A Cascade–Cas3-enabled method called TRIM3 is reported that generates large deletions by targeting a randomly integrated transposon, enabling facile generation of a genome-reduced mutant library and represents a new avenue for large-scale genome modifications and the development of improved bioprocessing hosts.
E. Fulk, R. M. Swart, Akira K Nakamura et al.· Nucleic Acids Research· 0 citations
This study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield and indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid and greenhouse gas emissions.
Dowan Kim, Torrey M Lind, Chen Ling et al.· bioRxiv· 1 citation
Efficient co-utilization of hexose and pentose sugars from lignocellulose is essential for microbial bioconversion, yet engineered catabolic pathways can be unstable or suboptimal in complex resource environments. Here, we use a Pseudomonas putida strain engineered to catabolize xylose and arabinose to examine how resource abundance, temporal availability, and subculturing shape evolutionary outcomes. Using an automated adaptive laboratory evolution (ALE) platform, we evolve the strain under simple single-substrate and complex multi-substrate selection pressures. These environments drive divergence between catabolic specialists and generalists. Weak or absent selection for xylose frequently leads to loss of xylose catabolism, whereas carbon-limited mixed-sugar environments promote stable retention and coordinated optimization of multiple catabolic pathways, enhancing growth and substrate utilization. Genomic, proteomic, and biochemical analyses show that pathway-specific fitness costs determine evolutionary stability. A generalist clone also shows improved indigoidine production from mixed sugars relative to the parental strain. Together, these findings show how resource dynamics shape fitness landscapes that govern catabolic specialization, generalization, evolutionary trade-offs, and engineering of bioconversion. Efficient co-utilization of sugars from lignocellulose is essential for microbial bioconversion. Here the authors perform laboratory evolution of P. putida to reveal how selection shapes retention or loss of catabolic pathways, offering design rules for biomanufacturing phenotypes.
Sunghwa Woo, H. Lim, B. Norton-Baker et al.· Nature Communications· 0 citations
Results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining the understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction.
Alissa C. Bleem, Tracy L. Hodges, Torrey M Lind et al.· bioRxiv· 2 citations
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