Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel ¹³C‐metabolite fingerprinting

Abstract

For rapid analysis of microbial metabolisms, (13)C-fingerprinting employs a set of tracers to generate unique labeling patterns in key amino acids that can highlight active pathways. In contrast to rigorous (13)C-metabolic flux analysis ((13)C-MFA), this method aims to provide metabolic insights without expensive flux measurements. Using (13)C-fingerprinting, we investigated the metabolic pathways in Rhodococcus opacus PD630, a promising biocatalyst for the conversion of lignocellulosic feedstocks into value-added chemicals. Specifically, seven metabolic insights were gathered as follows: (1) glucose metabolism mainly via the Entner-Doudoroff (ED) pathway; (2) lack of glucose catabolite repression during phenol co-utilization; (3) simultaneous operation of gluconeogenesis and the ED pathway for the co-metabolism of glucose and phenol; (4) an active glyoxylate shunt in acetate-fed culture; (5) high flux through anaplerotic pathways (e.g., malic enzyme and phosphoenolpyruvate carboxylase); (6) presence of alternative glycine synthesis pathway via glycine dehydrogenase; and (7) utilization of preferred exogenous amino acids (e.g., phenylalanine). Additionally, a (13)C-fingerprinting kit was developed for studying the central metabolism of non-model microbial species. This low-cost kit can be used to characterize microbial metabolisms and facilitate the design-build-test-learn cycle during the development of microbial cell factories.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence

“…The EDP offers the possibility of directing carbon toward pyruvate, which can be converted into acetyl‐CoA and enter the TCA cycle, where biomass precursors are formed. The EDP consists of two enzymes, 6PG dehydratase ( edd ) and 2‐keto‐3‐deoxyphosphogluconate (KDPG) aldolase ( eda ), and branches off from the competing OPPP after the glucose‐6‐phosphate dehydrogenase ( zwf ) produces 6PG (Hollinshead et al, ). The KDPG aldolase produces a molecule of GAP and a molecule of pyruvate.…”

Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and 13C metabolic flux analysis

“…Since low dilution rate mimics the late stationary phase because of carbon source limitation, this drives E. coli cells to reduce its glycolytic flux and scavenge any possible carbon sources including acetate [37]. Similar to E. coli growing at the low dilution rate, other slow growing bacteria with weak glycolytic fluxes, such as Corynebacterium and Rhodococcus [38, 39], also showed no CCR without acetate overflow.…”

Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system

“…Moreover, in addition to measuring labeling of protein-bound amino acids, it may be advantageous to measure 13 C-labeling of carbohydrates (McConnell and Antoniewicz, 2016), fatty acids (Crown et al, 2015b), nucleosides (Miranda-Santos et al, 2015), glycogen and RNA (Guzman et al, 2014; Long et al, 2016a), and intracellular metabolites (Ahn and Antoniewicz, 2013; Millard et al, 2014). Finally, a practical consideration is the cost associated with performing 13 C-MFA studies: the cost of tracers (Table 1), analytical instruments, sample preparation, analysis time, and the cost of performing parallel labeling experiments vs. single tracer experiments (Hollinshead et al, 2016). All of these factors should be considered collectively to determine the best or most convenient strategy for completing a 13 C-MFA study.…”