Metabolic Engineering Of Starmerella Bombicola For Producing Acid-type Sophorolipid

Sophorolipid is a kind of biosurfactant,which not only has the general properties of solubilization,emulsifying,wetting,foaming,dispersing,reducing surface tension,but is extremely resistant to the environment.S.bombicola can produce sophorolipids by fermendation,but sophorolipids have different types such as acid type,lactone type and acetylated type,and various structures.This study intends to construct a strain of S.bombicola engineered with high acid-producing sophorolipid through metabolic engineering.Main results in this paper research were as follows:?1?Using the bioinformatics tool,a possible peroxisome synthesis factor PEX10 was isolated from the genome of S.bombicola and its function was identified.The PEX10 gene deletion strain was constructed.The gene is deficient,which has no effect on the metabolism of dodecane and glucose,but it seriously hinders the utilization of rapeseed oil by the cells,so that the production of sophorolipids is also greatly reduced.?2?SBLE gene deletion strain and SBLE overexpression strain were constructed by gene knockout and gene overexpression.And the SBLE gene transcription level was detected by real-time PCR?qPCR?.The SBLE transcription rate of the SBLE overexpression strain was more than 30 times that of the wild-type strain,and it was not detected in the SBLE deficient strain.The fermentation results showed that only the acid-type sophorolipid was detected in the fermentation product of the SBLE gene-deficient strain,The fermentation product of the SBLE overexpressing strain was mainly lactone type sophorolipid,and no acid type sophorolipid was detected.?3?The hygromycin gene recycling system was constructed through rational design.Under the control of S.bombicola galactose-inducible promoter,combined with?-Rec serine recombinase,construct a hygromycin resistance screening marker ejection system,the system mainly includes hygromycin resistance expression cassette,Rec serine recombinase and two SIX recombination sites.The construction of the system enables self-excising of the hygromycin resistance marker and multiple use of hygromycin labeling,thereby enabling multi-gene editing of S.bombicola.?4?Several recombinant strains with high acid-producing sophorolipids were constructed based on the Rec-six gene editing system of the hygromycin resistance marker gene recycling system.the key gene of the synthetic lactone-type sophorolipid,the lactamase gene SBLE,was knocked out to obtain a strain produced only acid-type sophorolipid.The engineering strain SBSL1??sble::six?further homologously overexpresses the glucosyltransferase gene UGTB and knocks out the peroxisome membrane transporter gene PXA1 to construct a yeast engineering strain with high acid-producing sophorolipids.Compared with the starting strain,the recombinant S.bombicola fermented oleic acid can synthesize a single acid-type sophorolipid,but no longer synthesizes lactone-type sophorolipids,and the yield of acid-type sophorolipids is increased from 20 g·L^-11 to 44 g·L^-1.