Disruption of the Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating .

is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 () gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker gene in the background of a lipid-accumulating strain overexpressing ATP citrate lyase () and diacylglycerol acyltransferase 1 () genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases- construct at the disrupted site in the genome of host cells. Importantly, we observed that the disruption of the gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased cellulolytic activity by 30%, whereas the "total newly formed FAME (fatty acid methyl esters)" increased by 16% in comparison with a random integrative cellulase-expressing mutant in the same YNB-Avicel medium. The disruption platform demonstrated in this study provides a potent tool for the further development of as a robust host for the expression of cellulases and other commercially important proteins.