Biology subjects

Collins, J. H.

Publications and source records attributed to Collins, J. H..

5 recordsLinked to original sources

bioRxiv2023

Phenotypic and genomic evidence for transparent cellulose, metabolic diversity, and stable cellulose production in the Acetobacteraceae

The Acetobacteraceae are a family of microbes that use sugars from fruits, beverages and fermented foods to overproduce bacterial nanocellulose (BNC), a living material with broad applications in medicine and industry. Yet, the family has few complete, contiguous genome sequences available. Here, three different strains - a high production strain NQ5, a metabolic engineering host NCIB 8034, and a new isolate DS12 from kombucha were characterized and complete de novo genomes assembled. Initial growth and yield experiments reveal a diversity of carbon source utilization profiles and BNC production rates, with NQ5 achieving the highest yield on glucose and DS12 having the narrowest utilization profile. All strains synthesize optically clear BNC. Genomic evidence assigns the DS12 isolate to Komagataeibacter nataicola, reassigns NCIB 8034 from Komagataeibacter xylinus to Komagataeibacter oboediens, and supports NQ5 as Novacetimonas hansenii. The bcs gene clusters that encode BNC synthesis are also diverse. The highest producing strain, N. hansenii NQ5, has fewer bnc copies than K. oboediens NQ5, indicating that copy number does not explain high productivity. Analysis also reveals the type and frequency of mobile genetic elements. Notably, N. hansenii NQ5 has a paucity of transposons relative to other strains, which could explain the BNC production stability of N. hansenii NQ5 in culture. Thus, this work argues that Acetobacteraceae are metabolically diverse, and provides genomic evidence explaining beneficial BNC production characteristics of N. hansenii NQ5. Therefore, this work provides evidence for selection of appropriate BNC production strains. IMPORTANCEBacterial cellulose is an important material for biomedical applications like wound dressings. This study defines important characteristics of microbes that produce bacterial cellulose, namely their ability to process different sugars and features of their genomes that make cellulose yield more consistent. These findings will aid in the development of better bacterial cellulose production processes.

microbiology↗

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↗

bioRxiv2023

A correlative study of the genomic underpinning of virulence traits and drug tolerance of Candida auris

Candida auris is an opportunistic fungal pathogen with high mortality rates that presents a clear and present threat to public health. The risk of C. auris infection is high because it can colonize the body, resist antifungal treatment, and evade the immune system. The genetic mechanisms for these traits are not well-known. Identifying them could lead to new targets for new treatments. To this end, we present an analysis of the genetics and gene expression patterns of C. auris carbon metabolism, drug resistance, and macrophage interaction. We chose to study two C. auris isolates simultaneously, one drug sensitive (B11220 from Clade II) and one drug resistant (B11221 from Clade III). Comparing the genomes, we found that B11220 was missing a 12.8 kb gene cluster encoding proteins related to alternative sugar utilization, possibly L-rhamnose. We show that B11221, which has the cluster, more readily assimilates and utilizes D-galactose and L-rhamnose. B11221 also exhibits increased adherence and drug resistance compared to B11220 when grown in these sugars. Transcriptomic analysis of both strains grown on glucose or galactose showed that genes associated with translation were upregulated in B11221, and the putative L-rhamnose gene cluster was upregulated when grown on D-galactose. These findings reinforce the growing evidence of a link between metabolism and tolerance. Since immune system evasion depends heavily on the cell surface, we characterized cell wall composition and macrophage evasion for the two strains. We found that B11221 has far less {beta}-1,3-glucan exposure, a key determinant of immune system evasion, and resists phagocytosis by macrophages compared to B11220. In a transcriptomic analysis of both strains co-cultured with macrophages we found that B11221 upregulates genes associated with early stages of growth and transcription factors that regulate transport. These key differences in growth and membrane composition could explain the resistance to phagocytosis and increased stress tolerance in general of B11221, and indicates another connection between metabolism and immune system evasion. Taken together, these data show that membrane composition, metabolism, and transport all correlate with colonization, drug resistance, and immune system evasion in C. auris.

microbiology↗

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↗

bioRxiv2020

Genetic Analyses of Blood Cell Structure for Biological and Pharmacological Inference

Thousands of genetic associations with phenotypes of blood cells are known, but few are with phenotypes relevant to cell function. We performed GWAS of 63 flow-cytometry phenotypes, including measures of cell granularity, nucleic acid content, and reactivity, in 39,656 participants in the INTERVAL study, identifying 2,172 variant-trait associations. These include associations mediated by functional cellular structures such as secretory granules, implicated in vascular, thrombotic, inflammatory and neoplastic diseases. By integrating our results with epigenetic data and with signals from molecular abundance/disease GWAS, we infer the hematopoietic origins of population phenotypic variation and identify the transcription factor FOG2 as a regulator of platelet -granularity. We show how flow cytometry genetics can suggest cell types mediating complex disease risk and suggest efficacious drug targets, presenting Daclizumab/Vedolizumab in autoimmune disease as positive controls. Finally, we add to existing evidence supporting IL7/IL7-R as drug targets for multiple sclerosis.

genetics↗