神经酰胺是应用于化妆品和药品的一类脂质分子,利用微生物代谢工程合成神经酰胺具有重要前景。巴斯德毕赤酵母(Pichia pastoris)是优良的工业宿主,目前尚无利用P.pastoris强化神经酰胺的研究。首先构建P.pastoris GS115ΔPAS_chr3_0329(G-K)作为底盘,然后在G-K中敲除神经酰胺合成负反馈基因PAS_chr4_0427(G-KO),并利用脂质组学技术探究神经酰胺的组成及含量变化。结果表明,与G-K相比,G-KO中神经酰胺在总脂质中相对含量增加了628.13%,达到9.32%;并促进合成更多具有不饱和长链碱基的神经酰胺。与此同时,G-KO中鞘脂相对含量增加,而磷脂相对含量降低。对P.pastoris中神经酰胺丰度最高的Cer(d18∶0)神经酰胺进行HPLC分析,显示G-KO中Cer(d18∶0)神经酰胺合成量达90.22 mg/L,相比G-K提升了872.20%。该研究证实P.pastoris可用于神经酰胺合成,PAS_chr4_0427缺失株G-KO具有良好的神经酰胺合成能力,具有工业发酵制备神经酰胺的潜力。
Ceramide is a class of lipid molecules used in cosmetics and pharmaceuticals.The synthesis of ceramide by microbial metabolic engineering has important prospects.Pichia pastoris is an excellent industrial host.At present, there is no report on the use of P.pastoris to enhance synthesis of ceramide.Firstly, P.pastoris GS115ΔPAS_chr3_0329 (G-K) was constructed as the chassis, and then the ceramide synthesis negative feedback gene PAS_chr4_0427 was knocked out in G-K to obtain G-KO, and the composition and content of ceramide were investigated by lipidomics.Results showed that compared with G-K, the relative content of ceramide in total lipid in G-KO increased by 628.13% to 9.32%.Moreover, the deletion of gene PAS_chr4_0427 promoted the synthesis of more ceramides with unsaturated long-chain bases.At the same time, the relative content of sphingolipids in G-KO increased, while the relative content of phospholipids decreased.HPLC analysis of Cer(d18∶0) ceramide with the highest ceramide abundance in P.pastoris showed that the content of Cer(d18∶0) ceramide in G-KO reached 90.22 mg/L, which was 872.20% higher than that of G-K.This study confirmed that P.pastoris could be used for ceramide synthesis.The PAS_chr4_0427 deletion strain G-KO has excellent ceramide synthesis ability and has the potential for industrial fermentation to prepare ceramide.
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