Search

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.

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.

The simplest and scientific method to determine the bioelectric activity of a plant root is the measurement of the Trans-Root Electric Potential (TRP) i.e., the electric potential difference between the xylem apoplast and the earth surrounding the root.

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.

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.

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

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.

The photoexcited and ground state chiralities of enantiopure oligopeptides bearing two pyrenyl groups (DD-2/LL-2: four methylene spacer between two pyrenyl groups and DD-1/LL-1: no methylene spacer thereof) in eight common organic solvents (CHCl3, CH2Cl2, MeOH, CH3CN, DMF, NMP, DMAc, and acetone) were investigated by means of circularly polarized luminescence (CPL) and circular dichroism (CD) spectroscopies. It was found that chiroptical signs of pyrene-origin CPL and CD signals largely depend on the nature of solvents and are susceptible to the methylene spacer. Actually, the excimer-origin CPL signs at 450–510 nm of DD-1/LL-1 in DMF were (–)/(+), respectively, while those in other solvents were oppositely (+)/(–), respectively. On the other hand, in DD-2/LL-2, the excimer-origin CPL signs were (–)/(+) in CH2Cl2 (and CHCl3), respectively, and (+)/(–) in MeOH/DMF/NMP/DMAc, respectively. The solvent dependent and the methylene spacer dependent CD sign inversion of DD-1/LL-1 and DD-2/LL-2 were also found. These results led to conclude that the solvent molecule and the methylene spacer collaboratively affect both the ground state chirality and the photoexcited chirality.

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.

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.

We prepared 16 novel chiral peptide–pyrene organic luminophores with different distances between the fluorescent pyrene groups and investigated their properties in CHCl3 at room temperature. The peptide–pyrene organic luminophores bearing two pyrene groups emit strong excimer circularly polarized luminescence (CPL) in the solution state. Two pendant pyrenyl groups in a series of chiral oligopeptides clearly revealed excimer CPL in chloroform at 480–490 nm (|gem| ≈ (2–8) × 10–3). When the distance between the two pyrenes increased, the sign of the CPL signals was inverted twice, while the sign of the corresponding circular dichroism (CD) signals was retained, (|gCD| ≈ (3–8) × 10–5).

The coordination of Eu(iii) (from Eu(iii)(hfa)3) by chiral P(iii) (from BINAP) led to circularly polarised luminescence in dilute solutions on the order of |gem| ≈ 10−2 at 5D0 → F1 and |gem| ≈ 10−2 at 5D0 → F2 transitions, i.e., in chloroform and acetone.

The circularly polarised luminescence and circular dichroism signs of the blue circularly polarised luminescence generated by two photoexcited pyrenes as puppets are controlled by the wire structure of the C2-symmetric binaphthyl as puppeteer. The experimental results, though at variance with theoretical simulation, offer a rational design in the photoexcited and ground state chiralities of luminophores.

Chiral organic compounds in the photoexcited state are potential chiral luminophores. Photoluminescence properties of these compounds can be measured using circularly polarised luminescence (CPL) spectroscopy. Axially chiral binaphthyl BINAP in CHCl3 solution or the KBr-dispersed state exhibits no significant CPL signal. Herein, we report the first CPL spectrum of BINAP by doping into a poly(methyl methacrylate) (PMMA) organic polymeric matrix. For comparison, CPL data of BINAPO, a structural analogue of BINAP, were collected. Contrarily to BINAP, BINAPO showed a clear CPL signal in all three states.

Circularly polarized luminescent properties of C2-symmetrical binaphthyl molecular systems bearing two achiral anthracenes as luminophores were tuned by solely exploiting photoexcited-state chirality transfer from the binaphthyl to anthracenes when open- or closed-type binaphthyl structure as non-luminophore were chosen.

A supramolecular benzothiophene heterocyclic host complex was successfully prepared by combining benzo[b]thiophene-3-acetic acid and chiral (1R,2S)-2-amino-1,2-diphenylethanol. Four types of n-alkyl alcohols could be stored as guests in one-dimensional channel-like cavities composed of 21-helical columnar network structures. The release temperatures of the stored guest alcohols could be multiply tuned according to the type of the benzothiophene unit in the complex.

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 β-sheet and the fifth α-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.

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.

Proteins are a major regulatory component in complex biological systems. Among them, DNA/RNA-binding proteins, the key components of the central dogma of molecular biology, and membrane proteins, which are necessary for both signal transduction and metabolite transport, are suggested to be the most important protein families that arose in the early stage of life. In this study, we computationally analyzed the whole proteome data of six model species to overview the protein diversity in the three domains of life (Bacteria, Archaea and Eukaryota), especially focusing on the above two protein families. To compare the protein distribution among the six model species, we calculated various protein profiles: hydropathy, molecular weight, amino acid composition and periodicity for each protein. We found a domain-specific distribution of the proteome based on 2D correlation analysis of hydropathy and molecular weight. Further, the merged protein distribution of Archaea and other domains revealed many membrane proteins localized in Bacteria-specific regions with a high ratio of hydropathy and many DNA/RNA-binding proteins localized in Eukaryotaspecific regions with a low ratio of hydropathy. Since about half of the proteins encoded in the genome are still functionally unknown, we further conducted Support Vector Machine (SVM)-based functional prediction using amino acid composition (CO score) and periodicity (PD score) as feature vectors to predict the overall number of DNA/RNA-binding proteins and membrane proteins in the proteome. Our estimation indicated that two functional categories occupy approximately 60% to 80% of the proteome, and further, the proportion of the two categories varied among the three domains of life, suggesting that the proteome has gone through different selective pressure during evolution.

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.

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.

Regulation of the in vivo yielding properties of the cell wall, namely cell wall extensibility ( ) and effective turgor (/*—10, of Vigna hypocotyl segments during adaptive recovery of growth after osmotic stress was investigated by successive measurements using the pressure-jump method. In the absence of IAA, cell wall extensibility ( ) showed no significant changes under osmotic stress but effective turgor (P—10 reduced to near zero, resulting in no growth recovery. In the presence of 10 //M IAA, the cell wall extensibility was slightly decreased even when distinct recovery of growth took place in response to osmotic stress caused by 60 mM sorbitol. The effective turgor, however, showed a characteristic increase in contrast to the wall extensibility. It is not wall extensibility but effective turgor that regulates the cell wall yielding during the adaptive recovery of growth. In excised hypocotyl segments, however, reduced turgor would not be expected to recover, as has been observed in hollow cylinders (Nakahori et al. 1990). The results presented here suggest that the IAA-dependent adjustment of the yield threshold plays an indispensable role in wall-yielding for rapid adaptive growth recovery under osmotic stress.

The growth responses to osmotic stress of hypocotyl sections of Vigna unguiculata were studied by the xylem perfusion method. Hypocotyl sections shrank upon exposure to osmotic stress. Sections showed no adaptive responses to osmotic stress when they were in an IAAdepleted condition as a result of perfusion with solutions that lacked IAA for 3-4 h. The correlation between the growth rate and the membrane potential of the xylem/symplast boundary (V*) was very limited in the absence of IAA. By contrast, hypocotyl sections showed distinct adaptive responses to osmotic stress after perfusion with solutions that contained 10 fiM IAA. In the presence of IAA, V* increased in the negative direction and growth resumed in spite of the osmotic stress. The growth rate was closely correlated with the xylem membrane potential. Hyperpolarization of the membranes of the xylem/symplast boundary always preceded the recovery of growth under osmotic stress. It appears that IAA is essential for the adaptive recovery of growth under osmotic stress and, moreover, that the xylem proton pump plays an indispensable role in modulating the growth of hypocotyl sections. This result confirms prediction of an earlier simulation study using the apoplast canal model [Katou and Furumoto (1986) Protoplasma 133: 174, Katou and Enomoto (1991) Plant Cell Physiol. 32: 343].

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.

学位の種類：工学 学位授与年月日：令和3年3月18日 主査：須藤,篤教授 報告番号：甲第1484号 学内授与番号：工第220号 Mimura, Y; Nishikawa, T; Fuchino, R; Nakai, S; Tajima, N; Kitamatsu, M; Fujiki, M; Imai, Y; Organic and Biomolecular Chemistry, Issue 21, 2017, Yuki Mimura, Sayaka Kitamura, Motohiro Shizuma, Mizuki Kitamatsu, Michiya Fujiki, Yoshitane Imai, Chmistry Select, Volume2, Issue26, September 11, 2017, Pages 7759-7764, Yuki Mimura, Sayaka Kitamura, Motohiro Shizuma, Mizuki Kitamatsu, Yoshitane Imai, Organic & Biomolecular Chemistry, 37,2018, Yuki Mimura, Yuki Motomura, Mizuki Kitamatsu, Yoshitane Imai, TETRAHEDRON LETTERS, Volume 61, Issue 39, 24 September 2020, Yuki Mimura,Yuki Motomura,Mizuki Kitamatsu,Yoshitane Imai, ASIAN JOURNAL OF ORGANIC CHEMISTRY, 30 December 2020, Yuki Mimura, Yuki Motomura, Mizuki Kitamatsu, Yoshitane Imai, Processes, Volume 8, Issue 12 掲載