Engineering cell metabolism for bioproduction not only consumes building blocks and energy molecules (e.g., ATP) but also triggers energetic inefficiency inside the cell. The metabolic burdens on microbial workhorses lead to undesirable physiological changes, placing hidden constraints on host productivity. We discuss cell physiological responses to metabolic burdens, as well as strategies to identify and resolve the carbon and energy burden problems, including metabolic balancing, enhancing respiration, dynamic regulatory systems, chromosomal engineering, decoupling cell growth with production phases, and co-utilization of nutrient resources. To design robust strains with high chances of success in industrial settings, novel genome-scale models (GSMs), (13)C-metabolic flux analysis (MFA), and machine-learning approaches are needed for weighting, standardizing, and predicting metabolic costs.

During microbial applications, metabolic burdens can lead to a significant drop in cell performance. Novel synthetic biology tools or multi-step bioprocessing (e.g., fermentation followed by chemical conversions) are therefore needed to avoid compromised biochemical productivity from over-burdened cells. A possible solution to address metabolic burden is Division of Labor (DoL) via natural and synthetic microbial consortia. In particular, consolidated bioprocesses and metabolic cooperation for detoxification or cross feeding (e.g., vitamin C fermentation) have shown numerous successes in industrial level applications. However, distributing a metabolic pathway among proper hosts remains an engineering conundrum due to several challenges: complex subpopulation dynamics/interactions with a short time-window for stable production, suboptimal cultivation of microbial communities, proliferation of cheaters or low-producers, intermediate metabolite dilution, transport barriers between species, and breaks in metabolite channeling through biosynthesis pathways. To develop stable consortia, optimization of strain inoculations, nutritional divergence and crossing feeding, evolution of mutualistic growth, cell immobilization, and biosensors may potentially be used to control cell populations. Another opportunity is direct integration of non-bioprocesses (e.g., microbial electrosynthesis) to power cell metabolism and improve carbon efficiency. Additionally, metabolic modeling and 13C-metabolic flux analysis of mixed culture metabolism and cross-feeding offers a computational approach to complement experimental research for improved consortia performance.

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO 2 and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a 13 C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A 13 C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO 2 fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate: ferredoxin oxidoreductase, and the CO 2 -anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO 2 to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80ϳ100 units for mixotrophic cultures grown on acetate and 200ϳ230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO 2 fixation and biomass growth rate; C. tepidum fixed more CO 2 and had a higher biomass yield (Y X/S , mole carbon in biomass/mole substrate) in pyruvate culture (Y X/S ‫؍‬ 9.2) than in acetate culture (Y X/S ‫؍‬ 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.Chlorobaculum tepidum is a representative green sulfur bacterium that is ecologically significant in global cycling of carbon, nitrogen, and sulfur (1, 2). The C. tepidum genome has been sequenced, and the genetic tools for creating C. tepidum mutant strains have been developed to make transposon-based mutations or targeted gene disruptions, which offer great potential to engineer C. tepidum for future applications (3). The annotated genome reveals unique aspects in carbon and energy metabolism in C. tepidum. Instead of using the Calvin-Benson cycle for CO 2 assimilation as in most photosynthetic organisms, C. tepidum captures energy from light and uses it along with electrons, primarily derived from oxidation of sulfur compounds, to drive the reductive tricarboxylic acid cycle (RTCA) 3 for synthesis of building block molecules (3). C. tepidum can grow mixotrophically with acetate or pyruvate as the organic carbon source (2). Although recent research has been performed on the carbon and energy metabolism of C. tepidum (4, 5), rigorous quantification of the metabolic pathway activities has not yet been achieved. To provide quantitative readout of the metabolic functions and regulatory mechanisms, this study has performed 13 C-assisted metabolic flux analysis of ...

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.

The acetyl-CoA "Wood-Ljungdahl" pathway couples the folatemediated one-carbon (C1) metabolism to either CO 2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO 2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyltetrahydrofolate (CH 3 -THF) for methionine biosynthesis, representing an unconventional strategy for generating CH 3 -THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.reductive dechlorination | 13 C isotope analysis | acetyl-CoA synthase