order to expand their use and vigorously develop green biological manufacturing, the technical route of bio-converting a carbon gas to synthesize various chemicals and energy substances has received wide attention.
known to use a carbon gas micro-organisms according to energy needs can be divided into chemical energy, light energy and electric energy self-support and other types.
gas clostridium is a main type of self-breeding bacteria, with hydrogen or other reductive compounds as energy donors, and its gas fermentation process matches traditional industrial fermentation equipment better, and has a good application prospect.
however, there are few natural product types of clostridium, low fermentation units and difficult genetic operation.
, it is urgent to develop an efficient molecular technology platform for its metabolic engineering design, transformation and synthetic biology research, and thus to create a more efficient and product diversification of artificial carbon sequestration system, to achieve a carbon gas biological conversion and utilization capacity.
to build an efficient and diversified poravirus cell factory, whether it is the enhanced synthesis of natural products or the adaptation expression of the heterogeneous pathway, the efficient integration and editing of gene clusters on chromosomes of large fragment metabolic pathways is an effective strategy that must be used, and its genetic stability is significantly better than the method of using free-nature granule carriers.
2016, the Jiang Weihong research team pioneered the establishment of CRISPR-Cas9-mediated genome editing technology (Huang et al., ACS Synth Biol, 2016) in Clostridium Difectoma Clostridium ljungdahlii.
this technique can be used for the deletion of genes on the pavailability of Clostridium clostridium or the integration of small fragments of DNA on chromosomes, but is limited by the lower homologous recombination efficiency, and can not achieve the purpose of introducing large fragments of target products to synthesize gene clusters on chromosomes.
to solve these problems, the study first used the CRISPR-Cas9 gene editing system to integrate attB sites from two heterogeneous phage integration systems on the plostle cocci chromosomal chromosome.
on this basis, by integrating the enzyme-mediated attP-attB recombination mechanism, the high-efficiency insertion of the exogenous gene cluster at the attB site in chromosomes is realized, thus realizing the synthesis of new products.
, the new method also achieves the integration of specific target gene clusters on chromosomes through the screening of two recombinations based on the "double-integration enzyme-double attP/attB" strategy and the screening of CRISPR-Cas9 directional cutting, and solves the defects that introduce other "impurity DNA" when using a single set of attP/attB systems, thus ensuring the stable expression of gene clusters.
finally, the authors successfully integrated a cluster of butyric acid synthesis gene from Clostridium cocitacoccal Clostridium coccora on the chromosomes of Clostridium coccid cocci.
in a single batch of gas fermentation, the yield of engineering bacteria butyric acid reached 1.01 g/L, the product concentration and synthesis rate are higher than the reported ceccoccal, and after more than 10 generations of continuous culture can still maintain this fermentation level, showing good genetic stability.
the above results make up for the deficiency of the existing genetic operation method of CLOSTella, which is very convenient for the construction of a diversified cell factory for this important industrial microorganism.
, because the phage site-specific recombination system is widely distributed and has a certain degree of universality, the method can also be extended to other Clostridium difficile or carbon sequestration microorganism transformation, thus effectively accelerating the application of the biological conversion and utilization of a carbon gas.
associate researcher Huang He is the first author of the paper, and researcher Gu Yang is the co-author of the paper.
the research was funded by the National Natural Science Foundation of China, the Shanghai Science and Technology Science and Technology Research Action Plan, the basic research field project, the Chinese Academy of Sciences Youth Innovation Promotion Association and other grants.
Source: Center for Excellence in Molecular Plant Science/Institute for Plant Physiology and Ecology.