Analysis of the response mechanism in lipid degradation of key gene LDH1 knockout strains of Saccharomyces cerevisiae under levulinic acid stress.

Abstract

Levulinic acid (LA) is the main toxic by-product in the production of fuel ethanol, and its large-scale emission adversely affect the ecological environment. In order to effectively remove LA from the liquid waste, microbial degradation methods are adopted but the challenge is that microorganisms cannot fully tolerate LA in the waste. Therefore, it is particularly important to explore the tolerance mechanism of microorganisms to LA. In this study, the whole-genome knockout library scanning and sensitive knockout strain identification were carried out. In addition, subcellular structures such as mitochondria, vacuoles, and endoplasmic reticulum as well as reactive oxygen species (ROS) accumulation were observed under a fluorescence microscopy after stained with fluorescent dyes such as 2'7'-DCF diacetate, Mito Tracker Green FM, Vacuole Membrane Marker MDY-64, and ER-Tracker Red dye. We also performed genomic sequencing on the wild-type strain and knockout strain. Through comparative genomic analysis, it's been found that the LDH1 (YBR204C) gene in Saccharomyces cerevisiae helps promote the clearance of intracellular reactive oxygen species, and the deletion of LDH1 leads to a more-than-two-fold down-regulation of genes related to cell membrane, cell wall, and cell cycle. By measuring the transcriptome and metabolome of the LDH1 knockout strain (ldh1Δ) under LA stress and comparing it with the wild-type strain BY4741, we found that under the condition of LDH1 knockout, the accumulation of NAD⁺ intermediates would be activated, disrupting normal cell functions and causing cell damage. The LDH1 gene knockout also affects the Methyl Cycle in the cell, which is closely related to the accumulation of ROS. These research results make it possible to create a new genetically modified strain of S. cerevisiae with desired higher tolerance which enhances degradation efficiency and reduces cost.