Metabolic flux analysis of Escherichia coli knockouts: lessons from the Keio collection and future outlook

Antoniewicz

2014

Anal. Chem.

We developed a set of methods for the quantification of four major components of microbial biomass using gas chromatography-mass spectrometry (GC/MS). Specifically, methods are described to quantify amino acids, RNA, fatty acids, and glycogen, which comprise an estimated 88% of the dry weight of Escherichia coli. Quantification is performed by isotope ratio analysis with fully 13C-labeled biomass as internal standard, which is generated by growing E. coli on [U-13C]glucose. This convenient, reliable, and accurate single-platform (GC/MS) workflow for measuring biomass composition offers significant advantages over existing methods. We demonstrate the consistency, accuracy, precision, and utility of this procedure by applying it to three metabolically unique E. coli strains. The presented methods will have widespread applicability in systems microbiology and bioengineering.

Section: Resultsmentioning

confidence: 99%

Quantifying Biomass Composition by Gas Chromatography/Mass Spectrometry

Antoniewicz

2014

Anal. Chem.

“…Flux analysis studies can yield important insights into cellular metabolism that cannot be obtained from genome analysis alone (Crown and Antoniewicz, 2013b; He et al, 2014; Long and Antoniewicz, 2014a). In the accompanying paper (Cordova and Antoniewicz, 2015), a detailed characterization of xylose metabolism of Geobacillus LC300 is described using the developed model and the recently established COMPLETE-MFA methodology (Crown and Antoniewicz, 2013a; Leighty and Antoniewicz, 2013).…”

Section: Discussionmentioning

confidence: 99%

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium

Cordova

Venkataramanan

et al. 2015

Metabolic Engineering

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h−1 on glucose and 1.52 h−1 on xylose (doubling time less than 30 minutes). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio’s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.

“…Mutations in metabolic enzymes force a rewiring of flux in the cell, the nature of which can inform our understanding of alternative pathways, kinetics, and regulation (1,2). Adaptive laboratory evolution (ALE) is a powerful approach by which a microbe is cultured continuously for many generations, typically achieving improved fitness (e.g., faster growth rate) through natural selection.…”

mentioning

confidence: 99%

“…Phosphoglucose isomerase (pgi) knockouts of Escherichia coli are of significant interest in metabolic engineering and have been the subject of many investigations (1). Pgi catalyzes the first reaction in glycolysis, the conversion of glucose 6-phosphate (G6P) to fructose 6-phosphate (F6P), which in the wild type during aerobic growth on glucose catabolizes ∼70% of glucose (18,19).…”

mentioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

Gonzalez

Feist

et al. 2017

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.