Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Activation of the root xylem proton pump by hydraulic signals from leaves under suppressed transpiration.

Long term field observations have revealed that the inhibition of transpiration by heavy rainfall promotes immediate positive shift in the trans-root electric potential (TRP), indicating activation of the xylem proton pump in the tree root system presumably participating in acropetal water transport. This phenomenon is indicative of signal transmission from the aerial part to the root system via change in the xylem hydraulic pressure. To test this hypothesis, we constructed a new device that enables the simultaneous recording of artificially applied xylem hydraulic pressure and the change in the TRP of tree saplings. With the application of artificial pressure to the xylem vessels (20-62 kPa), TRP shifted towards positive potential by 20-80 mV, which indicates the activation of the proton pump in the root xylem. The reaction was observed in 11 tree species, six deciduous and five evergreen, although only during the resting phase of the xylem proton pump (May to October) when the transpiration rates were high. Contrastingly the application of tension (negative pressure) produced no reaction. Simultaneous determination of the two components of the TRP, i.e. Vps (electric membrane potential difference across root surface cell membrane) and Vpx (electric membrane potential difference between root symplast and xylem vessel), are performed using the intra-cellular micro-electrode technique throughout the four seasons. Application of excess xylem hydraulic pressure had no significant effect on Vps, while it brought about hyper-polarisation of Vpx except during the winter season, most significantly during summer when transpiration is vigorous and the xylem pump is in a resting state. Such effect of excess xylem pressure was, however, not observed under anoxia.

Light-inducible flux control of triosephosphate isomerase on glycolysis in Escherichia coli.

An engineering tool for controlling flux distribution on metabolic pathways to an appropriate state is highly desirable in bioproduction. An optogenetic switch, which regulates gene expression by light illumination is an attractive on/off switchable system, and is a promising way for flux control with an external stimulus. We demonstrated a light-inducible flux control between glycolysis and the methylglyoxal (MGO) pathway in Escherichia coli using a CcaS/CcaR system. CcaR is phosphorylated by green light and is dephosphorylated by red light. Phosphorylated CcaR induces gene expression under the cpcG2 promoter. The tpiA gene was expressed under the cpcG2 promoter in a genomic tpiA deletion strain. The strain was then cultured with glucose minimum medium under green or red light. We found that tpiA messenger RNA level under green light was four times higher than that under red light. The repression of tpiA expression led to a decrease in glycolytic flux, resulting in slower growth under red light (0.25 hr -1 ) when compared to green light (0.37 hr -1 ). The maximum extracellular MGO concentration under red light (0.2 mM) was higher than that under green light (0.05 mM). These phenotypes confirm that the MGO pathway flux was enhanced under red light.

Circular dichroism and circularly polarised luminescence of bipyrenyl oligopeptides, with piperidines added in the peptide chains.

Upon combining chiral peptides (the most basic chiral source) with pyrene moieties, we found that chiral oligopeptides bearing two-pendant pyrenyl units exhibited circularly polarised luminescence (CPL) originating from intramolecular excimers at 450-490 nm in various solvents, and the sign of their CPL signals depended on the type of solvent employed. The CPL and circular dichroism signs and intensities could be tuned by the introduction of a piperidine unit into the chiral peptide chain; thus, the obtained structure could be considered a practical Lock ON-OFF system for oligopeptide luminophores.

Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus.

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth beta-sheet and the fifth alpha-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.

Identification of a rate-limiting step in a metabolic pathway using the kinetic model and in vitro experiment.

Identification of the rate-limiting step in a metabolic pathway is an important challenge in metabolic engineering for enhancing pathway flow. Although specific enzyme activities (Vmax) provide valuable clues for the identification, it is time-consuming and difficult to measure multiple enzymes in the pathway because different assay protocols are required for each enzyme. In the present study, we propose a method to simultaneously determine the Vmax values of multiple enzymes using a kinetic model with a time course of the intermediate concentrations through an in vitro experiment. To demonstrate this method, nine glycolysis reactions for converting glucose-6-phosphate (G6P) to pyruvate in Escherichia coli were considered. In a reaction mixture containing G6P and cofactors, glycolysis was initiated by adding a crude cell extract obtained from stationary phase cells. The Vmax values were optimized to minimize the difference between the measured and simulated time-courses using a kinetic model. Metabolic control analysis using the kinetic model with the estimated Vmax values revealed that fructose bisphosphate aldolase (FBA) was the rate-limiting step in the upper part of glycolysis. The addition of FBA in the reaction mixture successfully increased the glycolytic flux in vitro. Furthermore, in vivo, the specific glucose consumption rate of an FBA overexpression strain was 1.4 times higher than that of the control strain during the stationary phase. These results confirmed that FBA was the rate-limiting step in glycolysis under the stationary phase. This approach provides Vmax values of multiple enzymes in a pathway for metabolic control analysis with a kinetic model.

Estimation of linear and cyclic electron flows in photosynthesis based on 13C-metabolic flux analysis.

Photosynthetic organisms produce ATP and NADPH using light as an energy source and further utilize these cofactors during metabolism. Photosynthesis involves linear and cyclic electron flows; as the cyclic electron flow produces ATP more effectively than the linear electron flow without NADPH, the cell efficiently adjusts ATP and NADPH production using the two different pathways. Nevertheless, direct measurement of ATP and NADPH production during photosynthesis has been difficult. In the present study, the photosynthetic ATP and NADPH production rates of Synechocystis sp. PCC 6803 under three different single peak wavelength lights (blue: 470 nm, R630: 630 nm, and R680: 680 nm) were evaluated based on 13C-metabolic flux analysis (13C-MFA) by considering the mass balance of ATP and NADPH between photosynthesis and metabolism. The ratios of ATP/NADPH production via photosynthesis were estimated as 3.13, 1.70, and 2.10 under blue, R630, and R680 light conditions, respectively. Moreover, the linear and cyclic electron flow ratios were estimated to be 1.1-2.2, 0.2-0.5, and 0.5-1.0 under blue, R630, and R680 light conditions, respectively. The predicted linear and cyclic electron flow ratios were consistent with the excitation ratio between photosystems I and II, as observed in the steady-state fluorescence spectra.

CHOTTO1, a double AP2 domain protein of Arabidopsis thaliana, regulates germination and seedling growth under excess supply of glucose and nitrate.

Arabidopsis chotto1 (cho1) mutants show resistance to (-)-R-ABA, an ABA analog, during germination and seedling growth. Here, we report cloning and characterization of the CHO1 gene. cho1 mutants showed only subtle resistance to (+)-S-ABA during germination. The cho1 mutation acts as a strong enhancer of the abi5 mutant, whereas the cho1 abi4 double mutant showed ABA resistance similar to the abi4 single mutant. This suggests that CHO1 and ABI4, but not ABI5, act in the same genetic pathway. Map-based cloning revealed that the CHO1 gene encodes a putative transcription factor containing double AP2 domains. The CHO1 gene was expressed predominantly in seed, with the strongest expression in imbibed seed. Induction of CHO1 expression was observed 4 h after seed imbibition and reached a maximum level at 24 h. Induction of CHO1 expression did not occur in the abi4 mutants, indicating that this is an ABI4-dependent process. Microarray experiments showed that a large number of genes involved in primary metabolism and the stress response were up-regulated in the cho1 mutant. Growth of abi4 and cho1 mutant seedlings was resistant to high concentrations of glucose. In addition, growth of cho1 mutant seedlings was partially resistant to excess nitrate (50 mM), as evident from their expanded green cotyledons. However, their growth was normal under moderate nitrate concentrations (< 10 mM). This nitrate response was specific to the cho1 mutants and was not observed in the abi4 mutants. Taken together, our results indicate that CHO1 regulates nutritional responses downstream of ABI4 during germination and seedling growth.

13C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in Escherichia coli.

Phenol is an important chemical product that can be used in a wide variety of applications, and it is currently produced from fossil resources. Fermentation production of phenol from renewable biomass resources by microorganisms is highly desirable for sustainable development. However, phenol toxicity hampers phenol production in industrial microorganisms such as Escherichia coli. In the present study, it was revealed that culturing E. coli in the presence of phenol not only decreased growth rate, but also biomass yield. This suggests that phenol affects the carbon flow of the metabolism, but the mechanism is unknown. To investigate the effect of phenol on the flux distribution of central carbon metabolism, 13C-metabolic flux analysis (13C-MFA) was performed on cells grown under different phenol concentrations (0, 0.1, and 0.15%). 13C-MFA revealed that the TCA cycle flux reduced by 25% increased acetate production from acetyl-CoA by 30% in the presence of 0.1% phenol. This trend of flux changes was emphasized at a phenol concentration of 0.15%. Although the expression level of citrate synthase, which catalyzes the first reaction of the TCA cycle, does not change regardless of phenol concentrations, the in vitro enzyme activity assay shows that the reaction was inhibited by phenol. These results suggest that the TCA cycle flux decreased due to phenol inhibition of citrate synthase; therefore, ATP could not be sufficiently produced by respiration, and growth rate decreased. Furthermore, since carbon was lost as acetate due to overflow metabolism, the biomass yield became low in the presence of phenol.

Proteome-wide prediction of novel DNA/RNA-binding proteins using amino acid composition and periodicity in the hyperthermophilic archaeon Pyrococcus furiosus.

Proteins play a critical role in complex biological systems, yet about half of the proteins in publicly available databases are annotated as functionally unknown. Proteome-wide functional classification using bioinformatics approaches thus is becoming an important method for revealing unknown protein functions. Using the hyperthermophilic archaeon Pyrococcus furiosus as a model species, we used the support vector machine (SVM) method to discriminate DNA/RNA-binding proteins from proteins with other functions, using amino acid composition and periodicities as feature vectors. We defined this value as the composition score (CO) and periodicity score (PD). The P. furiosus proteins were classified into three classes (I-III) on the basis of the two-dimensional correlation analysis of CO score and PD score. As a result, approximately 87% of the functionally known proteins categorized as class I proteins (CO score + PD score > 0.6) were found to be DNA/RNA-binding proteins. Applying the two-dimensional correlation analysis to the 994 hypothetical proteins in P. furiosus, a total of 151 proteins were predicted to be novel DNA/RNA-binding protein candidates. DNA/RNA-binding activities of randomly chosen hypothetical proteins were experimentally verified. Six out of seven candidate proteins in class I possessed DNA/RNA-binding activities, supporting the efficacy of our method.

Metabolomics approach for enzyme discovery.

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism.

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.