Effects of n-butanol production on metabolism and the photosystem in Synecococcus elongatus PCC 7942 based on metabolic flux and target proteome analyses.

Although n-butanol (BuOH) is an ideal fuel because of its superior physical properties, it has toxicity to microbes. Previously, a Synechococcus elongatus PCC 7942 derivative strain that produces BuOH from CO2 was developed by introducing six heterologous genes (BUOH-SE strain). To identify the bottleneck in BuOH production, the effects of BuOH production and its toxicity on central metabolism and the photosystem were investigated. Parental (WT) and BUOH-SE strains were cultured under autotrophic conditions. Consistent with the results of a previous study, BuOH production was observed only in the BUOH-SE strain. Isotopically non-stationary 13C-metabolic flux analysis revealed that the CO2 fixation rate was much larger than the BuOH production rate in the BUOH-SE strain (1.70 vs 0.03 mmol gDCW-1 h-1), implying that the carbon flow for BuOH biosynthesis was less affected by the entire flux distribution. No large difference was observed in the flux of metabolism between the WT and BUOH-SE strains. Contrastingly, in the photosystem, the chlorophyll content and maximum O2 evolution rate per dry cell weight of the BUOH-SE strain were decreased to 81% and 43% of the WT strain, respectively. Target proteome analysis revealed that the amounts of some proteins related to antennae (ApcA, ApcD, ApcE, and CpcC), photosystem II (PsbB, PsbU, and Psb28-2), and cytochrome b6f complex (PetB and PetC) in photosystems decreased in the BUOH-SE strain. The activation of photosynthesis would be a novel approach for further enhancing BuOH production in S. elongatus PCC 7942.

Transomics data-driven, ensemble kinetic modeling for system-level understanding and engineering of the cyanobacteria central metabolism.

In silico kinetic modeling is an essential tool for rationally designing metabolically engineered organisms based on a system-level understanding of their regulatory mechanisms. However, an estimation of enzyme parameters has been a bottleneck in the computer simulation of metabolic dynamics. In this study, the ensemble-modeling approach was integrated with the transomics data to construct kinetic models. Kinetic metabolic models of a photosynthetic bacterium, Synechocystis sp. PCC 6803, were constructed to identify engineering targets for improving ethanol production based on an understanding of metabolic regulatory systems. A kinetic model ensemble was constructed by randomly sampling parameters, and the best 100 models were selected by comparing predicted metabolic state with a measured dataset, including metabolic flux, metabolite concentrations, and protein abundance data. Metabolic control analysis using the model ensemble revealed that a large pool size of 3-phosphoglycerate could be a metabolic buffer responsible for the stability of the Calvin-Benson cycle, and also identified that phosphoglycerate kinase (PGK) is a promising engineering target to improve a pyruvate supply such as for ethanol production. Overexpression of PGK in the metabolically engineered PCC 6803 strain showed that the specific ethanol production rate and ethanol titers at 48 h were 1.23- and 1.37-fold greater than that of the control strain. PGK is useful for future metabolic engineering since pyruvate is a common precursor for the biosynthesis of various chemicals.

Comparative analysis of fermentation and enzyme expression profiles among industrial Saccharomyces cerevisiae strains.

Industrial diploid strains of Saccharomyces cerevisiae are selected from natural populations and then domesticated by optimizing the preferred properties for producing products such as bread, wine, and sake. In this study, for comparing the fermentation performance of various industrial yeasts, seven diploid strains of S. cerevisiae, namely, BY4947 (laboratory yeast derived from S288C), Kyokai7 and Kyokai9 (sake yeasts), Red Star and NBRC0555 (bread yeasts), and QA23 and EC1118 (wine yeasts), were cultivated in a synthetic medium. The fermentation profiles of the seven yeast strains showed significant differences. The specific ethanol production rates of sake yeasts (Kyokai7 and Kyokai9) and wine strains (QA23 and EC1118) were higher and lower than those of laboratory strains, respectively. Targeted proteome analysis was also conducted to investigate the variation in the expression of metabolism-related enzymes. The expression profiles of central metabolism-related enzymes showed considerable variations among the industrial strains. Upregulation of the TCA cycle in wine strains was observed both in the synthetic and grape-juice media. These results suggested that these variations should be consequences of complex interactions between the domestication process, genetic polymorphism, and environmental factors such as the fermentation conditions.

Comparative Targeted Proteomics of the Central Metabolism and Photosystems in SigE Mutant Strains of Synechocystis sp. PCC 6803.

A targeted proteome analysis was conducted to investigate the SigE dependent-regulation of central metabolism in Synechocystis sp. PCC 6803 by directly comparing the protein abundance profiles among the wild type, a sigE deletion mutant (ΔsigE), and a sigE over-expression (sigEox) strains. Expression levels of 112 target proteins, including the central metabolism related-enzymes and the subunits of the photosystems, were determined by quantifying the tryptic peptides in the multiple reaction monitoring (MRM) mode of liquid-chromatography⁻triple quadrupole mass spectrometry (LC⁻MS/MS). Comparison with gene-expression data showed that although the abundance of Gnd protein was closely correlated with that of gnd mRNA, there were poor correlations for GdhA/gdhA and glycogen degradation-related genes such as GlgX/glgX and GlgP/glgP pairs. These results suggested that the regulation of protein translation and degradation played a role in regulating protein abundance. The protein abundance profile suggested that SigE overexpression reduced the proteins involved in photosynthesis and increased GdhA abundance, which is involved in the nitrogen assimilation pathway using NADPH. The results obtained in this study successfully demonstrated that targeted proteome analysis enables direct comparison of the abundance of central metabolism- and photosystem-related proteins.