Publications

Tuberculosis (TB), caused by *Mycobacterium tuberculosis*, is one of the world’s deadliest diseases, currently responsible for ~1.5 million deaths per year and rising. Recently, rifampicin-resistant *M. tuberculosis* was designated as a critical priority pathogen status by the World Health Organization. However, few controlled laboratory studies are available that systematically assess the molecular drivers of antibiotic resistance development in TB. In this study, we paired laboratory-directed evolution and population-level deep-sequencing approaches to map the evolutionary pathways taken by *M. tuberculosis* to develop resistance to first- and last-line therapies (rifampicin and linezolid) and then characterized *de novo* resistance mutation occurrence over time. We demonstrated that the majority of *M. tuberculosis* populations readily acquire mutations in genes commonly found in rifampicin- and linezolid-resistant clinical isolates (*rpoB* and *rplC*). However, we also identified mutations in six genes, mostly present in subpopulations (17–41%) and not previously linked to rifampicin or linezolid resistance, including four associated with rifampicin resistance (Rv0052, *ppsD*, *ppsE* and *mptC*) and two associated with linezolid resistance (*glpK* and echA12). The *ppsD*, *glpK* and *mptC* mutations were also identified in published individual sequencing reads of antibiotic-resistant clinical isolates. Further investigation of the identified resistance determinants *ppsD/E* established that mutations in these genes appear to mediate resistance across multiple species, with an *Escherichia coli* mutant of the ortholog (*fabF*), representing a shared domain featured in PpsD and PpsE, phenotypically displaying increased antibiotic tolerance to low-level rifampicin. This study highlights the power of using controlled laboratory studies to uncover minority variants in populations of *M. tuberculosis*. These outcomes will lead to improved diagnosis of antibiotic resistance emergence in TB, to optimize management and treatment of TB infections, and ultimately to minimize patient deaths.

Dairy food waste (DFW) is a major global issue, with estimates indicating that almost 20% of production is wasted in some regions. Such losses result in economic and environmental challenges but also create opportunities to recover valuable nutrients, reduce costs, and support a more circular dairy industry. This review explores the sources and composition of DFW and highlights strategies to transform it into valuable bioproducts through yeast fermentation, focusing on lactose-fermenting species as near-term platforms and *Saccharomyces cerevisiae* as the longer-term alternative. Key DFW streams, particularly cheese whey and its derivatives, offer nutrient-rich substrates for bioproduction. Yeast fermentation can convert these into value-added products, such as microbial biomass, AA, vitamins, minerals, fatty acids, and enzymes with direct applications in dairy cattle nutrition. Despite this potential, challenges persist, including the inability of industrially preferred yeasts to natively metabolize lactose, the variability in DFW composition, microbial contamination, and economic, logistical, and regulatory barriers. Emerging approaches such as strain engineering, adaptive evolution, and biofoundry-based synthetic biology offer promising solutions. By integrating bioproduction into the dairy cycle, the industry has the potential to reduce DFW, lower greenhouse gas emissions, and create new revenue streams.

The ability to generate genomic diversity expands opportunities for understanding and engineering biology. Here, we demonstrate on-demand generation of diversity in bacterial genome configurations and its application to probing physiology under altered genome organization. We engineered the fast-growing bacterium Vibrio natriegens to enable large-scale stochastic duplications, translocations, inversions, and deletions, producing populations with a wide range of genome arrangements. We investigated phenotypic robustness to genome reconfigurations and found that distinct genome organizations can support stable physiology, indicating that bacteria may tolerate chromosomal alterations more readily than previously appreciated. Our work provides a framework for advancing the understanding and engineering of bacterial genomes and suggests that genome reconfigurations may contribute to phenogenetic drift, allowing for evolutionary exploration while preserving phenotype.