Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli

Abstract

Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic robustness, regulation, and areas of kinetic limitation. In this study, whole-genome sequencing and high-resolution 13C-metabolic flux analysis were performed on 10 adaptively evolved pgi knockouts of Escherichia coli. Pgi catalyzes the first reaction in glycolysis, and its loss results in major physiological and carbon catabolism pathway changes, including an 80% reduction in growth rate. Following adaptive laboratory evolution (ALE), the knockouts increase their growth rate by up to 3.6-fold. Through combined genomic–fluxomic analysis, we characterized the mutations and resulting metabolic fluxes that enabled this fitness recovery. Large increases in pyridine cofactor transhydrogenase flux, correcting imbalanced production of NADPH and NADH, were enabled by direct mutations to the transhydrogenase genes sthA and pntAB. The phosphotransferase system component crr was also found to be frequently mutated, which corresponded to elevated flux from pyruvate to phosphoenolpyruvate. The overall energy metabolism was found to be strikingly robust, and what have been previously described as latently activated Entner–Doudoroff and glyoxylate shunt pathways are shown here to represent no real increases in absolute flux relative to the wild type. These results indicate that the dominant mechanism of adaptation was to relieve the rate-limiting steps in cofactor metabolism and substrate uptake and to modulate global transcriptional regulation from stress response to catabolism.

Controlled replication of ‘Candidatus Liberibacter asiaticus‘ DNA in citrus leaf discs

“…L. asiaticus ’ encodes a nearly complete glycolytic pathway, but is missing glucose 6‐phosphate isomerase ( pgi , EC 5.3.1.9; Duan et al , ; Fagen et al , ). Based on mutational analysis in Escherichia coli (Charusanti et al , ; Long et al , ), loss of pgi can have significant negative implications for utilization of glucose with corresponding re‐arrangements of metabolic flux. Because the genome(s) of ‘ Ca.…”

“…'Ca. L. asiaticus' is either able to metabolize glucose, predicted from metabolic pathway reconstruction (Fagen et al, 2014b), and analysis of E. coli mutants with defects in pgi (Charusanti et al, 2010;Long et al, 2018), or responds to a glucose-dependent alteration in leaf physiology, such as synthesis of ATP. Ability of 'Ca.…”

Lipid availability influences the metabolic maturation of human pluripotent stem cell-derived cardiomyocytes

“…Cellular physiology must guide the development of differentiation media that promote mature cell performance. Metabolic flux analysis has long been used to identify industrially-relevant cellular bottlenecks (Long et al, 2018) and now is being applied to understand alterations in hPSCs , Badur et al, 2015 and cancer (Cantor et al, 2017). Metabolic requirements of non-traditional substrates that support cellular growth is an emerging concept (Pavlova et al, 2018, Sousa et al, 2016 and has already been used to improve cellular differentiation (Beyer et al, 2018).…”

LC‐MS/MS‐based metabolic profiling of Escherichia coli under heterologous gene expression stress

“…Up to eight times decrease in the level of L-citrulline suggests an elevated synthesis and concomitant utilization of L-arginine in E. coli cells under stress. [29][30][31][32] We suggest that the alterations in carbohydrate metabolism were used to promote antibiotic stress-resisting events in bacterial cells. 25 Our results show 2.52 times increase in oxoglutarate concentration among the ampicillin-resistant cells.…”