Gene expression of the yeast Wickernhamomyes anomalus CCMA 0358 during biosurfactant production using kitchen waste oil as substrate and identification of bacteria present in tea tissue culture (Camellia sinensis) through metagenomics analysis

Abstract

Biosurfactants are secondary metabolites, of an amphiphilic nature, with potential for application in detergents, drugs, cosmetics and in the food industry. The aim of this study was to purify and identify the structure of the biosurfactant produced by the yeast Wickerhamomyces anomalus (CCMA 0358) using kitchen waste oil (KWO) as a carbon source and its isolated fractions, as well as to evaluate changes in gene expression during the production of the biosurfactant. The biosurfactant was produced in a bioreactor, using previously optimized culture medium, with a different carbon source, for 12h. The crude biosurfactant and its isolated and purified fractions (F1 and F2) were characterized by Thin Layer Chromatography (TLC), Liquid Chromatography coupled to a Mass Spectrum (LC-MS) and Nuclear Resonance Magnetic (NMR). It was expected to find a glycolipid biosurfactant, however, our spectrum shows that this biosurfactant does not have a sugar chain in its structure, but our spectrum suggests that it is a triglyceride. The analysis of gene expression based on RNA-seq identified more than 6,000 expressed genes, in which 829 are expressed differently between control samples (without KWO) and treatment (with KWO). The precursor enzymes to produce biosurfactants were presented in this study, having greater expression in precursors of the lipid and fatty acid metabolism pathways, where one of the fractions of the biosurfactants is produced. The crude biosurfactant, F1 and F2 were also evaluated against the Gram-positive bacterium Bacillus cereus and showed good antibacterial activity with MIC of 120, 240 and 60 μg/mL respectively and MBC of 480, 240 and 60 μg/mL, respectively. Tea (Camellia sinensis) is one of the most important drinks in the world and provides several secondary metabolites that are responsible for its rich taste and health benefits. In in vitro cultures, numerous losses due to bacterial contamination can occur, but several studies indicate that endophytes have a positive effect on plants. Therefore, this study shows the population of bacteria found in two varieties of tea tissue culture, using the metagenomic technique. In total, 17 bacterial DNAs have been identified, 10 of these species have been identified in both varieties, including Klebsiella pneumoniae, which can fix atmospheric nitrogen in a form that can be used by plants, Cronobacter sakazakii often found associated with plants and rhizosphere and is related to the solubilization of mineral phosphate and production of indole acetic acid and Paenibacillus spp. where many have proven to be important for agricultural and horticultural, industrial and medical applications. This study is the first to correlate microorganisms associated with the in vitro cultivation of C. sinensis.