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.     

ATP in current biotechnology: Regulation,applications and perspectives

Adenosine tri-phosphate (ATP), the most important energy source for metabolic reactions and pathways, plays a vital role in the growth of industrial strain and the production of target metabolites. In this review, current advances in manipulating ATP in industrial strains, including altering NADH availability, and regulating NADH oxidation pathway, oxygen supply, proton gradient, the electron transfer chain activity and the F₀F₁-ATPase activity, are summarized and discussed. By applying these strategies, optimal product concentrations, yields and productivity in industrial biotechnology have been achieved. Furthermore, the mechanisms by which ATP extends the substrate utilization spectra and enhances the ability to challenge harsh environmental stress have been elucidated. Finally, three critical issues related to ATP manipulation have been addressed.     

Energy intake,oxidative stress and antioxidant in mice during lactation

Reproduction is the highest energy demand period for small mammals, during which both energy intake and expenditure are increased to cope with elevated energy requirements of offspring growth and somatic protection. Oxidative stress life history theory proposed that reactive oxygen species(ROS) were produced in direct proportion to metabolic rate, resulting in oxidative stress and damage to macromolecules. In the present study, several markers of oxidative stress and antioxidants activities were examined in brain, liver, kidneys, skeletal muscle and small intestine in non-lactating(Non-Lac) and lactating(Lac) KM mice. Uncoupling protein(ucps) gene expression was examined in brain, liver and muscle. During peak lactation, gross energy intake was 254% higher in Lac mice than in Non-Lac mice. Levels of H2O2 of Lac mice were 17.7% higher in brain(P<0.05), but 21.1%(P<0.01) and 14.5%(P<0.05) lower in liver and small intestine than that of Non-Lac mice. Malonadialdehyde(MDA) levels of Lac mice were significantly higher in brain, but lower in liver, kidneys, muscle and small intestine than that of Non-Lac mice. Activity of glutathione peroxidase(GSH-PX) was significantly decreased in brain and liver in the Lac group compared with that in the Non-Lac group. Total antioxidant capacity(TAOC) activity of Lac mice was significantly higher in muscle, but lower in kidneys than Non-Lac mice. Ucp4 and ucp5 gene expression of brain was 394% and 577% higher in Lac mice than in Non-Lac mice. These findings suggest that KM mice show tissuedependent changes in both oxidative stress and antioxidants. Activities of antioxidants may be regulated physiologically in response to the elevated ROS production in several tissues during peak lactation. Regulations of brain ucp4 and ucp5 gene expression may be involved in the prevention of oxidative damage to the tissue.     

Energy metabolism during diapause in Culex pipiens mosquitoes

Diapause in overwintering adult female Culex pipiens mosquitoes plays an important role in the transmission of West Nile and other encephalitis-inducing flaviviruses. To investigate the dynamic metabolic processes that control Cx. pipiens diapause, we used radioactive tracer techniques with [¹⁴C]-glucose to investigate the metabolic fate and flux of glucose in adult mosquitoes reared under diapause (18 °C, short day) and non-diapause (27 °C, long day) conditions. We found that by 72 h post-¹⁴C-labeling of 1-day-old mosquitoes, the diapause-destined mosquitoes had converted 46% more ¹⁴C-labled glucose into ¹⁴C-labled lipid than mosquitoes reared under non-diapausing conditions. When 5-day-old mosquitoes were fed [¹⁴C]-glucose, and then switched to water only, the non-diapausing mosquitoes oxidized nearly three times more ¹⁴C-labled glycogen and lipid by day 7 than diapausing-mosquitoes. This increased energy expenditure in non-diapausing mosquitoes is most likely due to temperature- and light-dependent increases in the basal metabolic rate. Amongst the diapausing-mosquitoes we analyzed over a subsequent 7-week period, we found that the amount of ¹⁴C-labeled glycogen decreased steadily for the first month of diapause, whereas, ¹⁴C-labeled-lipid levels were not significantly decreased until after day 35 of diapause, indicating that flux through glycogenolysis is higher than lipolysis during the first month of diapause. Lastly, our analysis revealed that 38% of the initial ¹⁴C-labled lipid that was synthesized during the adult pre-diapause phase was still present following the first gonotrophic cycle. About 33% of this remaining ¹⁴C-labeled lipid was localized to the newly developed eggs, suggesting that lipid sparing processes during a minimal 7-week long diapause may enhance egg production.     

Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress

Environmental stress generally disturbs cellular homeostasis. Researchers have hypothesized that chilling injury is linked to a shortage of ATP. However, previous studies conducted on insects exposed to nonfreezing low temperatures presented conflicting results. In this study, we investigated the mitochondrial bioenergetics of Drosophila melanogaster flies exposed to chronic cold stress (4 °C). We assessed mitochondrial oxygen consumption while monitoring the rate of ATP synthesis at various times (0, 1, 2, and 3 days) during prolonged cold stress and at two assay temperatures (25 and 4 °C). We compared organelle responses between cold-susceptible and cold-acclimated phenotypes. Continuous exposure to low temperature provoked temporal declines in the rates of mitochondrial respiration and ATP synthesis. Respiratory control ratios (RCRs) suggested that mitochondria were not critically uncoupled. Nevertheless, after 3 days of continuous cold stress, a sharp decline in the mitochondrial ATP synthesis rate was observed in control flies when they were assayed at low temperature. This change was associated with reduced survival capacity in control flies. In contrast, cold-acclimated flies exhibited high survival and maintained higher rates of mitochondrial ATP synthesis and coupling (i.e., higher RCRs). Adaptive changes due to cold acclimation observed in the whole organism were thus manifested in isolated mitochondria. Our observations suggest that cold tolerance is linked to the ability to maintain bioenergetics capacity under cold stress.     

Energy budget,oxidative stress and antioxidant in striped hamster acclimated to moderate cold and warm temperatures

The mechanism of the rate of living-free radical theory suggests that higher rate of oxidative metabolism results from greater rate of mitochondria oxidative phosphorylation, leading to a consequent increase in production of free radicals. However, the relation between metabolic rate and oxidative stress is tissue dependent in animals acclimated to cold temperatures. Here we examined oxidative stress, reflected by changes of antioxidant activity and other related markers, in striped hamsters acclimated to moderate cold (15 °C), room (23 °C) or warm temperature (30 °C) for 6 weeks, by which either higher or lower metabolic rate was induced experimentally. Energy intake and the rate of metabolism and nonshivering thermogenesis were increased at 15 °C, but decreased at 30 °C compared with that at 23 °C. Effects of temperatures on the markers of both oxidative stress and antioxidant activities were rarely significant. The percentages of positive correlation between the 11 tissues (brain, BAT, liver, heart, lung, kidneys, stomach, small and large intestine, caecum and skeletal muscle) were 14.5% (8/55) for catalase (CAT), 7.3% (4/55) for the capacity of inhibition of hydroxyl free radical (CIH), 5.5% (3/55) for activities of superoxide dismutase (SOD), 1.8% (1/55) for total antioxidant capacity (T-AOC), 4.3% (2/46) for H₂O₂ and 11.1% (4/36) for the capacity of inhibition of hydroxyl free radical (CIH). This indicated that the tissue-dependent changes of both oxidative stress and antioxidant activity were less consistent among the different tissues. Finally the data from this study were less consistent with the prediction of the mechanism of the rate of living-free radical theory.     

Energy metabolism of Bdellovibrio bacteriovorus

The P/O ratio of Bdellovibrio bacteriovorus, strain Bd 109 Sa, was evaluated by two different methods based on the determination of energy-rich phosphate bonds and either NADH oxidation or oxygen-uptake. P/O values calculated on the basis of NADH oxidation were up to 6, which has to be regarded as being overestimated. P/O values calculated from energy-rich phosphate bonds and oxygen uptake were around 2. The P/O values determined for Escherichia coli B were similar. The loss of phosphorylation efficiency at one site is discussed.The ATP pool turnover rate of Bdellovibrio was 8/min during endogenous respiration and 24/min during substrate respiration. The corresponding values in Escherichia coli B were 3/min and 38/min.This study was performed at the University of Hamburg (Institut für Allgemeine Botanik, Abteilung Mikrobiologic).     

多轮次胁迫驯化及多因子复合筛选丁醇高产菌

【目的】从陕西省石泉县玉米地土壤中分离获得一株产丁醇菌株并提高其丁醇耐受性和丁醇产量。【方法】采用自行设计的多因子复合筛选方法和丁醇胁迫驯化处理,在获得丁醇高产菌株的同时提高菌株的丁醇耐受性。【结果】野生菌株D64经多轮次丁醇胁迫驯化处理和多因子复合筛选,分离获得突变株T64,其丁醇耐受性明显提高,能在丁醇浓度为20 g/L的复合筛选培养基上正常生长,发酵7%玉米醪丁醇产量由13.35 g/L提高到15.18 g/L,总溶剂(丙酮、丁醇、乙醇)达到21.8 g/L。【结论】采用长时间且丁醇浓度呈梯度渐进增加的胁迫驯化方式,可使菌种在丁醇的环境中不断进化并有效地提高菌株对丁醇的耐受性。多因子复合筛选方法较其他单一因子筛选方法更为有效,能较快获得丁醇高产菌。     

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications

Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T _m of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive γ-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.     

Harnessing yeast subcellular compartments for the production of plant terpenoids

The biologically and commercially important terpenoids are a large and diverse class of natural products that are targets of metabolic engineering. However, in the context of metabolic engineering, the otherwise well-documented spatial subcellular arrangement of metabolic enzyme complexes has been largely overlooked. To boost production of plant sesquiterpenes in yeast, we enhanced flux in the mevalonic acid pathway toward farnesyl diphosphate (FDP) accumulation, and evaluated the possibility of harnessing the mitochondria as an alternative to the cytosol for metabolic engineering. Overall, we achieved 8- and 20-fold improvement in the production of valencene and amorphadiene, respectively, in yeast co-engineered with a truncated and deregulated HMG1, mitochondrion-targeted heterologous FDP synthase and a mitochondrion-targeted sesquiterpene synthase, i.e. valencene or amorphadiene synthase. The prospect of harnessing different subcellular compartments opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds.     

The time course of acclimation to the stress of triose phosphate use limitation

Triose phosphate utilisation (TPU) limits the maximum rate at which plants can photosynthesise. However, TPU is almost never found to be limiting photosynthesis under ambient conditions for plants. This, along with previous results showing adaptability of TPU at low temperature, suggest that TPU capacity is regulated to be just above the photosynthetic rate achievable under the prevailing conditions. A set of experiments were performed to study the adaptability of TPU capacity when plants are acclimated to elevated CO₂ concentrations. Plants held at 1500 ppm CO₂ were initially TPU limited. After 30 h they no longer exhibited TPU limitations but they did not elevate their TPU capacity. Instead, the maximum rates of carboxylation and electron transport declined. A timecourse of regulatory responses was established. A step increase of CO₂ first caused PSI to be oxidised but after 40 s both PSI and PSII had excess electrons as a result of acceptor-side limitations. Electron flow to PSI slowed and the proton motive force increased. Eventually, non-photochemical quenching reduced electron flow sufficiently to balance the TPU limitation. Over several minutes rubisco deactivated contributing to regulation of metabolism to overcome the TPU limitation.     

Metabolic and evolutionary engineering research in Turkey and beyond

This review discusses metabolic engineering research with an emphasis on evolutionary (whole cell and protein) engineering, which is an inverse metabolic engineering approach. For each section on metabolic, inverse metabolic and evolutionary engineering research, a general review of the major global studies in the literature is made and research examples from Turkey are given and discussed. It is expected that with the rapid development in systems biology and the novel powerful analytical technologies to identify the genetic basis of cellular phenotypes, metabolic and evolutionary engineering research will become widespread and increasingly important in Turkey, following global scientific trends.     

Improving metabolic efficiency of the reverse beta-oxidation cycle by balancing redox cofactor requirement

Previous studies have made many exciting achievements on pushing the functional reversal of beta-oxidation cycle (r-BOX) to more widespread adoption for synthesis of a wide variety of fuels and chemicals. However, the redox cofactor requirement for the efficient operation of r-BOX remains unclear. In this work, the metabolic efficiency of r-BOX for medium-chain fatty acid (C₆-C₁₀, MCFA) production was optimized by redox cofactor engineering. Stoichiometric analysis of the r-BOX pathway and further experimental examination identified NADH as a crucial determinant of r-BOX process yield. Furthermore, the introduction of formate dehydrogenase from Candida boidinii using fermentative inhibitor byproduct formate as a redox NADH sink improved MCFA titer from initial 1.2 g/L to 3.1 g/L. Moreover, coupling of increasing the supply of acetyl-CoA with NADH to achieve fermentative redox balance enabled product synthesis at maximum titers. To this end, the acetate re-assimilation pathway was further optimized to increase acetyl-CoA availability associated with the new supply of NADH. It was found that the acetyl-CoA synthetase activity and intracellular ATP levels constrained the activity of acetate re-assimilation pathway, and 4.7 g/L of MCFA titer was finally achieved after alleviating these two limiting factors. To the best of our knowledge, this represented the highest titer reported to date. These results demonstrated that the key constraint of r-BOX was redox imbalance and redox engineering could further unleash the lipogenic potential of this cycle. The redox engineering strategies could be applied to acetyl-CoA-derived products or other bio-products requiring multiple redox cofactors for biosynthesis.     

Viability staining and detection of metabolic activity of sourdough lactic acid bacteria under stress conditions

World Journal of Microbiology and Biotechnology - Forty-one strains of lactic acid bacteria isolated from wheat sourdoughs were exposed to acid, osmotic and oxidative stresses. Live/Dead®...     

Recognition of Cytosolic DNA Attenuates Glucose Metabolism and Induces AMPK Mediated Energy Stress Response

Both viral infection and DNA transfection expose single-stranded or double-stranded DNA to the cytoplasm of mammalian cells. Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway. Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types. Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1. Our results suggest that in concert with but independent of innate immune response, recognition of cytosolic DNA induced cellular energy stress potentially functions as a metabolic barrier to viral replication.     

The xanthophyll cycle and acclimation of Pinus ponderosa and Malva neglecta to winter stress

Seasonal differences in the efficiency of open PSII units (F _v/F _m), leaf pigment composition and xanthophyll cycle conversion (Z+A)/(V+A+Z), leaf adenylate status, and photosynthetic capacity were investigated in Pinus ponderosa (Ponderosa pine) and Malva neglecta. In P. ponderosa, acclimation to winter involved a lower photosynthetic capacity, higher carotenoid to chlorophyll ratio, persistent reductions in F _v/F _m corresponding to persistent retention of Z+A, and no change in foliar ATP/ADP ratios. In contrast, M. neglecta characterized in winter exhibited higher rates of photosynthesis than in summer with no change in carotenoid to chlorophyll ratio, while small nocturnally persistent reductions in F _v/F _m were observed exclusively on colder winter nights when nocturnal retention of Z+A, and high ATP/ADP ratios were also present. Upon removal of winter-stressed leaves or needles from the field to room temperature, a portion of F _v/F _m relaxed within 15 min of warming and recovery was completed within 5 h in M. neglecta but required 100 h in P. ponderosa. In M. neglecta, the entire recovery of F _v/F _m correlated with decreases in the foliar ATP/ADP ratio, while in P. ponderosa this ratio remained unchanged. Possible ATP-dependent forms of sustained (Z+A)-dependent energy dissipation are discussed including a nocturnally retained pH gradient on cold winter nights. The slow recovery in pine involved not only retention of Z+A, but apparently also a persistent engagement of Z+A for energy dissipation via an unidentified mechanism. Received: 15 May 1998 / Accepted: 9 November 1998     

Emerging roles of phosphoinositide-associated membrane trafficking in plant stress responses

Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell. These barriers are formed by assembly of lipids and protein in aqueous environments. Lipids not only serve as building blocks for membrane construction, but also possess regulatory functions in cellular activities. These regulatory lipids are non-uniformly distributed in membrane systems; their temporal and spatial accumulation in specific membranes decodes environmental cues and changes cellular activity accordingly. Phosphoinositides (PIs) are phospholipids that exert regulatory effects. In recent years, research on PIs roles in regulating plant growth, development, and responses to environmental stress is increasing. Several reviews have been published on the composition of PIs, intermolecular transferring of PIs by lipid kinases (phosphatases) or PI-PLCs, subcellular localization, and specially their functions in plant developments. Herein, we review the crucial regulatory functions of PIs in plant stress responses, with a particular focus on PIs involved in membrane trafficking.     

miR396-GRF模块参与植物逆境胁迫响应的研究进展

miR396-GRF模块是一类新型高效的植物生长发育调控模块。该模块中miR396通过负调控生长调节因子GRF,影响植物生长发育过程中的信号传导通路,并广泛参与植物对干旱、盐害、温度、UV-B等非生物胁迫,以及胞囊线虫和病原菌等造成的生物胁迫的响应过程。本文综述了miR396-GRF模块的调控网络和其参与植物抗逆响应的作用机理,并探讨了相关研究动态及存在的问题,旨在为进一步推动GRF相关研究提供理论参考。