AUTHOR=Liu Jun , Zhao Guoqin , Zhang Xinyu , Song Xin 

TITLE=Identification of Four Secreted Aspartic Protease-Like Proteins Associated With Sophorolipids Synthesis in Starmerella bombicola CGMCC 1576

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.737244

DOI=10.3389/fmicb.2021.737244

ISSN=1664-302X

ABSTRACT=<p>The non-pathogenic yeast <italic>Starmerella bombicola</italic> CGMCC 1576 is an efficient producer of sophorolipids (SLs). The lactonic SLs are mainly produced with yeast extract, and the acidic SLs are mainly produced with ammonium sulfate. Naturally produced SLs are a mixture of various lactonic and acidic SLs. Usually, the SL mixture is not well separated technically, and the separation cost is relatively high. In order to reduce the cost of separation, four secreted aspartic protease-like proteins were identified through proteomic analysis of fermentation broth of <italic>S. bombicola</italic> under different nitrogen source conditions. The coding genes of the four proteins, namely, <italic>sapl1</italic>, <italic>sapl2</italic>, <italic>sapl3</italic>, and <italic>sapl4</italic>, are of high sequence similarity (above 55%) and included in a gene cluster. The expression of the four genes was significantly upregulated on (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> compared with that on yeast extract. The four genes were deleted together to generate a strain Δ<italic>sapl</italic>. The titer of SLs in Δ<italic>sapl</italic> reached 60.71 g/L after 5 days of fermentation using (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> as the nitrogen source and increased by 90% compared with the wild-type strain. The concentration of acidic SLs was 55.84 g/L, accounting for 92% of the total SLs. The yield of SLs from glucose (g/g) by Δ<italic>sapl</italic> was 0.78, much higher than that by wild-type strain (0.47). However, no increase of SLs production was observed in Δ<italic>sapl</italic> under yeast extract condition. Compared with that of the wild-type strain, the expression levels of the key genes for SLs synthesis were all upregulated to varying degrees in Δ<italic>sapl</italic> under (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> conditions, and particularly, the expression level of <italic>ugta1</italic> encoding UDP glucosyltransferase was upregulated by 14.3-fold. The results suggest that the <italic>sapl</italic> gene cluster is negatively involved in the production of SLs in the case of (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> by restraining the expression of the key genes involved in SLs synthesis. The Δ<italic>sapl</italic> strain is an excellent producer of high-titer and high-yield acidic SLs.</p>