| Surfactants are one of the most important chemicals. Because of their abilities of promoting liquefaction, emulsification and diffusion, surfactants are widely used in many industrial sectors, such as cosmetics, detergents, textiles, foods and pharmaceu-ticals. What’s more, for their properties of antisticking, defoaming, anti-corrosion, and anti-static, surfactans can be also applied in some specific fine chemical industries. But synthetic surfactants can be hardly degraded by living organisms, and some components are harmful to the environment. In the last decades, with the increasing awareness to environmental problems, more attentions have been diverted to biosur-factants due to their low toxicity, good biodegradability, and environmental friendli-ness. Sophorolipids are one of the most promising biosurfactants with the highest yields, and may replace the synthetic surfactants in the future.The main research contents and results are as follows:1. Identification, characterization of a monooxygenase Mo A responsible for the metabolism of a sophorolipid moleculeStrain Starmerella bombicola, a sophorolipid-producing yeast strain, was isolated from oil-containing wastewater by our laboratory and was originally identified as Wickerhamiella domercqiae var. Sophorolipid through physiological and biochemical identification test. Nowadays, It was reclassified to be S. bombicola based on se-quence analysis. The yeast S. bombicola CGMCC 1576 can produce abundant sophorolipids (SLs) including almost the equal proportion of acidic and lactonic SLs.In this study, a monooxygenase MoA responsible for the metabolism of a sophoro-lipid molecule, C18:2 diacetylated acidic sophorolipid (C18:2 DASL) was identified, through genomic analysis, protein modeling and gene knock-out strategy. The yield and compositions of SLs produced by the deletion mutant △moA changed dramatical-ly. In HPLC chromatogram, the UV absorption area of C18:2 DASL (one major acid-ic sophorolipid component) increased from 9.84×106 mAUxs to 34.26×106 mAUxs, by an increase of 248.17%, when oleic acid was used as the hydrophobic carbon source. When linoleic acid was used as the hydrophobic carbon source, compared with wild type and △moA, the content of C18:2 DASL produced by the overexpressed strain Peno::moA decreased significantly. Furthermore, the MoA enzyme was heter-ologously expressed in Escherichia coli JM109 (DE3) with a recombinant plasmid named pMAL-c2x-moA, and the purified enzyme was obtained through a MBP (maltose-binding protein) affinity chromatography column. The purified C18:2 DASL and C18:1 DASL were used as the substrates in the catalytic reaction of MoA enzyme, respectively. It turned to be that C18:1 DASL still remained in the MoA reaction sys-tem, but C18:2 DASL disappeared.2. The transport and catalytic capacity of a long-chain fatty acid transporter ALCSIt is generally believed that fatty acid transport is performed by protein-mediated and diffusional methods. So far, three kinds of membrane-bound fatty acid transport proteins have been found in higher eukaryotic cells, such as a rat adipocyte membrane protein, fatty acid translocase (FAT); a membrane-bound fatty acid-binding protein (FABPpm) (classified as mitochondrial aspartate aminotransferase); and a fatty acid transport protein (FATP) in 3T3-L1 adipocytes. The sophorolipid-producing yeast S. bombicola has an excellent ability to utilize exogenous long-chain fatty acids, howev-er, the performance of this process is still not clear.In this study, we found a gene encoding a long-chain acyl-CoA synthetase (ALCS), which was putatively identified as a membrane-bound long chain fatty acid transport protein and was found to contribute to the uptake of the long chain fatty acids. Dis-ruption of ales resulted in an impaired growth of the alcs-deleted mutant in MM me-dium containing different fatty acids (C12:0, C14:0, C16:0, C18:0, C22:0 and C24:0) as the sole carbon source and led to a dramatic decrease in the uptake of the boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823), a fluorescent long-chain fatty acid analogue. The absence of this alcs gene caused obvious phenotype changes. Compared with the wild-type strain, the yield and compositions of SLs produced by the deletion mutant of Aalcs::six changed markedly, almost no lactonic SLs were produced by Aalcs::six mutant, and the acidic SLs were composed of medium-chain SLs. To characterize the ALCS enzyme, this enzyme was heterologously expressed in E. coli JM109 (DE3) with a recombinant plasmid named pMAL-c2x-alcs, and the enzyme was purified through a MBP (maltose-binding protein) affinity chromatog-raphy column and confirmed to be of homogeneity by SDS-PAGE. The recombinant enzyme could catalyze the formation of long chain acyl-CoA when the long chain fatty acids and coenzyme A (CoA) were used as substrates.3. Enhancement of sophorolipids yields by overexpressing sophorolipids synthe-sis gene cluster through RecET recombinationUp to now, some genes contributing to the synthesis of SLs have been found, such as the terminal or subterminal fatty acid-hydroxylation enzyme (CYP52-M1), two UDP-activated glucose-dependent transferases (UGTA1 and UGTB1), a SLs acetyl transferase (AT), a SLs transport protein MDR, and the mysterious SL lactonization enzyme LIP1. What’s more, it was found that there was a biosynthetic gene cluster for SLs and the gene cluster contained almost all the above genes except lip1, in S. bom-bicola.In order to meet the increasing demands of SLs as chemicals in many industrial ar-eas and as potential medicines in pharmaceutical industry, it is essential to construct some mutants with the abilities of producing the SLs with higher yield and betfer bio-logical activities. In this study, a mutant with two copies of gene cluster for sophoro-lipid (SL) synthesis was constructed using the RecET system and named as OE-5. According to the anthrone method, the yield of SLs produced by the OE-5 mutant in-creased from 56.9 g/L to 79.6 g/L by an increase of 41.7%, and the yield of lactonic SLs increased from 23.1 g/L to 36.4 g/L by an increase of 57.6%. High performance liquid chromatography (HPLC) results indicated that the compositions of SLs mixture from OE-5 mutant and wild type were almost similar, but the UV absorption area of every SL peak from OE-5 mutant was higher than that from the wild type. Especially one kind of major acidic content and two kinds of major lactonic contents: C18:2NASL, C18:2 DLSL and C18:l DLSL, increased respectively by 806.59%, 35.20% and 62.52%, when oleic acid was used as the hydrophobic carbon source.4. The study of regulons about sophorolipids synthesisThrough the transcriptional analysis, we found an acidic extracellular protease (AxpA) in S. bombicola was markedly up-regulated on the fermentation medium sup-plemented with ammonium sulfate as the nitrogen source. We also found three other homologues named AxpB, AxpC, and AxpD in the genome of S. bombicola by local Blast analysis. A deletion mutant of these four acidic extracellular proteases was con-structed by homologous recombination using hph as the resistant marker. Compared with wild-type strain, the yield of SLs from the deletion mutant △axp::six was pro-moted when cultured on the fermentation with ammonium sulfate as the sole nitrogen source.Moreover, we also found a protein (PalA) responsible for the synthesis of SLs, reg-ulation of pH balance and calcium homeostasis. For further analysis of this protein, we constructed the knock-out mutant of target gene palA. The disruption of palA gene caused a growth defect in the presence of high concentrations of calciumchloride (0.75 M) or alkaline environment (pH 9.5). What’s more, the transcriptional levels of some key genes associated with sophorolipids synthesis, such as cyp52m1, ugta1, ugtbl, slat, and mdr, presented dramatically down-regulated when palA gene was de-leted. It was supposed that the PalA protein was localized in the cytoplasm through fluorescence microscopy and the fused recombinant protein PalA-EGFP. We guessed that the PalA protein was responsible for the signal transduction in the SLs synthetic pathway.5. The construction of a self-excising hph marker systemAs we know, the SLs producer S. bombicola has a resistant tolerance to many kinds of antibiotics, such as pyrithiamine, zeocin and G418. Among so many antibiotics, only hygromycin B has been applied to be a selection agent of S. bombicola success-fully. Besides the auxotrophic marker orotidine-5’-phosphate decarboxylase (ura3 gene), only the hph resistance marker was used for the genetic engineering of S. bom-bicola. Once the existing resistance markers have been used, the bare genetic selective markers will limit the future genetic modification of the SL-producing yeast.We constructed a self-excising hph marker system successfully, using the β serine recombinase and an inducible promoter of galactokinase (galk). The coding sequence of target gene was disrupted through replacing with the self-excising hph marker cas-sette. After induction by galactose, the deletion mutant lost its hph resistance cassette. In this way, we obtained a deletion mutant without any heterogeneous resistance maker. The construction of self-excising hph marker system makes it possible to use the hph resistance marker more than one time. This strategy makes complex study of multiple genes possible. It is a very promising tool in the study of SLs synthesis. |
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