Development and physiological characterization of cellobiose‐consuming Yarrowia lipolytica

Abstract

Yarrowia lipolytica is a promising production host for a wide range of molecules, but limited sugar consumption abilities prevent utilization of an abundant source of renewable feedstocks. In this study we created a Y. lipolytica strain capable of utilizing cellobiose as a sole carbon source by using endogenous promoters to express the cellodextrin transporter cdt-1 and intracellular β-glucosidase gh1-1 from Neurospora crassa. The engineered strain was also capable of simultaneous co-consumption of glucose and cellobiose. Although cellobiose was consumed slower than glucose when engineered strains were cultured with excess nitrogen, culturing with limited nitrogen led to cellobiose consumption rates comparable to those of glucose. Under limited nitrogen conditions, the engineered strain produced citric acid as a major product and we observed greater citric acid yields from cellobiose (0.37 g/g) than glucose (0.28 g/g). Culturing with a sole carbon source of either glucose or cellobiose induced additional differences on cell physiology and metabolism and a link is suggested to evasion of glucose-sensing mechanisms through intracellular creation and consumption of glucose. We ultimately applied this cellobiose-utilization system to produce citric acid from bioconversion of crystalline cellulose through simultaneous saccharification and fermentation (SSF).

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica

“…This phenotype is often found in the many biomassdegrading fungi that tend to produce base-level cellulases regardless of growth conditions (64,65). While native Y. lipolytica could grow on cellobiose as a sole carbon source, the cellobiose assimilation was inefficient compared to that seen with native N. crassa (14), native A. niger (66), recombinant S. cerevisiae (67), or recombinant Y. lipolytica (62), likely due to the limitation of the cellobiose transporter. With the knowledge of putative cellobiose transporters and BGL genes identified in this study, further optimization of the cellobiose degradation pathway is highly feasible.…”

“…S1 in the supplemental material). Since studies by other groups did not investigate cell growth of the native Y. lipolytica after 2 days (34,62), the xylose and cellobiose degradation pathways were not activated in those studies. In our study, since the adaptation period was conducted for less than 15 generations, which was sufficient to achieve relatively stable cell growth, the adapted phenotypes of the native Y. lipolytica were reversible (results not shown).…”

Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica

“…We have previously described how the overexpression of endogenous or heterologous BGLs allows Y. lipolytica to grow in cellobiose [40,42]. Engineered strains were able to grow on cellulose with the help of a commercial cellulose cocktail [40,42]. In another study, a group of strains able to grow in cellulose was constructed [58].…”

“…Cellobiose is released, which can be converted into its glucose subunits by BGLs. We have previously described how the overexpression of endogenous or heterologous BGLs allows Y. lipolytica to grow in cellobiose [40,42]. Engineered strains were able to grow on cellulose with the help of a commercial cellulose cocktail [40,42].…”

“…Two alternative metabolic engineering strategies have generated strains that are able to consume cellobiose as efficiently as they consume glucose. In the first, the Neurospora crassa cellodextrin transporter CDT-1 and the intracellular b-glucosidase (BGL) GH1-1 were coexpressed, which generated strains that produced 50% more citric acid from cellobiose than from glucose [42]. In the second, two endogenous Y. lipolytica BGLs were overexpressed -the intracellular Bgl1 and the extracellular Bgl2; the strains generated could produce microbial lipids from cellobiose [40].…”