Developing next-generation tools for metabolic engineering in non-conventional yeast Yarrowia lipolytica
Date
2023
Authors
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Publisher
University of Delaware
Abstract
Metabolic engineering is a promising alternative to producing chemicals over chemical synthesis and plant extraction. Metabolic engineering primarily involves changing the flux to produce target molecules by changing the genetic information. The non-conventional yeast yarrowia lipolytica has been emerging as an industrial workhorse due to its high lipid flux and the ability to use alternative feedstock. However, the genetic tools are minimal compared to the model yeast. Building efficient tools accelerates metabolic engineering. In this work, we developed three tools for metabolic engineering in non-conventional yeast Y. lipolytica. ☐ The first tool involves engineering Yarrowia lipolytica for multiplexed gene editing to enhance naringenin synthesis. We found traditional CRISPR-Cas9 toxic for multiplexed gene editing, while the nickase-based cytosine base editor showed no toxicity, making it a preferable alternative. We increased multiplexed editing efficiency by optimizing the expression of gRNA and Base editor. We extended multiplexed base editing to an NHEJ-competent strain by a co-selection approach, which edits the target loci and loci corresponding to the canavanine resistance gene. We increased heterologous natural product naringenin production by performing cytosine base editor-based multiplexed knockouts in the malonyl-CoA and tyrosine biosynthetic pathways, identifying new targets to enhance malonyl-CoA flux with different carbon sources. ☐ The second tool involves fine-tuning the base editor for functional genomics. We evaluated the base editor for genome-wide knockout-based functional genomics. We identified that expanding the editing window or relaxing the PAM requirement increases the target scope of the cytosine base editor. We identified an efficient cytosine base editor by screening for cytosine deaminases and changed the editing window with scaffold engineering through a SpyTag. We replaced the NGG PAM using SpCas9 with NG PAM using SpCas9 to expand the target scope of base editors further and increased the editing efficiency with a co-selection approach. ☐ The third tool was a library-based promoter engineering to regulate the expression of genes. We randomized a TATA Box region through a degenerate library and performed FACS to isolate variants showing different expression levels. Then, we took a library approach to balance a deoxyviolacein metabolic pathway. We identified that changing the expression of the deoxyviolacein metabolic pathway genes increases the production of deoxyviolacein. ☐ Overall, this research provides strategies to advance synthetic biology and metabolic engineering in Yarrowia lipolytica.
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Keywords
Deoxyviolacein, Yarrowia lipolytica, SpyTag, Base editor, Metabolic engineering