Abstract
Yarrowia lipolytica 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 Y. lipolytica 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 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 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 in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.
Highlights
Yarrowia lipolytica has a six-decade-long history of industrial application for the production of nutritional products, organic acids, and erythritol
It was reported that the overexpression of Mus musculus ATP citrate lyase (ACL) enhanced the lipid accumulation in Y. lipolytica (Elshourbagy et al, 1992; Zhang et al, 2014), mouse ACL (NM_001199296) was selected for overexpression in Y. lipolytica in this study because of the relative simplicity
We successfully demonstrated the targeting of the Snf1 site for coexpression of three cellulase genes fused with the recyclable selection markers HisG–URA3– HisG in Y. lipolytica
Summary
Yarrowia lipolytica has a six-decade-long history of industrial application for the production of nutritional products (e.g., including omega-3 and omega-6 fatty acids, and carotenoids), organic acids (especial citric acid), and erythritol (see recent reviews Sitepu et al, 2014; Madzak, 2015; Zhu and Jackson, 2015). One hinderance for fulfilling the full potential of these natural Yarrowia strains is that they cannot utilize cellulosic biomass as a carbon source, which contributes to the relatively high cost for biofuel production To overcome this hurdle, consolidated bioprocessing (CBP), in which microorganisms are engineered for the direct conversion of cellulosic biomass to biofuels and biochemicals has been proposed using Clostridium thermocellum (Lynd et al, 2005; Maki et al, 2009), Caldicellulosiruptor bescii (Chung et al, 2014), S. cerevisiae (Fan et al, 2012; Olson et al, 2012; Sun et al, 2012; Yamada et al, 2013; Kricka et al, 2014), Kluyveromyces marxianus (Chang et al, 2013), and Myceliophthora thermophila (Li et al, 2020). We and other workers have explored the potential of Y. lipolytica to serve as a CBP organism by screening and expressing fungal
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