Background Synechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium characterized to date. Its genome was found to be 99.8% identical to S. elongatus 7942 yet it grows twice as fast. Current genome-to-phenome mapping is still poorly performed for non-model organisms. Even for species with identical genomes, cell phenotypes can be strikingly different. To understand Synechococcus 2973’s fast-growth phenotype and its metabolic features advantageous to photo-biorefineries, 13C isotopically nonstationary metabolic flux analysis (INST-MFA), biomass compositional analysis, gene knockouts, and metabolite profiling were performed on both strains under various growth conditions.ResultsThe Synechococcus 2973 flux maps show substantial carbon flow through the Calvin cycle, glycolysis, photorespiration and pyruvate kinase, but minimal flux through the malic enzyme and oxidative pentose phosphate pathways under high light/CO2 conditions. During fast growth, its pool sizes of key metabolites in central pathways were lower than suboptimal growth. Synechococcus 2973 demonstrated similar flux ratios to Synechococcus 7942 (under fast growth conditions), but exhibited greater carbon assimilation, higher NADPH concentrations, higher energy charge (relative ATP ratio over ADP and AMP), less accumulation of glycogen, and potentially metabolite channeling. Furthermore, Synechococcus 2973 has very limited flux through the TCA pathway with small pool sizes of acetyl-CoA/TCA intermediates under all growth conditions.ConclusionsThis study employed flux analysis to investigate phenotypic heterogeneity among two cyanobacterial strains with near-identical genome background. The flux/metabolite profiling, biomass composition analysis, and genetic modification results elucidate a highly effective metabolic topology for CO2 assimilatory and biosynthesis in Synechococcus 2973. Comparisons across multiple Synechococcus strains indicate faster metabolism is also driven by proportional increases in both photosynthesis and key central pathway fluxes. Moreover, the flux distribution in Synechococcus 2973 supports the use of its strong sugar phosphate pathways for optimal bio-productions. The integrated methodologies in this study can be applied for characterizing non-model microbial metabolism.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0958-y) contains supplementary material, which is available to authorized users.

BackgroundGlycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden–Meyerhof–Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism.ResultsOverexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium).ConclusionsWe engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0630-y) contains supplementary material, which is available to authorized users.

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593-1602. © 2017 Wiley Periodicals, Inc.

Cyanobacteria (blue-green algae) play profound roles in ecology and biogeochemistry. One model cyanobacterial species is the unicellular cyanobacterium Synechocystis sp. PCC 6803. This species is highly amenable to genetic modification. Its genome has been sequenced and many systems biology and molecular biology tools are available to study this bacterium. Recently, researchers have put significant efforts into understanding and engineering this bacterium to produce chemicals and biofuels from sunlight and CO2. To demonstrate our perspective on the application of this cyanobacterium as a photosynthesis-based chassis, we summarize the recent research on Synechocystis 6803 by focusing on five topics: rate-limiting factors for cell cultivation; molecular tools for genetic modifications; high-throughput system biology for genome wide analysis; metabolic modeling for physiological prediction and rational metabolic engineering; and applications in producing diverse chemicals. We also discuss the particular challenges for systems analysis and engineering applications of this microorganism, including precise characterization of versatile cell metabolism, improvement of product rates and titers, bioprocess scale-up, and product recovery. Although much progress has been achieved in the development of Synechocystis 6803 as a phototrophic cell factory, the biotechnology for “Compounds from Synechocystis” is still significantly lagging behind those for heterotrophic microbes (e.g., Escherichia coli).

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.