Biology subjects

Keating, K. W.

Publications and source records attributed to Keating, K. W..

3 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

Systematic part transfer by extending a modular toolkit to diverse bacteria

It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection for E. coli. This enabled testing of a library of DNA constructs across the Pseudomonadota - Escherichia coli, Pseudomonas putida, Cupriavidus necator, and Komagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms - meaning that the same parts can be used to program E. coli, P. putida, C. necator, and K. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently in K. nataicola than in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, making it possible to create a small number of parts sets that can span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.

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↗