Leqian Liu

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolyticas native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. We further complete direct conversion of lipid content into biodiesel. Tri-level metabolic control results in saturated cells containing upwards of 90% lipid content and titres exceeding 25 g l(-1) lipids, which represents a 60-fold improvement over parental strain and conditions. Through this rewiring effort, we advance fundamental understanding of lipogenesis, demonstrate non-canonical environmental and intracellular stimuli and uncouple lipogenesis from nitrogen starvation. The high titres and carbon-source independent nature of this lipogenesis in Y. lipolytica highlight the potential of this organism as a platform for efficient oleochemical production.

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

ABSTRACT The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast Yarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported for Y. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters in Y. lipolytica are enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

Frontiers of yeast metabolic engineering: diversifying beyond ethanol and Saccharomyces

Microbial systems provide an attractive, renewable route to produce desired organic molecules such as fuels and chemicals. While attention within the field of metabolic engineering has mostly focused on Escherichia coli, yeast is a potent host and growing host for industrial products and has many outstanding, biotechnologically desirable native traits. Thus, there has been a recent shift in focus toward yeast as production hosts to replace E. coli. As such, products have diversified in yeast beyond simply ethanol. Additionally, nonconventional yeasts have been considered to move beyond Saccharomyces cerevisiae. This review highlights recent advances in metabolic engineering of yeasts for producing value-added chemical compounds including alcohols, sugar derivatives, organic acids, fats, terpenes, aromatics, and polyketides. Furthermore, we will also discuss the future direction of metabolic engineering of yeasts.

An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica.

Lipogenic organisms provide an ideal platform for biodiesel and oleochemical production. Through our previous rational metabolic engineering efforts, lipogenesis titers in Yarrowia lipolytica were significantly enhanced. However, the resulting strain still suffered from decreased biomass generation rates. Here, we employ a rapid evolutionary metabolic engineering approach linked with a floating cell enrichment process to improve lipogenesis rates, titers, and yields. Through this iterative process, we were able to ultimately improve yields from our prior strain by 55% to achieve production titers of 39.1g/L with upwards of 76% of the theoretical maximum yield of conversation. Isolated cells were saturated with up to 87% lipid content. An average specific productivity of 0.56g/L/h was achieved with a maximum instantaneous specific productivity of 0.89g/L/h during the lipid production phase in fermentation. Genomic sequencing of the evolved strains revealed a link between a decrease/loss of function mutation of succinate semialdehyde dehydrogenase, uga2, suggesting the importance of gamma-aminobutyric acid assimilation in lipogenesis. This linkage was validated through gene deletion experiments. This work presents an improved host strain that can serve as a platform for efficient oleochemical production.

Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica

The complete biosynthetic replacement of petroleum transportation fuels requires a metabolic pathway capable of producing short chain n-alkanes. Here, we report and characterize a proof-of-concept pathway that enables microbial production of the C5 n-alkane, pentane. This pathway utilizes a soybean lipoxygenase enzyme to cleave linoleic acid to pentane and a tridecadienoic acid byproduct. Initial expression of the soybean lipoxygenase enzyme within a Yarrowia lipolytica host yielded 1.56 mg/L pentane. Efforts to improve pentane yield by increasing substrate availability and strongly overexpressing the lipoxygenase enzyme successfully increased pentane production three-fold to 4.98 mg/L. This work represents the first-ever microbial production of pentane and demonstrates that short chain n-alkane synthesis is conceivable in model cellular hosts. In this regard, we demonstrate the potential pliability of Y. lipolytica toward the biosynthetic production of value-added molecules from its generous fatty acid reserves.

Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant

Lipogenic organisms represent great starting points for metabolic engineering of oleochemical production. While previous engineering efforts were able to significantly improve lipid production in Yarrowia lipolytica, the lipogenesis landscape, especially with respect to regulatory elements, has not been fully explored. Through a comparative genomics and transcriptomics approach, we identified and validated a mutant mga2 protein that serves as a regulator of desaturase gene expression and potent lipogenesis factor. The resulting strain is enriched in unsaturated fatty acids. Comparing the underlying mechanism of this mutant to other previously engineered strains suggests that creating an imbalance between glycolysis and the TCA cycle can serve as a driving force for lipogenesis when combined with fatty acid catabolism overexpressions. Further comparative transcriptomics analysis revealed both distinct and convergent rewiring associated with these different genotypes. Finally, by combining metabolic engineering targets, it is possible to further engineer a strain containing the mutant mga2 gene to a lipid production titer of 25g/L.

Draft Genome Sequence of the Oleaginous Yeast Yarrowia lipolytica PO1f, a Commonly Used Metabolic Engineering Host.

ABSTRACT The draft genome sequence of the oleaginous yeast Yarrowia lipolytica stain PO1f, a commonly used metabolic engineering host, is presented here. The approximately 20.3-Mb genome sequence of PO1f will greatly facilitate research efforts in metabolic engineering of Yarrowia lipolytica for value-added chemical production.

Effect of polypeptide from Chlamys farreri on UVB-induced ROS/NF-κB/COX-2 activation and apoptosis in HaCaT cells

Polypeptide from Chlamys farreri (PCF) is a novel marine polypeptide compound isolated from gonochoric Chinese scallop Chlamys farreri, this study we further investigate the mechanisms of PCF exerting its anti-apoptotic effect. The results indicated that PCF, ROS scavenger NAC and NF-kappaB inhibitor MG132 effectively inhibited UVB-induced HaCaT cells apoptosis. PCF (2.84mM) showed potential ROS scavenging activities in a kinetic process. PCF (1.42-5.69mM) dose-dependently increased the expressions of Cu, Zn-SOD, CAT and GPx meanwhile decreased the expressions of p-NF-kappaB/p65 and COX-2 in UVB-induced HaCaT cells. Additionally, pretreatment with NAC significantly declined the generation of ROS and the expression of p-NF-kappaB/p65. We concluded that ROS, NF-kappaB and COX-2 are involved in UVB-induced HaCaT cells apoptosis, PCF exerts its protective effects via scavenging ROS, increasing the expression of antioxidative enzymes and inhibition the activation of NF-kappaB and COX-2.

From Pathways to Genomes and Beyond: The Metabolic Engineering Toolbox and Its Place in Biofuels Production

Abstract Concerns about the availability of petroleum-derived fuels and chemicals have led to the exploration of metabolically engineered organisms as novel hosts for biofuels and chemicals production. However, the complexity inherent in metabolic and regulatory networks makes this undertaking a complex task. To address these limitations, metabolic engineering has adapted a wide-variety of tools for altering phenotypes. In this review, we will highlight traditional and recent metabolic engineering tools for optimizing cells including pathway-based, global, and genomics enabled approaches. Specifically, we describe these tools as well as provide demonstrations of their effectiveness in optimizing biofuels production. However, each of these tools provides stepping stones towards the grand goal of biofuels production. Thus, developing methods for largescale cellular optimization and integrative approaches are invaluable for further cell optimization. This review highlights the challenges that still must be met to accomplish this goal.