Integrating omics into genome engineering workflows

Collins, Joseph H.

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Integrating omics into genome engineering workflows

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Engineered hosts are becoming ever more prevalent as cell factories for fuels, chemicals, and pharmaceuticals. With myriad possible hosts and methods available, larger integrated workflows are needed for genetic engineers. This work aims to enable nonconventional host engineering by developing two omics based approaches; an end-to-end sequencing and genome assembly workflow for engineered yeasts and a systems and synthetic biology approach to domesticate the red yeast Xanthophyllomyces dendrorhous. Yeast genomes can be assembled from sequencing data, but genetic engineering changes often fail to be resolved with accuracy, completeness, and contiguity. Further, searching for engineered sequences in sequence data is currently a manual process. To overcome these challenges, we created an integrated and automated workflow that achieves accurate whole genome and plasmid sequences of engineered yeasts, automatically annotating synthetic biology parts. 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. Xanthophyllomyces dendrorhous, a basidiomycete red yeast, is a promising platform organism due to its naturally high metabolic flux through the terpenoid pathway, a foundational pathway for production of carotenoids, anti-cancers, anti-inflammatories, and antimicrobials. In addition, X. dendrorhous has a known response to light - increased production of carotenoids. We sought to understand and exploit natural optogenetic regulatory parts to enable X. dendrorhous as a terpenoid cell factory. Our initial investigation involved exposure to light at various wavelengths using a LED apparatus, which showed that ultraviolet (UV) light significantly increased carotenoid production. Transcriptomics were then done to identify genes with significant differential expression in response to these various light wavelengths. Genes with significant differential expression were identified under UV and blue light, whereas red and green light did not elicit any genes. We then extracted the promoter sequences for light induced genes and tested their expression using the NanoLuc luciferase. We show that we can increase or decrease bioluminescence of NanoLuc with these new X. dendrorhous optogenetic parts. In sum, this work is a blueprint for building integrated workflows to fully capitalize on the unique metabolism of a given nonconventional host, and to alleviate engineering roadblocks. Our automated genome sequencing and assembly pipeline demonstrates how to build and evaluate whole genome sequencing workflows for genetic engineers, and establishes methods to identify genetic engineering from whole genome sequencing data. We envision that the demonstrated transcriptomics-guided workflow to identify and harness optogenetic parts in X. dendrorhous will be a key strategy for engineering nonconventional organisms in the future.

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