Omics driven onboarding of the carotenoid producing red yeast Xanthophyllomyces dendrorhous CBS 6938

Abstract

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Genetic part discovery has never been more necessary, as advances in synthetic biology increase the number of tractable organisms that need tunable gene expression for genetic circuits and metabolic pathways. Therefore, approaches are needed to assess a tractable organism and obtain a convenient set of genetic parts to support future research. Here, we describe a genomic and transcriptomic approach to derive a modular integrative part library with constitutive and regulated promoters in the basidiomycete yeast Xanthophyllomyces dendrorhous CBS 6938. X. dendrorhous is currently the sole biotechnologically relevant organism in the Tremellomycete family - it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Particularly for this yeast, there are not large libraries of parts from related organisms that could be transferred. They must be derived. To do this, a contiguous genome was first obtained through combined short read and long read sequencing. Then, differential gene expression (DGE) analysis using transcriptomics was performed, comparing oxidative stress and exposure to different wavelengths of light. This revealed a set of putative light-responsive regulators that mediate a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was then used to derive 26 constitutive and regulated gene expression elements from the genome. The gene expression elements were designed to be compatible with a new modular cloning system for X. dendrorhous which includes integration sites, terminators, selection markers, and reporters. Each element was characterized by luciferase assay of an integrated gene expression cassette. Notably, a novel promoter from a hypothetical gene that has 9-fold activation upon UV exposure was characterized. This study defines an advanced modular genetic part collection for engineering the basidiomycete X. dendrorhous CBS 6938 while simultaneously discovering potential targets for increasing tetraterpenoid biosynthesis. Further, it demonstrates that -omics-to-parts workflows can simultaneously provide useful genomic data and advance genetic tools for nonconventional microbes, particularly those without a related model organism. This approach will be broadly useful in current efforts to engineer diverse microbes. KEY POINTSO_LIOmics-to-parts can be applied to non-model organisms for rapid "onboarding". C_LIO_LI26 promoters native to X. dendrorhous were identified. C_LIO_LIOmics revealed unique photobiology in X. dendrorhous. C_LI