High-Efficiency Multiplexed Cytosine Base Editors for Natural Product Synthesis in Yarrowia lipolytica.

Yarrowia lipolytica is an industrial host with a high fatty acid flux. Even though CRISPR-based tools have accelerated its metabolic engineering, there remains a need to develop tools for rapid multiplexed strain engineering to accelerate the design-build-test-learn cycle. Base editors have the potential to perform high-efficiency multiplexed gene editing because they do not depend upon double-stranded DNA breaks. Here, we identified that base editors are less toxic than CRISPR-Cas9 for multiplexed gene editing. We increased the editing efficiency by removing the extra nucleotides between tRNA and gRNA and increasing the base editor and gRNA copy number in a Ku70 deficient strain. We achieved five multiplexed gene editing in the ΔKu70 strain at 42% efficiency. Initially, we were unsuccessful at performing multiplexed base editing in NHEJ competent strain; however, we increased the editing efficiency by using a co-selection approach to enrich base editing events. Base editor-mediated canavanine gene (CAN1) knockout provided resistance to the import of canavanine, which enriched the base editing in other unrelated genetic loci. We performed multiplexed editing of up to three genes at 40% efficiency in the Po1f strain through the CAN1 co-selection approach. Finally, we demonstrated the application of multiplexed cytosine base editor for rapid multigene knockout to increase naringenin production by 2-fold from glucose or glycerol as a carbon source.

Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica.

Yarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR-Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

Heterologous biosynthesis of natural product naringenin by co-culture engineering.

Co-culture engineering is an emerging approach for microbial biosynthesis of a variety of biochemicals. In this study, E. coli-E. coli co-cultures were developed for heterologous biosynthesis of the natural product naringenin. The co-cultures were composed of two independent E. coli strains dedicated to functional expression of different portions of the biosynthetic pathway, respectively. The co-culture biosynthesis was optimized by investigating the effect of carbon source, E. coli strain selection, timing of IPTG induction and the inoculation ratio between the co-culture strains. Compared with the mono-culture strategy, the utilization of the designed co-cultures significantly improved the naringenin production, largely due to the reduction of metabolic stress, employment of proper hosts for improving pathway enzyme activities, and flexible adjustment of the relative biosynthetic strength between the co-culture strains. The findings of this study extend the applicability of co-culture engineering in complex natural product biosynthesis.