Marsan, C. B. | bioRxiv Science

bioRxiv2023

Transcriptomics elucidates metabolic regulation and functional promoters in the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938

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

synthetic biology↗

bioRxiv2020

Verification of genetic engineering in yeasts with nanopore whole genome sequencing

Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.

synthetic biology↗