| Surfactants are amphipathic compounds with both hydrophilic and hydrophobic moieties and have a wide range of applications.Most surfactants in use today are petroleum-based and chemically synthesized.However,such chemically-synthesized surfactants are derived from nonrenewable oil resources and difficult to break down through the action of microorganisms,and thus cause significant ecological problems.Biosurfactants have several advantages when compared to their chemically produced counterparts.Such advantages involve non-toxicity,excellent foaming activity,biodegradability,biocompatibility,eco-friendliness,effectiveness and stability at extreme environments(active in broad range of pH,temperature and salt concentrations),and long storage time.Therefore,biosurfactants attract increasing interests from researchers.Sophorolipids(SLs)are an important biosurfactant with the highest yield among all the biosurfactants and will be one of the most potential sustainable substitutes for chemical surfactants.The key enzymes involved in the SLs synthesis pathway have been identified,but there are few reports on the proteins that affect SLs synthesis or the transcription factors that regulate SLs synthesis.In addition,the pH of the fermentation broth not only affects the titers of SLs,but also affects the compositions of SLs.The relationship between pH-related proteins and SLs synthesis has not been reported yet.Therefore,the research on the proteins related to SLs synthesis will help to understand the regulation mechanism of SLs synthesis and can be used to guide the genetic manipulation of S.bombicola.The research on transcription factors can reveal the regulation mechanism of SLs synthesis in S.bombicola and provide a theoretical basis for constructing engineered strains with high SLs production.The main research contents and results of this paper are as follows:1.Screening of sophorolipids synthesis related proteins and transcription factorsWhen pH value was lower than 2.0,S.bombicola mainly produces acidic SLs.When pH was adjusted to 3.0 or 3.5,a large amount of lactonic SLs will be produced.The key genes for SLs synthesis have been identified,however,the regulatory mechanism of SLs synthesis pathway has not yet been studied.Through bioinformatics analysis combined with S.bombicola genome data,two pH-related proteins Bro1 and Rlp which affect SLs synthesis in S.bombicola were identified.Four secreted aspartic protease-like proteins were identified through proteomic analysis of fermentation broth of S.bombicola under the condition of ammonium sulfate or yeast extract as the nitrogen source,and the effect of Sapl proteins on the synthesis of SLs was analyzed.In addition,transcription factor genes were predicted to construct a library of transcription factor deletion strains by analyzing the fungal transcription factor database and S.bombicola genome sequencing data.Ninety strains were deleted,and the SLs fermentations of mutant strains were analyzed.It was for the first time that 12 transcription factors involved in SLs synthesis were mined.2.The study of the function of Brol and Rlp proteinTo identify the function of the protein Bro1 in S.bombicola,the deletion,overexpression and complementary mutant strains were constructed.The deletion mutant no longer produced SLs.However,there is no increase in the titer of SLs in the overexpression strain of bro1,which indicated that bro1 is indispensable for the synthesis of SLs.Transcriptome analysis indicated that the expression levels of the key enzyme genes of SLs biosynthetic pathway were significantly down-regulated in the?bro1,especially the expression level of cyp52ml that encodes the first rate-limiting enzyme in SLs synthesis pathway was downregulated 13 folds and the expression of fatty acid ?-oxidation-related enzymes was also down-regulated.This indicates that Bro1 was essential for SLs synthesis.The titer of SLs in ?rlp reached 76.5 g/L,which was increased by 17.9%compared with that of the wild-type strain.Combining the results of transcriptomics analysis and real-time fluorescent quantitative PCR,it was found that the expression levels of the key genes sble and UGPase for SLs synthesis in ?rlp were 2.1 and 3.9 times higher than those of the wild-type strain,respectively.UDP-glucose was a key precursor for the synthesis of SLs.Therefore,the deletion of the rlp gene brought the increase in the production of SLs by allowing more carbon fluxes to flow to the synthesis of SLs.3.Functional analysis of transcription factors related to sophorolipids synthesisTwelve transcription factors related to SLs synthesis were screened in S.bombicola.Among them,the SLs production of 6 deleted strains of transcription factor genes increased to varying degrees,and the other 6 deleted strains of transcription factors no longer produced SLs.The transcription factor ztfl with the highest increase in SLs production in deleted strains,and the genes swi3 and ada2 with the largest increase in the production of overexpression strains were used for transcriptomics analysis.It was found that the deleted strain of ztfl promoted the metabolism of sucrose or starch metabolism into glucose,and increased the synthesis of pyruvate precursor substances.The metabolism of pyruvate can provide acetyl-CoA and ATP for the synthesis of SLs.The expression levels of key genes responsible for SLs synthesis were all significantly down-regulated in ?swi3 and ?ada2.In the overexpression strains of swi3 and ada2,the expression of an esterase gene and one gene encoding ATPdependent kinase yfh7 were significantly up-regulated.Yfh7 was one of the P-loop proteins with unknown function and related to many cellular processes,such as signal transduction,metabolism and regulation.The esterase was involved in the lactonization of acidic SLs.The expressions of proteases related to amino acid transport,carbohydrate transport and metabolism were down-regulated in the overexpression of swi3.The changes in SLs production in the overexpression of ada2 may affect fatty acid metabolism,amino acid metabolism,and pyruvate metabolism.4.Multiple-gene knockout to construct engineered strains with high-producing SLsIn order to construct a high-yield SLs engineered strain,by weakening the competitive pathway with SLs synthesis,more carbon and energy can be used for the synthesis of SLs.Proteins and transcription factors that affect SLs synthesis were used as targets.The deletion strain of rlp was constructed with hygromycin resistance selfdeleting markers.The hygromycin gene was induced to delete in ?rlp.On this basis,three double knockout strains ?rlp?ztf1,?rlp?leu3 and ?rlp?gcl were constructed by the deletion of transcription factor genes.Among them,the production of SLs in ?rlp?gcl was the same as that of the single knockout strain ?rlp,and the highest total SLs production was achieved by ?rlp?leu3,which reached 93.99 g/L.The?rlp?gcl was selected as the starting strain of three-gene knockout strain,ztf1 was knocked out in ?rlp?leu3,and a knockout strain(?rlp?leu3?ztf1)with three genes knocked out was obtained.The titer of total SLs was 97.44 g/L in ?rlp?leu3?ztf1,which was increased by 3.67%compared to that of ?rlp?leu3,and the carbon source conversion rate(yield,g/g)which represents the ratio of the titer of SLs to the total amount of added substrate(glucose and oleic acid)was 0.70,which was higher than the reported 0.68(calculated based on the consumed carbon source).The titer of total SLs in ?rlp?leu3?ztf1 increased by 50.15%.This shows that the multi-gene knockout strategy has been proven to be effective in constructing high-producing SLs engineered strains and the obtained engineered strain can be used for reduced-cost large-scale production of SLs. |
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