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.

This study investigated the metabolism of Pseudomonas aeruginosa PAO1 during its biofilm development via microscopy imaging, gene expression analysis, and 13C-labeling. First, dynamic labeling was employed to investigate glucose utilization rate in fresh biofilms (thickness 40∼60 micrometer). The labeling turnover time of glucose-6-P indicated biofilm metabolism was substantially slower than planktonic cells. Second, PAO1 was cultured in continuous tubular biofilm reactors or shake flasks. Then 13C-metabolic flux analysis of PAO1 was performed based on the isotopomer patterns of proteinogenic amino acids. The results showed that PAO1 biofilm cells during growth conserved the flux features as their planktonic mode. (1) Glucose could be degraded by two cyclic routes (the TCA cycle and the Entner-Doudoroff-Embden-Meyerhof-Parnas loop) that facilitated NAD(P)H supplies. (2) Anaplerotic pathways (including pyruvate shunt) increased flux plasticity. (3) Biofilm growth phenotype did not require significant intracellular flux rewiring (variations between biofilm and planktonic flux network, normalized by glucose uptake rate as 100%, were less than 20%). (4) Transcription analysis indicated that key catabolic genes in fresh biofilm cells had expression levels comparable to planktonic cells. Finally, PAO1, Shewanella oneidensis (as the comparing group), and their c-di-GMP transconjugants (with different biofilm formation capabilities) were 13C-labeled under biofilm reactors or planktonic conditions. Analysis of amino acid labeling variances from different cultures indicated Shewanella flux network was more flexibly changed than PAO1 during its biofilm formation.

Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. Initially developed for proteomic analysis, the parallel reaction monitoring (PRM) technique exploiting high-resolution MS2 fragment ion data has shown high promise for targeted metabolite quantification. Notably, MS1 ion intensity data acquired independently as part of each PRM scan cycle are often underutilized in the PRM assay. In this study, we developed an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids and shikimate pathway-related metabolites on an orthogonal QqTOF system. Concentration curve assessment revealed that exploiting both MS1 and MS2 scans in PRM analysis afforded higher sensitivity, wider dynamic range and better reproducibility than relying on either scan mode for quantification. Furthermore, Skyline was incorporated into our workflow to process the MS1/MS2 ion intensity data, and eliminate noisy signals and transitions with interferences. This integrated MS1/MS2 PRM approach was applied to targeted metabolite quantification in engineered E. coli strains for understanding of metabolic pathway modulation. In addition, this new approach, when first implemented in a dynamic C-labeling experiment, showed its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationaryC-labeling metabolism analysis.

Our recent C-metabolic flux analysis ( C-MFA) study indicates that energy metabolism becomes a rate-limiting factor for fatty acid overproduction in E. coli strains (after "Push-Pull-Block" based genetic modifications). To resolve this bottleneck, Vitreoscilla hemoglobin (VHb, a membrane protein facilitating O transport) was introduced into a fatty-acid-producing strain to promote oxygen supply and energy metabolism. The resulting strain, FAV50, achieved 70% percent higher fatty acid titer than the parent strain in micro-aerobic shake tube cultures. In high cell-density bioreactor fermentations, FAV50 achieved free fatty acids at a titer of 7.02 g/L (51% of the theoretical yield). In addition to "Push-Pull-Block-Power" strategies, our experiments and flux balance analysis also revealed the fatty acid over-producing strain is sensitive to metabolic burden and oxygen influx, and thus a careful evaluation of the cost-benefit tradeoff with the guidance of fluxome analysis will be fundamental for the rational design of synthetic biology strains. Biotechnol. Bioeng. 2017;114: 463-467. © 2016 Wiley Periodicals, Inc.

Cyanobacterium offers a promising chassis for phototrophic production of renewable chemicals. Although engineered cyanobacteria can achieve similar product carbon yields as heterotrophic microbial hosts, their production rate and titer under photoautotrophic conditions are 10 to 100 folds lower than those in fast growing E. coli. Cyanobacterial factories face three indomitable bottlenecks. First, photosynthesis has limited ATP and NADPH generation rates. Second, CO 2 fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has poor efficiency. Third, CO 2 mass transfer and light supply are deficient within large photobioreactors. On the other hand, cyanobacteria may employ organic substrates to promote phototrophic cell growth, N 2 fixation, and metabolite synthesis. The photo-fermentations show enhanced photosynthesis, while CO 2 loss from organic substrate degradation can be reused by the Calvin cycle. In addition, the plasticity of cyanobacterial pathways (e.g., oxidative pentose phosphate pathway and the TCA cycle) has been recently revealed to facilitate the catabolism. The use of cyanobacteria as "green E. coli" could be a promising route to develop robust photobiorefineries.