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

Jun Liu,Xinyu Zhang,Guoqin Zhao,Xin Song

doi:10.3389/fmicb.2021.737244

Abstract

The non-pathogenic yeast Starmerella bombicola 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 S. bombicola under different nitrogen source conditions. The coding genes of the four proteins, namely, sapl1, sapl2, sapl3, and sapl4, are of high sequence similarity (above 55%) and included in a gene cluster. The expression of the four genes was significantly upregulated on (NH4)2SO4 compared with that on yeast extract. The four genes were deleted together to generate a strain Δsapl. The titer of SLs in Δsapl reached 60.71 g/L after 5 days of fermentation using (NH4)2SO4 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 Δsapl was 0.78, much higher than that by wild-type strain (0.47). However, no increase of SLs production was observed in Δsapl 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 Δsapl under (NH4)2SO4 conditions, and particularly, the expression level of ugta1 encoding UDP glucosyltransferase was upregulated by 14.3-fold. The results suggest that the sapl gene cluster is negatively involved in the production of SLs in the case of (NH4)2SO4 by restraining the expression of the key genes involved in SLs synthesis. The Δsapl strain is an excellent producer of high-titer and high-yield acidic SLs.

Highlights

Biosurfactants are amphiphilic microbial molecules with hydrophilic and hydrophobic moieties
The synthesis of SLs begins with the hydroxylation of fatty acids under the catalysis of cytochrome P450 family Cyp52m1 (Van Bogaert et al, 2009), the acidic SLs were formed by the catalysis of two UDP glucosyltransferases (Ugta1 and Ugtb1), and the substrates of two UDP glucose (UDPG) were produced by glucose metabolism (Saerens et al, 2011a,c)
When yeast extract was used as the nitrogen source, a large amount of lactonic SLs was produced during the fermentation of the wild-type stain of S. bombicola

Summary

Introduction

Biosurfactants are amphiphilic microbial molecules with hydrophilic and hydrophobic moieties. Biosurfactants have drawn more and more attentions of different industries due to their great advantages than their petroleum-based chemically synthesized counterparts, such as structural diversity, extensive foaming activities, low toxicity, greater biodegradability, bioavailability, biocompatibility, ecological acceptability, environmental friendliness, ability to function in wide ranges of pH, temperature, and salinity, as well as greater selectivity and lower critical micelle concentration (CMC). They exhibit good application prospects in the fields of antibacterial, antiviral, antitumor, and other pharmacology and immunity (Singh and Cameotra, 2004; Santos et al, 2016; Gaur et al, 2019; Valotteau et al, 2019). A protein Bro essential for SLs synthesis in Starmerella bombicola was revealed (Liu et al, 2020)

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