Metabolic Engineering Of Yarrowia Lipolytica For 5-aminolevulinic Acid Production

5-aminolevulinic acid(5-ALA)is the direct precursor of porphyrin pathway.The downstream products of porphyrin pathway include heme,chlorophyll and vitamin B12,which are very important for cell growth and metabolism.5-ALA has unique photosensitivity.In recent years,5-ALA is widely used in agriculture and medicine.Compared with traditional chemical methods,production of 5-ALA by microbial fermentation is green,environmentally friendly,highly sustainable,and has a mild reaction.At present,the biosynthesis of 5-ALA is concentrated in bacterial hosts such as coli and Corynebacterium glutamicum,showing certain industrial production prospects.Nevertheless,these strains still have limitations including the utilization efficiency of cheap substrates,insufficient 5-ALA synthesis intensity,and poor tolerance to environmental stress,and the production efficiency of 5-ALA is still far from commercial demand.To break the technological bottleneck,the potential of new platform microorganisms and biosynthetic strategies for 5-ALA production must be explored.Non-conventional microorganisms with unique physiological and metabolic properties play a key role in industrial biotechnology and may be more suitable as metabolic engineering hosts for the industrial production of 5-ALA.Yarrowia lipolytica is an important non-conventional yeast with the advantages of broad substrate utilization,high tolerance to acidic conditions and high safety.With the rapid development of synthetic biology and gene editing technology,Y.lipolytica has been constructed as a cell factory for biofuels,organic acids,fatty acids and their derivatives.As a strictly aerobic and oleaginous microorganism,Y.lipolytica has a high metabolic flux in the TCA cycle,and it can naturally secrete TCA cycle intermediates including citric acid,isocitrate and α-ketoglutarate.Therefore,Y.lipolytica may be an ideal host for 5-ALA with succinyl-CoA as a precursor.In addition,Y.lipolytica has strong acid-base tolerance,and can grow and metabolize normally under low pH conditions,without adding acid-base regulators during the fermentation process,reducing the risk of bacterial contamination,and helping to maintain product stability.At present,there is no research report on the production of 5-ALA in Y.lipolytica.This paper plans to introduce exogenous 5-ALA synthesis-related genes into the succinate dehydrogenase SDH-deficient Y.lipolytica strain constructed in the early stage of the research group to achieve 5-ALA synthesis,and to improve 5-ALA production by means of metabolic pathway modification,fermentation optimization,etc.,and construct high-efficiency 5-ALA synthetic strains.First,by comparing the effects of overexpression of endogenous and Saccharomyces cerevisiae 5-ALA synthase genes on 5-ALA accumulation in Y.lipolytica,SchemⅠ from S.cerevisiae was selected for 5-ALA synthesis;The most suitable strain PGC62 for 5-ALA production was screened out of Y.lipolytica strains,and by introducing SthemA of Salmonella typhimurium and EchemL of Escherichia coli,the C4 and C5 synthetic pathways were co-expressed in the engineering strain of Y.lipolytica to obtain The engineering strain PGC62-I4L;finally,after optimization of fermentation conditions,the high yield of 5-ALA with yeast as the host was achieved for the first time in fed-batch fermentation,and the yield was about 2216.8 mg/L.In order to further improve the synthesis efficiency of 5-ALA,researches on the regulation of metabolic pathways were carried out in this paper.Due to the strong metabolic capacity of the tricarboxylic acid cycle and the porphyrin pathway downstream of 5-ALA,using CRISPR interference(CRISPRi)technology to inhibit the expression levels of genes encoding succinyl-CoA synthase and 5-ALA dehydratase has no effect.Therefore,we focused our engineering strategy on enhancing overexpression of the 5-ALA biosynthetic pathway.By comparing the catalytic activity of 5-ALA synthase from different species in Y.lipolytica,RpahemO from Rhodopseudomonas palustris with stronger 5-ALA synthesis ability was obtained.The multi-copy integration of this gene was achieved in the PGC62 strain using a homology-independent random genome integration method and based on fermentation broth color screening.In shake flask fermentation,the 5-ALA yield of RpahemO high expression integration strain PGC62mcO reached 3541.8 mg/L.To further enhance intracellular 5-ALA synthase activity,pyridoxal 5’-phosphate kinase and pyridoxal 5’-phosphate transporter were overexpressed in PGC62mcO to enhance cofactor supply.However,this method did not improve 5-ALA production.Our study shows that,compared with the metabolic regulation of the upstream and downstream pathways,enhancing the strength of the C4 pathway is the most effective strategy to improve the 5-ALA synthesis efficiency and obtain 5-ALA high-producing strains in Y.lipolytica.