Evolutionary Engineering Improved d-Glucose/Xylose Cofermentation of Yarrowia lipolytica

Abstract

Background: Yarrowia lipolytica is considered as a promising biorefinery chassis for production of microbial lipids, the important precursors of advanced biofuels. Unfortunately, wild Yarrowia lipolytica is unable to consume xylose, the major pentose in lignocellulosic hydrolysates. A recombinant strain Yarrowia lipolytica yl-XYL+ can utilize xylose to produce microbial lipids efficiently, but its xylose uptake is severely delayed in the presentence of D-glucose. Therefore, it is critical to develop cofermenting D-glucose and xylose strains and study the underlying mechanisms. Results: In this study, an adaptive laboratory evolution (ALE) is performed to engineering the strains in the medium containing xylose and D-glucose analog 2-deoxyglucose (dG). After four stages of evolution over a total of 64 days, we obtained for the first time a strain of Y. lipolytica (yl-XYL+*04*10) with derepressed xylose metabolism. Xylose uptake kinetics showed that it could efficiently utilize xylose in the presence of 10 g/L dG or D-glucose. Transcriptional profiling analysis revealed that relative expression level of YALI0_C04730g and YALI0_D00363g (both encoding xylosespecific transporter) was significantly up-regulated. Besides, we found that missense mutations N373T and G270A in YALI0_E23287g (encoding a D-glucose transporter) and YALI0_E15488g (encoding a hexokinase) respectively. Conclusions: These results indicate that these are important gene targets responsible for improved xylose utilization in the evolved Yarrowia lipolytica. Our work provides a new approach for breeding Yarrowia lipolytica and paved the way for future pentose metabolic engineering.

Efficient production of the β-ionone aroma compound from organic waste hydrolysates using an engineered Yarrowia lipolytica strain

“…The resulting strains promise improved utilization of xylose in the production of chemical compounds and fuel ( Wei et al, 2020a , b ; Sun et al, 2021 ). Additional metabolic engineering approaches ( Ledesma-Amaro et al, 2016 ; Rodriguez et al, 2016 ; Wu et al, 2019 ), strain mating ( Li and Alper, 2020 ), lab adaptive evolution ( Zhou et al, 2020 ) and the use of artificial chromosomes have all been used to enhance xylose utilization. Although it appears that Y. lipolytica utilizes arabinose in a similar way to xylose, only a few studies have investigated arabinose fermentation by this yeast ( Tsigie et al, 2011 ; Ryu and Trinh, 2018 , 2021 ; Spagnuolo et al, 2018 ).…”

Optimized conversion of wheat straw into single cell oils by Yarrowia lipolytica and Lipomyces tetrasporus and synthesis of advanced biofuels

“…The higher affinity for glucose with respect to other carbon sources available in the medium is a well-known process, already documented for many oleaginous species due to the carbon catabolite repression [40]. This mechanism leads, in some cases, to diauxic growth phenomena that affect the metabolism of the secondary sugars and further limits the conversion efficiency during the fermentative process [41]. Some authors also found that the slow transport rate of xylose limits the downstream pathway; consequently, the enhancement of the transport rate is necessary for improving the cell growth rate and the xylose conversion efficiency [42,43].…”

The Future of Biomass Utilization Technologies Special Issue Editorial

“…Thus, as the energy demand and serious environmental concerns increase, the technologies including torrefaction, catalytic conversion, gasification, and hydrothermal liquefaction (HTL) have attracted more and more attention and have developed rapidly in recent years. A variety of reactions, including hydrogenation, oxidation, deoxygenation (hydrodeoxygenation, decarboxylation and decarbonylation), catalytic cracking, dehydrogenation, aromatization, reforming, pyrolysis, ketonization, and so on have been achieved. − Various products including biochar, bio-oil, hydrogen, aromatics, CO 2 , CO, CH 4 and other gaseous hydrocarbons have been obtained. − …”