Abstract
Yarrowia lipolytica, known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica. Three fungal cellulases, Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract-peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L-1, respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ∼4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain, suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. This is the first study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains.
Highlights
As one of the most abundant renewable resources today, lignocellulosic biomass is under development worldwide to produce biofuels and chemicals
The single-colony purified transformants were tested for their ability to hydrolyze cellulose by growth on Phosphoric acid swollen cellulose (PASC)-yeast extractpeptone-dextrose (YPD) agar for 6 days followed by Congo Red staining
The results indicate that the timeline profiling of cell mass weight of the parent control strains and cellulase-expressing transformants follows a similar trend as their growth curves illustrated in Figure 5, confirming that the co-expression of CBH I-cellobiohydrolase II (CBH II)-endoglucanase II (EG II) in YL165-1 comes at a small cost for the cells in terms of slightly reduced growth rate the late growth phase
Summary
As one of the most abundant renewable resources today, lignocellulosic biomass is under development worldwide to produce biofuels and chemicals. One key bottleneck hindering biofuels development is the high cost of conversion of feedstocks to sugars due to the general recalcitrance of plant cell walls (Himmel et al, 2007). To overcome this hurdle, a process strategy of cellulase production, cell wall polymer hydrolysis, and sugar fermentation in a single step termed consolidated bioprocessing (CBP) has been proposed (Lynd et al, 2005). The resultant strains were shown to grow efficiently on industrial cellulose pulp, which is mostly amorphous, but limited growth on recalcitrant crystalline cellulose was noted (Guo et al, 2017)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
