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.

Photoreactions of Ozone-Tetrahydrothiophene, Ozone-Pyrrolidine, and Ozone-Thiazolidine Complexes Studied Using Matrix-Isolation IR and Visible Absorption Spectroscopies.

The photoreactions of molecular complexes composed of O3 and three 5-membered heterocyclic compounds, tetrahydrothiophene (THT), pyrrolidine (PyD), and thiazolidine (TAD), are systematically investigated using matrix-isolation infrared (IR) and UV-visible spectroscopies. Two visible-light absorption bands appear in the visible spectra obtained for O3-THT and O3-PyD, whereas four bands are observed for O3-TAD, which contains both N and S atoms in the heterocyclic ring. Upon visible-light irradiation, O3-THT and O3-PyD form their corresponding oxide derivatives, tetrahydrothiophene-1-oxide and pyrrolidine-N-oxide. Although two O3-TAD complexes with different photoreactivities are detected, both structures form thiazolidine-1-oxide upon combining with O and S atom in the heterocyclic ring, but not thiazolidine-N-oxide. The mechanism of formation of these oxide compounds can be explained by the stability of the oxide compound in the triplet state formed via the combination of O(3P) and the paired ring molecule.

Visible-light-Induced Reaction of an Ozone-Trimethylamine Complex Studied by Matrix-Isolation IR and Visible Absorption Spectroscopies.

A visible-light-induced reaction of an O3-trimethylamine (TMA) complex isolated in low-temperature noble-gas matrices is investigated by infrared (IR) and visible absorption spectroscopies using the DFT calculation. The complex isolated in a Ne matrix yields trimethylamine-N-oxide (TMAO) upon irradiation (λ ≥ 800 nm) by dissociation of O3. When the wavelength of radiation is changed to λ = 455 nm, two stable conformers of dimethylaminomethanol (DMAM) are recognized besides TMAO. In an Ar matrix, DMAM and not TMAO is mainly produced upon λ = 455 nm irradiation. The photoreaction mechanism of the O3-TMA complex with a single collision reaction between O(3P) and TMA in the gas phase to produce OH and CH2N(CH3)2 radicals is discussed.

Effect of precise control of flux ratio between the glycolytic pathways on mevalonate production in Escherichia coli.

Mevalonate is a useful metabolite synthesized from three molecules of acetyl-CoA, consuming two molecules of NADPH. Escherichia coli ( E. coli) catabolizes glucose to acetyl-CoA via several routes, such as the Embden-Meyerhof-Parnas (EMP) and the oxidative pentose phosphate (oxPP) pathways. Although the oxPP pathway supplies NADPH, it is disadvantageous in terms of acetyl-CoA supply, compared with the EMP pathway. In this study, the optimal flux ratio between the EMP and oxPP pathways on the mevalonate yield was investigated. Expression level of pgi was controlled by isopropyl ß-D-1-thiogalactopyranoside (IPTG) inducible promoter in an engineered mevalonate-producing E. coli strain. The relationship between the flux ratio and mevalonate yield was evaluated by changing the flux ratio by varying IPTG concentration. At the stationary phase, the mevalonate yield was maximum at an EMP flux of 39.7%, and was increased by 25% compared with that with no flux control (EMP flux of 70.4%). The optimal flux ratio was consistent with the theoretical value based on the mass balance of NADPH. The flux ratio between EMP and oxPP pathways affects the synthesis fluxes of mevalonate and acetate from acetyl-CoA. Fine tuning of the flux ratio would be necessary to achieve an optimized production of metabolites that require NADPH.

Exploring an Artificial Metabolic Route in Fusarium sporotrichioides: Production and Characterization of 7-Hydroxy T-2 Toxin.

An artificial metabolic route to an unnatural trichothecene was designed by taking advantage of the broad substrate specificities of the T-2 toxin biosynthetic enzymes of Fusarium sporotrichioides. By feeding 7-hydroxyisotrichodermin, a shunt pathway metabolite of F. graminearum, to a trichodiene synthase-deficient mutant of F. sporotrichioides, 7-hydroxy T-2 toxin (1) was obtained as the final metabolite. Such an approach may have future applications in the metabolic engineering of a variety of fungal secondary metabolites. The toxicity of 7-hydroxy T-2 toxin was 10 times lower than that of T-2 toxin in HL-60 cells.

Conversion of Mevalonate to Isoprenol Using Light Energy in Escherichia coli without Consuming Sugars for ATP Supply.

Bioconversion of key intermediate metabolites such as mevalonate into various useful chemicals is a promising strategy for microbial production. However, the conversion of mevalonate into isoprenoids requires a supply of adenosine triphosphate (ATP). Light-driven ATP regeneration using microbial rhodopsin is an attractive module for improving the intracellular ATP supply. In the present study, we demonstrated the ATP-consuming conversion of mevalonate to isoprenol using rhodopsin-expressing Escherichia coli cells as a whole-cell catalyst in a medium that does not contain energy cosubstrate, such as glucose. Heterologous genes for the synthesis of isoprenol from mevalonate, which requires three ATP molecules for the series of reactions, and a delta-rhodopsin gene derived from Haloterrigena turkmenica were cointroduced into E. coli. To evaluate the conversion efficiency of mevalonate to isoprenol, the cells were suspended in a synthetic medium containing mevalonate as the sole carbon source and incubated under dark or light illumination (100 µmol m-2 s-1). The specific isoprenol production rates were 10.0 ± 0.9 and 20.4 ± 0.7 µmol gDCW-1 h-1 for dark and light conditions, respectively. The conversion was successfully enhanced under the light condition. Furthermore, the conversion efficiency increased with increasing illumination intensity, suggesting that ATP regenerated by the proton motive force generated by rhodopsin using light energy can drive ATP-consuming reactions in the whole-cell catalyst.

The effect of long-term past glycemic control on executive function among patients with type 2 diabetes mellitus.

OBJECTIVE: Patients with type 2 diabetes mellitus (T2DM) show more executive dysfunction than nondiabetics. However, how long poor glycemic control affects executive function remains unclear. Thus, we aimed to investigate the relationships in a cross-sectional study. METHODS: We studied 118 T2DM outpatients (age, ≥ 60 years; excluding history of stroke, dementia and severe hypoglycemia). HbA1c values were recorded every ≤ 12 weeks for ≥ 5 years. All patients underwent verbal-fluency tests (reflecting executive function) and Mini-Mental State Examination (MMSE). The correlation between past glycemic control values and both cognitive tests scores was investigated. As markers of past glycemic control, we used average hemoglobin A1c (HbA1c) values and glycemic control variability [coefficient of variation (CV) of HbA1c values (HbA1c-CV)]. RESULTS: Verbal-fluency tests scores correlated with HbA1c-CV, but not with average HbA1c values, after adjusting for age, years of education and sex. Verbal-fluency tests scores correlated with HbA1c-CV for the past 5 years, best compared with HbA1c-CV for past < 5 years. MMSE scores were also related to only HbA1c-CV for the past 3 years in an adjustment model. CONCLUSIONS: Five-year HbA1c variability affected executive function in T2DM patients, but not average HbA1c values. Long-term longitudinal studies may be required.

Development of microsatellite markers for the wind cave-associated shrub Lonicera alpigena subsp. glehnii (Caprifoliaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the wind cave-associated shrub Lonicera alpigena subsp. glehnii to conduct phylogeographic studies on the species. METHODS AND RESULTS: Based on the sequence data obtained by 454 sequencing, a total of 81 primer pairs were designed and 18 successfully amplified the microsatellite regions. These markers were highly variable (i.e., average number of alleles per locus = 6.2 [range = 2-15]; average expected heterozygosity per locus = 0.489 [range = 0.149-0.729]). Cross-species amplification of the primers was tested in 10 congeneric taxa (L. caerulea var. emphyllocalyx, L. chamissoi, L. chrysantha, L. gracilipes var. glandulosa, L. japonica, L. kurobushiensis, L. morrowii, L. ramosissima, L. sachalinensis, and L. strophiophora), and six to 11 primers amplified the microsatellite markers. CONCLUSIONS: The microsatellite markers developed in this study will be useful for phylogeographic studies and conservation genetics of L. alpigena subsp. glehnii as well as congeneric species.

Predicting the ability of elderly diabetes patients to acquire the insulin self-injection technique based on the number of animal names recalled.

AIMS/INTRODUCTION: To our knowledge, no studies have reported that cognitive tests can be used to evaluate whether or not patients can acquire the insulin self-injection technique. We investigated whether or not the number of animal names recalled in 1 min by elderly diabetes patients could be used as a predictor of the patients' ability to acquire the insulin self-injection technique within 1 week. MATERIALS AND METHODS: We enrolled 57 inpatients with type 2 diabetes aged >60 years who were starting insulin therapy. We carried out the Mini-Mental State Examination and verbal fluency tests, which included recalling animal names and common nouns starting with the letters 'a,' 'ka' and 'shi' (Japanese letters). We used 12 checkpoints for insulin self-injection to judge the patients' levels of acquisition of the technique. The most predictive cognitive test was determined by multivariate logistic regression analysis. RESULTS: In the present study, multivariate logistic analysis showed that the number of animal names recalled was the most reliable predictor of the ability to acquire the insulin self-injection technique within 1 week. A figure of 11 animal names predicted a successful acquisition, with a sensitivity of 73% and a specificity of 91% being observed (area under the curve 0.87, 95% confidence interval 0.76-0.97, P < 0.01). CONCLUSIONS: The number of animal names recalled in 1 min was the most useful indicator of the ability of elderly diabetes patients to learn to manage insulin self-injection therapy within 1 week. The cut-off value was 11 animal names.