Establishment Of Chemically Inducible Chromosomal Evolution Method In Corynebacterium Glutamicum

5-aminolevulinic acid(ALA),a non-protein amino acid,is an important precursor for the tetrapyrrole synthesis,such as porphyrin compounds,heme and vitamin B12.In recent years,ALA has drawn increasing attention as a photodynamic chemical,which has been widely applied in many fields,such as medicine,agriculture,and the food industry.ALA not only has potential applications in tumor localization and photodynamic therapy of a variety of cancers,but also can be used as a biodegradable herbicide and insecticide that is harmless to crops,humans and animals.In view of the ALA has a very broad application prospects and the current ALA market supply,so the synthesis of ALA research is of great significance.The production of ALA is mainly synthesized through chemical methods.However,there are many disadvantages in the chemically synthetic methods,such as complicated synthesis process,low recovery rate,high production cost and a series of environmental pollution problems.In recent years,with the development of microbial technology and metabolic engineering,microbes can be used as cell factories to synthesize ALA,which not only shortens the process of ALA production,but also has great potential for production.More importantly,the problem of environmental pollution caused by chemical synthesis is avoided.Thus,ALA biosynthesis has drawn intensive attention and will undoubtedly become the trend of future research.In living organisms,there are two major biosynthetic pathways of 5-ALA:C4 pathway and the C5 pathway.The metabolice regulation of C5 pathway is complicated and it is difficult to improve ALA yield.However,the C4 pathway has a short metabolic regulation and it has been widely researched.Research reports on ALA biosynthesis has mainly focused on Escherichia coli as engineering strain.In recent years,Corynebacterium glutamicum is used as an engineering strain to produce ALA.Corynebacterium glutamicum is a Gram-positive soil bacterium which has been widely used in large-scale industrial production of amino acids.Corynebacterium glutamicum,as a food safety strain,avoids the health and safety problems caused by the fermentation of Escherichia coli.Therefore,we consider taking Corynebacterium glutamicum 13032 as an engineering strain,using the genetic manipulation tools to construct its metabolic engineering and produce ALA through C4 pathway.There are still few studies in this area,which makes the research innovative.At present,the progress of inserting and overexpression of the heterologous genes involved in the synthesis of ALA that has mainly relied on plasmid-based expression systems.But fundamental issues concerning plasmid genetic instability have not been adequately resolved.In order to avoid the problem of plasmid instability,we found a chemically inducible chromosomal evolution(CIChE)technology that has been successfully explored in the Escherichia coli by referring to the literature.The genome of the microorganism can be evolved through the passage,and the number of copies of the heterologous genes on the genome is increased continuously.In this study,we first attempted to establish the chemically inducible chromosomal evolution method in the Corynebacterium glutamatosum,thereby constructing a recombinant strain with high genetic stability.The overall design idea of this study was taking Corynebacterium glutamicum 13032 as the engineering strain,using genomic integration technology and chemically inducible chromosomal evolution technology to construct a recombinant ALA production strain turough C4 pathway.In this paper,we first inserted the codon-optimized ALAS(hemA)gene from Rhodobacter capsulatus SB 1003 into Corynebacterium glutamicum 13032.Then the method of chemically inducible chromosomal evolution was established in the recombinant Corynebacterium glutamicum.In the process of evolution,the metabolic pressure of cells was increasing.RecA gene was deleted after the evolution reached a certain extent.hemA copy numbers were measured by qPCR and ALA production of CIChE strains were detected by shake flask fermentation.The highest ALA accumulation rate was 3g/L.ALA is an intermediate product of heme synthesis pathway.In order to further increase the yield of ALA,it is essential to reduce the metabolic flux of the downstream heme synthesis pathway.HemB encoding ALA dehydratase is the key gene for ALA to transform heme pathway.In this paper,we attempted to weaken the expression of hemB to reduce the metabolic flux of heme synthesis pathway.We used C.glutamicum genomic editing tools to mutate the active site of hemB.The results showed that the enzyme activity of the strain that successfully mutated hemB was reduced to 0.2%-3%,almost blocking heme synthesis pathway,and the ALA yield of the hemB mutant strain decreased seriously.The hemB mutation site selected in this paper was the active site and is critical to HemB enzyme activity.Mutation of this active site had a severe inhibitory effect on HemB enzyme activity.It is possible to try to mutate other sites or dynamically regulate the entire metabolic pathway downstream of ALA.