Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow—it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.

Plant-derived phenylpropanoids, in particular phenylpropenes, have diverse industrial applications ranging from flavors and fragrances to polymers and pharmaceuticals. Heterologous biosynthesis of these products has the potential to address low, seasonally dependent yields hindering ease of widespread manufacturing. However, previous efforts have been hindered by the inherent pathway promiscuity and the microbial toxicity of key pathway intermediates. Here, in this study, we establish the propensity of a tripartite microbial co-culture to overcome these limitations and demonstrate to our knowledge the first reported de novo phenylpropene production from simple sugar starting materials. After initially designing the system to accumulate eugenol, the platform modularity and downstream enzyme promiscuity was leveraged to quickly create avenues for hydroxychavicol and chavicol production. The consortia was found to be compatible with Engineered Living Material production platforms that allow for reusable, cold-chain-independent distributed manufacturing. This work lays the foundation for further deployment of modular microbial approaches to produce plant secondary metabolites.

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.Yet, applying WGS is a challenge because of the diversity of genetic backgrounds, the variety of engineering features, and the current scale of yeast strain engineering. Myriad laboratory strains of the baker's yeast Saccharomyces cerevisiae 9, 24, 25 and nonconventional yeasts like Yarrowia lipolytica 26-28 and Komagataella phaffii (formerly Pichia pastoris) 29, 30 are used to create yeast cell factories, so there are many potential genetic backgrounds. Methods of yeast engineering leave myriad sequence features behind, including standard plasmid sets with standard expression parts 31-34 , high efficiency transformation 35-37 , homologous recombination 10, 38-40 , gene knockouts using the Cre recombinase system 41 , and genome editing using RNAguided endonucleases 7,11,42,[44][45][46] . Furthermore, the scale of yeast engineering is increasing both in the...

Genomics has become the primary way to explore microbial diversity, because genetic tools are currently difficult to develop in non-model organisms. Here, we demonstrate that -omics can be leveraged to accelerate genetic tool development for the basidiomycete yeastXanthophyllomyces dendrorhousCBS 6938, the sole biotechnologically relevant organism in the Tremellomycete family. First, we sequence the genome. Then, we perform transcriptomics under a variety of conditions, focusing on light and oxidative stress. This data not only reveals novel photobiology and metabolic regulation, it also allows derivation of constitutive and regulated gene expression parts. Our analysis ofX. dendrorhousphotobiology shows for the first time that a complex system of white-collar and cryptochrome homologs mediate response to ultraviolet light (UV). Our analysis of metabolic regulation shows that UV activates DNA repair, aromatic amino acid and carotenoid biosynthesis and represses central carbon metabolism and the fungal-like apoptotic pathway. Thus,X. dendrorhousshows a dynamic response toward biosynthetic pathways for light-absorbing compounds and survival and away from energy production. We then define a modular cloning system, including antibiotic selections, integration sites, and reporter genes, and use the transcriptomics to derive strong constitutive and regulated promoters. Notably, we discover a novel promoter from a hypothetical gene that has 9-fold activation upon UV exposure. Thus, -omics-to-parts workflows can simultaneously provide useful genomic data and advance genetic tools for non-model microbes, particularly those without a closely related model organism. This approach will be broadly useful in current efforts to engineer diverse microbes.