Yoshihiro Toya

Flux analysis and metabolomics for systematic metabolic engineering of microorganisms.

Rational engineering of metabolism is important for bio-production using microorganisms. Metabolic design based on in silico simulations and experimental validation of the metabolic state in the engineered strain helps in accomplishing systematic metabolic engineering. Flux balance analysis (FBA) is a method for the prediction of metabolic phenotype, and many applications have been developed using FBA to design metabolic networks. Elementary mode analysis (EMA) and ensemble modeling techniques are also useful tools for in silico strain design. The metabolome and flux distribution of the metabolic pathways enable us to evaluate the metabolic state and provide useful clues to improve target productivity. Here, we reviewed several computational applications for metabolic engineering by using genome-scale metabolic models of microorganisms. We also discussed the recent progress made in the field of metabolomics and (13)C-metabolic flux analysis techniques, and reviewed these applications pertaining to bio-production development. Because these in silico or experimental approaches have their respective advantages and disadvantages, the combined usage of these methods is complementary and effective for metabolic engineering.

13C-metabolic flux analysis in heterologous cellulase production by Bacillus subtilis genome-reduced strain

The great potential of Bacillus subtilis to produce biomaterials would be further enhanced by the development of strains with deletions of non-essential genomic regions. Here, using stationary (13)C-metabolic flux analysis ((13)C-MFA), we investigated the metabolism during cellulase production by the genome-reduced B. subtilis strain MGB874. We transformed MGB874 and wild-type strains with the heterologous cellulase gene, and cultured these on a synthetic medium containing glucose as carbon source. The addition of glutamate and the genome reduction enhanced cellulase production, which led us to use (13)C-MFA to assess the effects of glutamate addition and gene deletions on metabolism. We found that there was a significant increase in the flux in the pentose phosphate (PP) pathway, whereas the fluxes of reactions from acetyl-CoA to α-ketoglutarate were repressed in the presence of glutamate. We hypothesize that the increase in the PP pathway flux was caused by the decrease of citrate synthase flux through the accumulation of glycolytic intermediates. Excess NADPH produced by the PP pathway may affect the increase in cellulase production. Furthermore, the fluxes on glycolysis and the acetate formation of the cellulase-producing wild-type strain were significantly larger than that of the cellulase-producing MGB874 strain when the strains were cultured with glucose and glutamate.

13C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli

Mevalonate (MVA) is used to produce various useful products such as drugs, cosmetics and food additives. An MVA-producing strain of Escherichia coli (engineered) was constructed by introducing mvaES genes from Enterococcus faecalis. The engineered strain produced 1.84xa0mmol/gDCW/h yielding 22% (C-mol/C-mol) of MVA from glucose in the aerobic exponential growth phase. The mass balance analysis revealed that the MVA yield of the engineered strain was close to the upper limit at the biomass yield. Since MVA is synthesized from acetyl-CoA using NADPH as a cofactor, the production of MVA affects central metabolism in terms of carbon utilization and NADPH requirements. The reason for this highly efficient MVA production was investigated based on 13C-metabolic flux analysis. The estimated flux distributions revealed that the fluxes of acetate formation and the TCA cycle in the engineered strain were lower than those in the control strain. Although the oxidative pentose phosphate pathway is considered as the NADPH generating pathway in E.xa0coli, no difference of the flux was observed between the control and engineered strains. The production/consumption balance of NADPH suggested that additional requirement of NADPH for MVA synthesis was obtained from the transhydrogenase reaction in the engineered strain. Comparison between the measured flux distribution and the ideal values for MVA production proposes a strategy for further engineering to improve the MVA production in E.xa0coli.

Investigation of useful carbon tracers for 13C-metabolic flux analysis of Escherichia coli by considering five experimentally determined flux distributions

The 13C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13C-labeled glucose for 13C-metabolic flux analysis (13C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli, the best fitted flux distribution and the 95% confidence interval were estimated by the 13C-MFA procedure. A comparison of the precision scores showed that [1, 2-13C]glucose and a mixture of [1-13C] and [U-13C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1-13C], and [U-13C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13C-MFA experiments.

Construction of a Genome-Scale Metabolic Model of Arthrospira platensis NIES-39 and Metabolic Design for Cyanobacterial Bioproduction

Arthrospira (Spirulina) platensis is a promising feedstock and host strain for bioproduction because of its high accumulation of glycogen and superior characteristics for industrial production. Metabolic simulation using a genome-scale metabolic model and flux balance analysis is a powerful method that can be used to design metabolic engineering strategies for the improvement of target molecule production. In this study, we constructed a genome-scale metabolic model of A. platensis NIES-39 including 746 metabolic reactions and 673 metabolites, and developed novel strategies to improve the production of valuable metabolites, such as glycogen and ethanol. The simulation results obtained using the metabolic model showed high consistency with experimental results for growth rates under several trophic conditions and growth capabilities on various organic substrates. The metabolic model was further applied to design a metabolic network to improve the autotrophic production of glycogen and ethanol. Decreased flux of reactions related to the TCA cycle and phosphoenolpyruvate reaction were found to improve glycogen production. Furthermore, in silico knockout simulation indicated that deletion of genes related to the respiratory chain, such as NAD(P)H dehydrogenase and cytochrome-c oxidase, could enhance ethanol production by using ammonium as a nitrogen source.

Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production. In the present study, a nitrogen starvation approach was applied on an ethanol producing strain for inhibiting the growth, since ethanol production competes with the cell growth. The effect of gene deletions in the glycogen and polyhydroxybutyrate (PHB) synthesis pathways was investigated. Measurements of intracellular glycogen and PHB revealed that the glycogen was accumulated under the nitrogen starvation condition and the gene deletion of glycogen synthesis pathway caused the accumulation of PHB. The ethanol producing strain harboring deletions for both the glycogen and the PHB synthesis pathways (ΔglgCΔphaCE/EtOH) produced ethanol at the specific rate of 240mgg (dry cell weight)-1u2009day-1 under the nitrogen starvation condition. In a high cell density culture (OD730=50) using this ΔglgCΔphaCE/EtOH strain, the ethanol production rates were 1.08 and 2.01gL-1u2009day-1 under light conditions of 40 and 80μmolm-2s-1, respectively.

SSDesign: Computational metabolic pathway design based on flux variability using elementary flux modes.

Metabolic pathway modification based on the stoichiometric model has been an effective approach for enhancing microbial bio‐production. The network of optimal pathways for “growth‐associated” and “non‐growth‐associated” production can be designed from the flux variability (solution space). The present study introduces a new computational method (solution space design [SSDesign]) that visually designs the gene knockout solution space. The smallest reaction sets that satisfy the mass balances of intermediates are called elementary flux nodes (EFMs). Because some of the EFMs necessarily occupy the outer boundary nodes of the flux solution space, the proposed SSDesign determines the area over which EFMs should be removed from the solution space of the parent strain, and explores the gene knockouts that will eliminate these undesirable EFMs. To evaluate the performance of SSDesign, the model was applied to growth‐associated and non‐growth‐associated succinate production in Escherichia coli. In the growth‐associated case, the deletion mutants that promoted succinate production at maximum biomass yield were predicted, and a candidate of ΔptsG ΔpykA,F ΔpflA has been experimentally confirmed as a succinate producer. Simply by changing the parameters, the gene knockout combinations yielding high growth yield were successfully predicted by SSDesign. In the non‐growth‐associated case, strong candidates for succinate production were the deletion mutants ΔpntAB ΔsfcA ΔpykA,F and ΔsfcA ΔmaeB ΔpykA,F Δzwf. According to the solution spaces, these strains allow high growth yield and inevitably produce succinate at zero biomass yield, since their metabolic pathways cannot sustain steady‐state without discarding succinate from the cell. Biotechnol. Bioeng. 2015;112: 759–768.

Enhanced dipicolinic acid production during the stationary phase in Bacillus subtilis by blocking acetoin synthesis

Bacterial bio-production during the stationary phase is expected to lead to a high target yield because the cells do not consume the substrate for growth. Bacillus subtilis is widely used for bio-production, but little is known about the metabolism during the stationary phase. In this study, we focused on the dipicolinic acid (DPA) production by B. subtilis and investigated the metabolism. We found that DPA production competes with acetoin synthesis and that acetoin synthesis genes (alsSD) deletion increases DPA productivity by 1.4-fold. The mutant showed interesting features where the glucose uptake was inhibited, whereas the cell density increased by approximately 50%, resulting in similar volumetric glucose consumption to that of the parental strain. The metabolic profiles revealed accumulation of pyruvate, acetyl-CoA, and the TCA cycle intermediates in the alsSD mutant. Our results indicate that alsSD-deleted B. subtilis has potential as an effective host for stationary-phase production of compounds synthesized from these intermediates. Graphical abstract Dipicolinic acid production in B. subtilis during the stationary phase. The deletion of alsSD enhanced the dipicolinic acid productivity by 1.4-fold.

Metabolic flux analysis of Synechocystis sp. PCC 6803 Δ nrtABCD mutant reveals a mechanism for metabolic adaptation to nitrogen-limited conditions

Metabolic flux redirection during nitrogen-limited growth was investigated in the Synechocystis sp. PCC 6803 glucose-tolerant (GT) strain under photoautotrophic conditions by isotopically non-stationary metabolic flux analysis (INST-MFA). A ΔnrtABCD mutant of Synechocystis sp. PCC 6803 was constructed to reproduce phenotypes arising during nitrogen starvation. The ΔnrtABCD mutant and the wild-type GT strain were cultured under photoautotrophic conditions by a photobioreactor. Intracellular metabolites were labeled over a time course using NaH13CO3 as a carbon source. Based on these data, the metabolic flux distributions in the wild-type and ΔnrtABCD cells were estimated by INST-MFA. The wild-type GT and ΔnrtABCD strains displayed similar distribution patterns, although the absolute levels of metabolic flux were lower in ΔnrtABCD. Furthermore, the relative flux levels for glycogen metabolism, anaplerotic reactions and the oxidative pentose phosphate pathway were increased in ΔnrtABCD. This was probably due to the increased expression of enzyme genes that respond to nitrogen depletion. Additionally, we found that the ratio of ATP/NADPH demand increased slightly in the ΔnrtABCD mutant. These results indicated that futile ATP consumption increases under nitrogen-limited conditions because the Calvin-Benson cycle and the oxidative pentose phosphate pathway form a metabolic futile cycle that consumes ATP without CO2 fixation and NADPH regeneration.

Identification of alcohol stress tolerance genes of Synechocystis sp. PCC 6803 using adaptive laboratory evolution

BackgroundSynechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.ResultsIsobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2xa0g/L isobutanol. These evolved strains grew on medium containing 5xa0g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.ConclusionsNovel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.