Biology subjects

Weintraub, S. J.

Publications and source records attributed to Weintraub, S. J..

2 recordsLinked to original sources

bioRxiv2025

Combinatorial metabolic engineering of alkane biosynthesis in the osmotolerant yeast Debaryomyces hansenii CBS 767

Debaryomyces hansenii is a promising yeast with diverse potential applications, one of which is producing oleochemicals from inexpensive biomass. Yet, combinatorial metabolic engineering in D. hansenii is not possible because, like many nonconventional organisms, key genomic data and genetic elements are missing. Here, we report phenotypic characterization, genomic integration loci, and modular genetic parts that together enable combinatorial metabolic engineering of an alkane pathway in D. hansenii. Phenotypic characterization revealed that D. hansenii produces lipids when grown on components of lignocellulosic and algal biomass in standard and saline media. Notably, D. hansenii produced 57.25% more lipids than Yarrowia lipolytica Po1f when grown on glucose. We designed genomic integration loci and derived gene expression elements from a resequenced D. hansenii CBS767 genome. While characterizing the integration sites, we also optimized the transformation procedure, validated selection markers, and determined fluorescent reporters. Using homology to known elements in S. cerevisiae, we derived 23 promoters and 24 terminators and made them compatible with a modular cloning standard so pathway constructs could be made with automated liquid handling. Flow cytometry measurements show that the promoters span an expression range of three orders of magnitude and the terminators span one order of magnitude. The new part collection was used to construct a genomically integrated combinatorial library of 18 alkane biosynthesis pathways. With no other genetic modifications, the best strain produced 38 mg/L of heptadecane. This is the highest titer observed in a microbe with no other modifications besides the biosynthetic genes. Thus, this work establishes D. hansenii as a cell factory for synthesizing oleochemicals from biomass while providing a blueprint for leveraging phenotyping, genomics, modular parts collections, and automated liquid handling for making cell factories from nonconventional organisms. Furthermore, the genetic parts collection now enables functional genetics for various applications in Debaryomyces yeasts.

synthetic biology↗

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↗