Yinjie J. Tang

Advances in analysis of microbial metabolic fluxes via 13C isotopic labeling

Metabolic flux analysis via (13)C labeling ((13)C MFA) quantitatively tracks metabolic pathway activity and determines overall enzymatic function in cells. Three core techniques are necessary for (13)C MFA: (1) a steady state cell culture in a defined medium with labeled-carbon substrates; (2) precise measurements of the labeling pattern of targeted metabolites; and (3) evaluation of the data sets obtained from mass spectrometry measurements with a computer model to calculate the metabolic fluxes. In this review, we summarize recent advances in the (13)C-flux analysis technologies, including mini-bioreactor usage for tracer experiments, isotopomer analysis of metabolites via high resolution mass spectrometry (such as GC-MS, LC-MS, or FT-ICR), high performance and large-scale isotopomer modeling programs for flux analysis, and the integration of fluxomics with other functional genomics studies. It will be shown that there is a significant value for (13)C-based metabolic flux analysis in many biological research fields.

Comparative Eco-Toxicities of Nano-ZnO Particles under Aquatic and Aerosol Exposure Modes

The antimicrobial activity of ZnO nanoparticles (NPs) was investigated under aquatic and aerosol exposure modes. ZnO NPs in aquatic media aggregated to micrometer-sized particles and did not interact with microorganisms effectively. Hence, the inhibition of microbial growth by nano-ZnO NPs (e.g., Mycobacterium smegmatis and Cyanothece 51142) in aquatic media was mainly attributable to dissolved zinc species. Shewanella oneidensis MR-1 and Escherichia coli were able to produce large amounts of extracellular polymeric substances, and their growth was not inhibited by ZnO NPs in aquatic media, even at high concentrations (>40 mg/L). On the other hand, when ZnO NPs were electrosprayed onto an E. coli biofilm so that NPs could be directly deposited onto the cell surface, the aerosol exposure dramatically reduced cellular viability. For example, an electrospray of ZnO NPs (20 nm) reduced the total number of viable E.coli cells by 57% compared to the control case, in which we electrosprayed only the buffer solution. However, electrospraying large-sized ZnO particles (480 nm) or nonsoluble TiO(2) NPs (20 nm) caused much less lethality to E. coli cells. The above observation implies that the aerosol method of exposing ZnO NPs to biological systems appears to have a much higher antimicrobial activity, and thus may lead to practical applications of employing a novel antimicrobial agent for airborne disease control.

Metabolic Burden: Cornerstones in Synthetic Biology and Metabolic Engineering Applications

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.

Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production

ABSTRACT Global warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacterium Synechocystis sp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl β-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employed in situ removal of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions.

Separation and mass spectrometry in microbial metabolomics

Measurements of low molecular weight metabolites have been increasingly incorporated in the characterization of cellular physiology, qualitative studies in functional genomics, and stress response determination. The application of cutting edge analytical technologies to the measurement of metabolites and the changes in metabolite concentrations under defined conditions have helped illuminate the effects of perturbations in pathways of interest, such as the tricarboxylic acid cycle, as well as unbiased characterizations of microbial stress responses as a whole. Owing to the complexity of microbial metabolite extracts and the large number of metabolites therein, advanced and high-throughput separation techniques in gas chromatography, liquid chromatography, and capillary electrophoresis have been coupled to mass spectrometry - usually high-resolution mass spectrometry, but not exclusively - to make these measurements.

Anti-microbial activities of aerosolized transition metal oxide nanoparticles

This study used the electrospray method to create airborne droplets of metal oxide nanoparticles (NPs) and examined their anti-microbial activities, employing Escherichia coli as a model microbial species. We tested the anti-microbial activities of six metal oxide NPs (NiO, ZnO, Fe(2)O(3), Co(3)O(4), CuO, and TiO(2)) in both an aqueous culture medium and an aerosol exposure mode (spraying the particles directly onto the cell surface). In the aqueous medium, the both NPs and stressed E. coli cells severely aggregated. Only NiO NPs (>20 mgL(-1)) showed significant growth inhibition of E. coli ( approximately 30%). In contrast to aqueous exposure, where the direct interactions between NPs and bacteria were limited, aerosol exposure of three metal oxide NPs to E. coli enhanced NP toxicity to cells and dramatically reduced cellular viability. Electrospraying NiO, CuO, or ZnO NPs (20 nm, 20 microg, in 10 min) reduced the total number of living E. coli by more than 88%, 77% and 71%, respectively (compared to the control experiments). However, TiO(2), Co(3)O(4), and Fe(2)O(3) NPs showed no significant antibacterial activities in either the aqueous exposure mode or the aerosol exposure mode. The above observations suggest the potential application of electrosprayed metal oxide NPs to disinfect airborne pathogens.

Phytotoxicity of Metal Oxide Nanoparticles is Related to Both Dissolved Metals Ions and Adsorption of Particles on Seed Surfaces

This study assesses the biological effects of nanoparticles (NPs) based on seed germination and root elongation tests. Lettuce, radish and cucumber seeds were incubated with various metal oxide NPs (CuO, NiO, TiO2, Fe2O3, Co3O4), of which only CuO and NiO showed deleterious impacts on the activities of all three seeds. The measured EC50 for seed germinations were: lettuce seed (NiO: 28 mg/L; CuO: 13 mg/L), radish seed (NiO: 401 mg/L; CuO: 398 mg/L), and cucumber seed (NiO: 175 mg/L; CuO: 228 mg/L). Phytotoxicity of TiO2, Fe2O3 and Co3O4 to the tested seeds was not significant, while Co3O4 NP solution (5 g/L) was shown to improve root elongation of radish seedling. Metal oxide NPs tended to adsorb on seed surfaces in the aqueous medium and released metal ions near the seeds. Therefore, metal oxide NPs had higher phytotoxicity than free metal ions of the equivalent concentrations. Further, the surface area-to-volume ratio of seeds may also affect NPs phytotoxicity, whereby small seeds (i.e., lettuce) were the most sensitive to CuO and NiO NPs in our experiments.

Central metabolic responses to the overproduction of fatty acids in Escherichia coli based on 13C-metabolic flux analysis.

We engineered a fatty acid overproducing Escherichia coli strain through overexpressing tesA (“pull”) and fadR (“push”) and knocking out fadE (“block”). This “pull‐push‐block” strategy yielded 0.17 g of fatty acids (C12–C18) per gram of glucose (equivalent to 48% of the maximum theoretical yield) in batch cultures during the exponential growth phase under aerobic conditions. Metabolic fluxes were determined for the engineered E. coli and its control strain using tracer ([1,2‐13C]glucose) experiments and 13C‐metabolic flux analysis. Cofactor (NADPH) and energy (ATP) balances were also investigated for both strains based on estimated fluxes. Compared to the control strain, fatty acid overproduction led to significant metabolic responses in the central metabolism: (1) Acetic acid secretion flux decreased 10‐fold; (2) Pentose phosphate pathway and Entner–Doudoroff pathway fluxes increased 1.5‐ and 2.0‐fold, respectively; (3) Biomass synthesis flux was reduced 1.9‐fold; (4) Anaplerotic phosphoenolpyruvate carboxylation flux decreased 1.7‐fold; (5) Transhydrogenation flux converting NADH to NADPH increased by 1.7‐fold. Real‐time quantitative RT‐PCR analysis revealed the engineered strain increased the transcription levels of pntA (encoding the membrane‐bound transhydrogenase) by 2.1‐fold and udhA (encoding the soluble transhydrogenase) by 1.4‐fold, which is in agreement with the increased transhydrogenation flux. Cofactor and energy balances analyses showed that the fatty acid overproducing E. coli consumed significantly higher cellular maintenance energy than the control strain. We discussed the strategies to future strain development and process improvements for fatty acid production in E. coli. Biotechnol. Bioeng. 2014;111: 575–585.

Anaerobic Central Metabolic Pathways in Shewanella oneidensis MR-1 Reinterpreted in the Light of Isotopic Metabolite Labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.

Photoautotrophic production of D-lactic acid in an engineered cyanobacterium

BackgroundThe world faces the challenge to develop sustainable technologies to replace thousands of products that have been generated from fossil fuels. Microbial cell factories serve as promising alternatives for the production of diverse commodity chemicals and biofuels from renewable resources. For example, polylactic acid (PLA) with its biodegradable properties is a sustainable, environmentally friendly alternative to polyethylene. At present, PLA microbial production is mainly dependent on food crops such as corn and sugarcane. Moreover, optically pure isomers of lactic acid are required for the production of PLA, where D-lactic acid controls the thermochemical and physical properties of PLA. Henceforth, production of D-lactic acid through a more sustainable source (CO2) is desirable.ResultsWe have performed metabolic engineering on Synechocystis sp. PCC 6803 for the phototrophic synthesis of optically pure D-lactic acid from CO2. Synthesis of optically pure D-lactic acid was achieved by utilizing a recently discovered enzyme (i.e., a mutated glycerol dehydrogenase, GlyDH*). Significant improvements in D-lactic acid synthesis were achieved through codon optimization and by balancing the cofactor (NADH) availability through the heterologous expression of a soluble transhydrogenase. We have also discovered that addition of acetate to the cultures improved lactic acid production. More interestingly, 13C-pathway analysis revealed that acetate was not used for the synthesis of lactic acid, but was mainly used for synthesis of certain biomass building blocks (such as leucine and glutamate). Finally, the optimal strain was able to accumulate 1.14 g/L (photoautotrophic condition) and 2.17 g/L (phototrophic condition with acetate) of D-lactate in 24 days.ConclusionsWe have demonstrated the photoautotrophic production of D-lactic acid by engineering a cyanobacterium Synechocystis 6803. The engineered strain shows an excellent D-lactic acid productivity from CO2. In the late growth phase, the lactate production rate by the engineered strain reached a maximum of ~0.19 g D-lactate/L/day (in the presence of acetate). This study serves as a good complement to the recent metabolic engineering work done on Synechocystis 6803 for L-lactate production. Thereby, our study may facilitate future developments in the use of cyanobacterial cell factories for the commercial production of high quality PLA.