Biology subjects

Hendry, J. I.

Publications and source records attributed to Hendry, J. I..

3 recordsLinked to original sources

bioRxiv2026

Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

synthetic biology↗

bioRxiv2024

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR/Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55 kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the WT. This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome to phenome relationships. ImportanceGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome editing tool and in this study we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

synthetic biology↗

bioRxiv2022

Proteome capacity constraints favor respiratory ATP generation

Cells face competing metabolic demands. These include efficient use of both limited substrates and limited proteome capacity, as well as flexibility to deal with different environments. Flexibility requires spare enzyme capacity, which is proteome inefficient. ATP generation can occur via fermentation or respiration. Fermentation is much less substrate-efficient, but often assumed to be more proteome efficient 1-3, thereby favoring fast-growing cells engaging in aerobic glycolysis 4-8. Here, however, we show that mitochondrial respiration is actually more proteome-efficient than aerobic glycolysis. Instead, aerobic glycolysis arises from cells maintaining the flexibility to grow also anaerobically. These conclusions emerged from an unbiased assessment of metabolic regulatory mechanisms, integrating quantitative metabolomics, proteomics, and fluxomics, of two budding yeasts, Saccharomyces cerevisiae and Issatchenkia orientalis, the former more fermentative and the latter respiratory. Their energy pathway usage is largely explained by differences in proteome allocation. Each organisms proteome allocation is remarkably stable across environmental conditions, with metabolic fluxes predominantly regulated at the level of metabolite concentrations. This leaves extensive spare biosynthetic capacity during slow growth and spare capacity of their preferred bioenergetic machinery when it is not essential. The greater proteome-efficiency of respiration is also observed in mammals, with aerobic glycolysis occurring in yeast or mammalian cells that maintain a fermentation-capable proteome conducive to both aerobic and anaerobic growth.

systems biology↗