Alkane Utilization, The Expression And Function Of Cytochrome P450 In Sophorolipid Synthesis In Wickerhamiella Domercqiae

Sophorolipids (SLs) are a kind of glycolipid biosurfactants mainly produced by several species of non-pathogenic yeasts. Different SLs have different surface and biological activities. Generally, lactonic SLs exhibit stronger biological activity and acidic SLs have better ability of foam formation and solubility. Because of their lower toxicity, good biodegradability and well biocompatibility among all biosurfactants, SLs have great application prospect in many sectors such as detergent, cosmetic, petroleum and environmental protection. Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 (W. domercqiae), a high SL-producing yeast strain, was isolated from sewage by author’s laboratory. The production of SLs by W. domercqiae can reach up to 130 g/L and W. domercqiae has better potentials for application. Therefore, a thorough study of the genes involved in the synthetic pathway of SLs in W. domercqiae will promote the development and applications of SLs.The main research aspects and results are as following:1. The alkane utilization of W. domercqiaeW. domercqiae was cultured in fermentation media containing different alkanes, including n-octane, n-decane, n-dodecane, n-tetradecane, n-hexadecane and n-octadecane. The results of SL production measured by Anthrone method and SL components analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that n-hexadecane was utilized best, n-tetradecane and n-octadecane were utilized better and n-octane, n-decane and n-dodecane were utilized weakly. In addition, we found that more lactonic SLs were produced in the presence of n-octane, n-decane, n-dodecane, n-tetradecane and n-hexadecane while more acidic SLs were produced from n-octadecane.2. The function of alkanes on expression levels of CYP450 genes in W. domercqiaeBy analyzing the results of W. domercqiae genome sequencing, seven new cytochrome P450 (CYP450) genes were found and named as CYP52-M1, CYP52-N1, CYP52-N2, CYP52-E1, CYP52-E2, CYP51 and CYP-X, respectively. W. domercqiae was cultured in fermentation media containing different alkanes, including n-octane, n-decane, n-dodecane, n-tetradecane, n-hexadecane and n-octadecane. The expression levels of CYP450 genes in W. domercqiae demonstrated that CYP52-M1, CYP52-E1, CYP52-E2, CYP52-N1 and CYP51 genes played important roles in alkane metabolism. Furthermore, CYP51 gene was constitutively expressed without the induction of alkanes, CYP52-M1, CYP52-E1, CYP52-E2 and CYP52-N1 genes were inducibly expressed. We also found that CYP52-N2 and CYP-X genes did not play the same important roles as other P450 genes in W. domercqiae in alkane metabolism.3. The function of CYP450 genes in W. domercqiaeCYP52-E1, CYP52-E2 and CYP52-N2 genes from W. domercqiae were successfully knocked out and respectively named as ΔCYP52-E1, ΔCYP52-E2 and ΔCYP52-N2. When wild strain W. domercqiae and its three deletion mutants ΔCYP52-E1, ΔCYP52-E2 and ΔCYP52-N2 were cultured in the fermentation media with oleic acid, the SL production of the three deletion mutants ΔCYP52-E1, ΔCYP52-E2 and ΔCYP52-N2 were lower than that of the wild strain, but much higher than that of ΔCYP52-M1. Therefore, it can be inferred that CYP52-E1, CYP52-E2 and CYP52-N2 maybe isoenzymes of CYP52-M1. In addition, CYP52-E1 and CYP51 failed to be heterologously expressed in prokaryotic and eukaryotic system. The expression levels of related genes can be detected, which demonstrated that the related genes were all transcribed in recombinant strains. However, target proteins were not induced. We speculated that related genes can probably be not translated and target proteins were difficult to be secreted or the two target proteins were difficult to be heterologously expressed.