Engineering Candida phangngensis – an oleaginous yeast from the Yarrowia clade – for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction

Authors: Quarterman, Joshua; Slininger, Patricia - Pat; Hector, Ronald - Ron; Dien, Bruce

Submitted to: Federation of European Microbiological Societies Yeast Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/15/2018
Publication Date: 9/21/2018
Citation: Quarterman, J.C., Slininger, P.J., Hector, R.E., Dien, B.S. 2018. Engineering Candida phangngensis – an oleaginous yeast from the Yarrowia clade – for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction. Federation Of European Microbiological Societies Yeast Research. 18(8):foy102. https://doi.org/10.1093/femsyr/foy102.
DOI: https://doi.org/10.1093/femsyr/foy102

Interpretive Summary: In this study, a Yarrowia type yeast was genetically engineered for robust growth and improved lipid production on cellulosic sugars. Yarrowia produces and stores lipids internally as droplets when grown on sugars in select types of media. In an earlier study, ARS researchers discovered that the recently isolated Yarrowia type yeast, named Candida phangngensis, produces approximately twice as much lipids as the most commonly reported upon Yarrowia yeast. Furthermore, these lipids show a composition predicted to make them suitable for use in biodiesel. However, economics will require using cellulosic sugars for cell growth, which is technically challenging because the process used to extract the sugars also creates chemicals that are toxic to microbes. In this study, sugars were manufactured in-house by pretreating a high biomass cultivar of switchgrass with dilute sulfuric acid, neutralizing it, and treating with commercial cellulase enzymes. Due to inhibitor sensitivity, C. phangngensis showed delayed growth in switchgrass hydrolysate cultures. It was hypothesized that growth could be improved by introducing a gene from distillers’ yeast to express an enzyme that was predicted to chemically alter the dominant inhibitory chemicals. Expressing that gene cut the lag time until growth by 68% by the expected mechanism. Introduction of a second gene, in this case encoding for the enzyme catalyzing the terminal step in lipid formation, enhanced lipid production by 33% in the hydrolysate. While production of lipids from hydrolysate sugars is a newly emerging research area, this work will be of broad interest to the agriculture processing community and supporting researchers.

Technical Abstract: Candida phangngensis is an ascomycetous yeast and a phylogenetic relative of the industrial workhorse Yarrowia lipolytica. In our previous work, the two known isolates of C. phangngensis showed a heightened propensity for de novo lipid synthesis in an undetoxified lignocellulosic biomass hydrolysate as compared to the model oleaginous yeast Y. lipolytica. Here, we report that genetic tools already established for use in the latter organism—including promoters, expression vectors, antibiotic resistance genes, a transformation protocol, and the Cre/lox system for marker recycle—can be transferred to the newer member of the Yarrowia clade with little or no need for modifications. Using these tools, we engineered C. phangngensis for improved cellulosic lipid production by introducing two heterologous yeast genes. First, overexpression of Saccharomyces cerevisiae ADH6 enhanced in situ detoxification of aldehyde fermentation inhibitors that are generated during biomass pretreatment (e.g. furfural). The primary mechanism of inhibitor tolerance in ScADH6 strains involved NADPH-specific reduction of the aldehyde to the less toxic alcohol form. Subsequently, Y. lipolytica DGA1 expression boosted lipid accumulation in C. phangngensis by pulling additional carbon flux into the triacylglycerol synthesis pathway. In acid-pretreated switchgrass hydrolysate cultures, the final engineered strain JQCP04 showed a 58% decrease in lag time, 32% increase in lipid titer, and 162% faster overall xylose consumption rate as compared to wild-type PT1-17. Furthermore, we expect that this study will generate new interest in the highly oleaginous yeast C. phangngensis, which is closely related to a safe, industrial species, and is shown here to be quite amenable for genetic manipulation.