毕赤酵母PAS_chr4_0427和PAS_chr2-2_0408基因缺失对神经酰胺合成的比较  

Comparison of the Effects of PAS_chr4_0427 and PAS_chr2-2_0408 Gene Deletions on Ceramide Synthesis in Pichia pastoris

作　　者：黄铭 吴佳欣 张目 胡晓清[1] HUANG Ming;WU Jiaxin;ZHANG Mu;HU Xiaoqing(School of Bioengineering,Jiangnan University,Wuxi 214122,China;Guangzhou Yuehui Cosmetics Co.Ltd.,Guangzhou 510000,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]广州悦荟化妆品有限公司,广东广州510000

出　　处：《现代食品科技》2025年第2期40-51,共12页Modern Food Science and Technology

基　　金：国家重点研发计划项目(2021YFC2100900)。

摘　　要：神经酰胺具有维护皮肤屏障、抗炎抗氧化等多种作用,其生物合成具有重要市场前景。目前神经酰胺主要利用酿酒酵母合成,而尚无利用重要工业宿主—巴斯德毕赤酵母(Pichia pastoris)合成的报道,该研究系首次通过基因改造P.pastoris GS115合成神经酰胺。通过CRISPR-Cas9技术依次敲除关键基因PAS_chr4_0427和PAS_chr2-2_0408,成功构建了GS115-O和GS115-OC两个突变株。与GS115相比,这两个突变株的最大生物量分别下降了17.03%和24.06%,而其神经酰胺的相对含量分别提升了3.80倍和4.75倍。在GS115-O和GS115-OC中合成了67种神经酰胺,包括28种植物神经酰胺,相较于GS115增加了5种;28种神经酰胺的相对含量增幅超过了100倍,最高达到了8533.98倍。此外,突变株中磷脂酸、甘油二酯、甘油三酯及特别是肌醇磷酸神经酰胺(PI-Cer)的含量显著增加,尤其是PI-Cer的增长幅度最大,这表明脂质代谢发生了显著变化,促进了神经酰胺向复杂鞘脂的转化。该研究表明GS115-O和GS115-OC是极具潜力的神经酰胺生产菌株。Ceramides play critical roles in maintaining skin barrier functions and exhibit anti-inflammatory and antioxidant properties;thus,their biosynthesis has substantial market potential.Ceramide synthesis has primarily been achieved using Saccharomyces cerevisiae,whereas there have been no reports on ceramide synthesis using the important industrial host Pichia pastoris.This research represents the first instance of ceramide synthesis via genetically modified P.pastoris GS115.The key genes PAS_chr4_0427 and PAS_chr2-2_0408 were sequentially targeted for deletion using CRISPR-Cas9 technology,leading to the construction of the mutant strains GS115-O and GS115-OC.The maximum biomasses of GS115-O and GS115-OC were reduced by 17.03%and 24.06%,respectively,compared with that of GS115,whereas the relative contents of ceramides in these mutants were increased by 3.80-and 4.75-fold,respectively.The analysis revealed the synthesis of 67 ceramide species,including 28 phytoceramides,using GS115-O and GS115-OC,marking an additional five species than that produced using GS115.Notably,the relative contents of 28 ceramide species increase by more than 100-fold,with the highest reaching 8533.98-fold.Furthermore,the contents of phosphatidic acid,diacylglycerols,triacylglycerols,and particularly inositol phosphoceramide were significantly increased in the mutants,with the inositol phosphoceramide content showing the most substantial increase.These results indicate that remarkable lipid metabolic alterations occur,accompanied by increased conversion of ceramides into complex sphingolipids.GS115-O and GS115-OC show great potential for ceramide production,including in industrial applications.

关 键 词：毕赤酵母 神经酰胺 CRISPR-Cas9技术 超高效合相色谱串联质谱 脂质组 

分 类 号：TQ920.6[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]