Metabolic engineering of yeasts for terpenoid production has mostly focused on the cytoplasm, whereas harnessing their organelles as subcellular factories has been overlooked. Herein, the farnesyl diphosphate synthetic pathway and α-bisabolene synthase were compartmentalized into the oleaginous yeast Yarrowia lipolytica's mitochondria to enable high-level α-bisabolene production. Through comprehensive metabolic engineering approaches, we exploited the potential and capability of the mitochondria as a subcellular factory to achieve 257.4 mg/L of α-bisabolene production from glucose. By combining mitochondrial and cytoplasmic engineering, we further boosted the α-bisabolene titer to 765.1 mg/L by utilizing waste cooking oil as the sole carbon source. Finally, the α-bisabolene titer of the resulting strain reached 1058.1 mg/L in a 5 L bioreactor, which is the highest titer in the engineered Y. lipolytica cell factory reported to date. Overall, our study has provided valuable insights into the mitochondrial engineering of Y. lipolytica for sustainable and green production of valuable compounds.
Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.
Background
In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.
Results
In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.
Conclusion
We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.
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