威克汉姆西弗酵母遗传操作系统的建立及其合成四乙酰基植物鞘氨醇的应用

Establishment and application of a genetic operating system in Wickerhamomyces ciferrii for the synthesis of tetraacetyl phytosphingosine

摘要:

威克汉姆西弗酵母(Wickerhamomyces ciferrii,W.c)是一种异宗生殖的非常规酵母，能够大量生产并分泌四乙酰基植物鞘氨醇(tetraacetyl phytosphingosine,TAPS)。由于其在TAPS生产方面的优异性能，本研究旨在构建W.c的遗传操作系统，以提升四乙酰基植物鞘氨醇(TAPS)的产量，并通过诱变和基因工程手段筛选高产菌株，从而为进一步开发鞘脂类代谢产物的工业生产奠定基础。本研究通过选取2个自主复制元件(CEN,2μ)并挖掘11个内源启动子元件以建立威克汉姆西弗酵母中的遗传操作系统，发现过量表达鞘脂代谢内部途径的Syr2和Lcb2能显著提高TAPS的产量。同时使用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)和流式细胞术建立了威克汉姆西弗酵母单倍体交配型鉴定方法，从双倍体ATCC 14091出发，筛选到5株单倍体菌株。其中a-型单倍体W.c 140生产TAPS水平最高，产量达到4.74 mg/g，滴度达到32.61 mg/L。使用常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术和本研究构建的遗传操作技术获得诱变菌株W.c 140-A9和W.c 140-A11及过表达重组菌株W.c 140 OELcb2和W.c 140 OESyr2。与出发株W.c 140相比，诱变株TAPS产量分别提高了61.39%和67.09%。过表达重组菌株产量提高至原来的2.24和2.56倍，达到10.60 mg/g和12.14 mg/g，超过了双倍体ATCC 14091的产量。本研究所构建的遗传操作系统和获得的出发菌株为后续在威克汉姆西弗酵母中建立更多遗传工程技术奠定了基础。

Abstract:

Wickerhamomyces ciferrii (W.c), an unconventional heterothallic yeast species, is renowned for its high production of tetraacetyl phytosphingosine (TAPS). Due to its excellent performance in TAPS production, this study aimed to construct a genetic operating system of W.c to enhance the production of TAPS and to screen high-yielding strains by mutagenesis and genetic engineering, thus laying the foundation for further development of industrial production of sphingolipid metabolites. In this study, we selected two autonomous replication elements (CEN, 2μ) and mined 11 endogenous promoter elements to establish a genetic operating system in W. ciferrii. The overexpression of Syr2 and Lcb2 in the sphingolipid metabolism pathway significantly increased the production of TAPS. Meanwhile, we established a method for the identification of haploid mating types of W. ciferrii by combining RT-PCR and flow cytometry. Five strains of W. ciferrii with different mating types constructed from the standard diploid W. ciferrii ATCC 14091 were screened out. A-type haploid W.c 140 showcased the highest production of TAPS with a yield of 4.74 mg/gand a titer of 32.61 mg/L. Mutant strains W.c 140-A9 and W.c 140-A11 were induced by atmospheric pressure room temperature plasma mutagenesis. The recombinant strains W.c 140 OELcb2 and W.c 140 OESyr2 with overexpression were constructed with the genetic operating system established in this study. The TAPS yields of the mutant strains increased by 61.39% and 67.09%, respectively, compared with that of starting strain W.c 140. The recombinant strains cultured in the LCBNB medium achieved yields of 10.60 mg/gand 12.14 mg/g, respectively, representing 2.24 and 2.56 times of that in strain W.c 140. Moreover, the yields of the two recombinant strains were significantly higher than that of the diploid strain ATCC 14091. The genetic operating system and the haploid strain W.c 140 established in this study provide a basis for the subsequent establishment of genetic engineering tools for W. ciferrii.