Proton Logic Gate Based on a Gramicidin-ATP Synthase Integrated Biotransducer.

Ion channels are membrane proteins that allow ionic signals to pass through channel pores for biofunctional modulations. However, biodevices that integrate bidirectional biological signal transmission between a device and biological converter through supported lipid bilayers (SLBs) while simultaneously controlling the process are lacking. Therefore, in this study, we aimed to develop a hybrid biotransducer composed of ATP synthase and proton channel gramicidin A (gA), controlled by a sulfonated polyaniline (SPA) conducting polymer layer deposited on a microelectrode, and to simulate a model circuit for this system. We controlled proton transport across the gA channel using both electrical and chemical input signals by applying voltage to the SPA or introducing calcium ions (inhibitor) and ethylenediaminetetraacetic acid molecules (inhibitor remover). The insertion of gA and ATP synthase into SLBs on microelectrodes resulted in an integrated biotransducer, in which the proton current was controlled by the flux of adenosine diphosphate molecules and calcium ions. Lastly, we created an XOR logic gate as an enzymatic logic system where the output proton current was controlled by Input A (ATP synthase) and Input B (calcium ions), making use of the unidirectional and bidirectional transmission of protons in ATP synthase and gA, respectively. We combined gA, ATP synthase, and SPA as a hybrid bioiontronics system to control bidirectional or unidirectional ion transport across SLBs in biotransducers. Thus, our findings are potentially relevant for a range of advanced biological and medical applications.

Metabolome analysis of metabolic burden in Escherichia coli caused by overexpression of green fluorescent protein and delta-rhodopsin.

Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E. coli strains have distinct metabolic capacities. In this study, two proteins were overexpressed in four E. coli strains (MG1655(DE3), W3110(DE3), BL21star(DE3), and Rosetta(DE3)), and their effects on metabolic burden were investigated. Metabolomic analysis showed that E. coli strains overexpressing green fluorescent protein had decreased levels of several metabolites, with a positive correlation between the number of reduced metabolites and green fluorescent protein expression levels. Moreover, nucleic acid-related metabolites decreased, indicating a metabolic burden in the E. coli strains, and the growth rate and protein expression levels were improved by supplementation with the five nucleosides. In contrast, two strains overexpressing delta rhodopsin, a microbial membrane rhodopsin from Haloterrigena turkmenica, led to a metabolic burden and decrease in the amino acids Ala, Val, Leu, Ile, Thr, Phe, Asp, and Trp, which are the most frequent amino acids in the delta rhodopsin protein sequence. The metabolic burden caused by protein overexpression was influenced by the metabolic capacity of the host strains and the sequences of the overexpressed proteins. Detailed characterization of the effects of protein expression on the metabolic state of engineered cells using metabolomics will provide insights into improving the production of target compounds.

Engineering yeast with a light-driven proton pump system in the vacuolar membrane.

BACKGROUND: The supply of ATP is a limiting factor for cellular metabolism. Therefore, cell factories require a sufficient ATP supply to drive metabolism for efficient bioproduction. In the current study, a light-driven proton pump in the vacuolar membrane was constructed in yeast to reduce the ATP consumption required by V-ATPase to maintain the acidification of the vacuoles and increase the intracellular ATP supply for bioproduction. RESULTS: Delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR. CONCLUSIONS: Yeast harboring the light-driven proton pump in the vacuolar membrane developed in this study are a potential optoenergetic cell factory suitable for various bioproduction applications.

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.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli.

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E. coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E. coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50 µmol m-2 s-1 at 37 °C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E. coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.

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.

Effect of ethanol on astaxanthin and fatty acid production in the red yeast Xanthophyllomyces dendrorhous.

AIM: The effects of detergent, ethanol and ethanol with plant meadowfoam oil on the growth of the red heterobasidomycete Xanthophyllomyces dendrorhous and on the production of astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) and fatty acids in this red yeast were investigated. METHODS AND RESULTS: Ethanol supplementation at a final concentration of 0.8% (v/v) caused an increase in the growth, astaxanthin production and fatty acid production of treated X. dendrorhous compared with untreated X. dendrorhous. Supplementation of meadowfoam oil with 0.8% ethanol further improved the growth and astaxanthin production of X. dendrorhous. Fatty acid compositions following supplementation with various concentrations of ethanol and oil were also analysed. With 0.8% ethanol supplementation, the ratio of linoleic acid (C18:2) and α-linolenic acid (C18:3ω3, ALA) decreased. Conversely, with 1.8% ethanol supplementation, the ALA ratio increased. CONCLUSIONS: Ethanol can serve as a promoting factor for coproduction of astaxanthin and fatty acids in X. dendrorhous, whereas simultaneous supplementation of ethanol and meadowfoam oil can cause further astaxanthin production. SIGNIFICANCE AND IMPACT OF STUDY: Astaxanthin is widely used in various functional products because of its antioxidant activity. This study shows that X. dendrorhous can coproduce astaxanthin and functional fatty acids at high levels following supplementation with ethanol.

A Method of Solubilizing and Concentrating Astaxanthin and Other Carotenoids.

The valuable marine carotenoid, astaxanthin, is used in supplements, medicines and cosmetics. In this study, crustacyanin, an astaxanthin-binding protein, was used to solubilize and concentrate astaxanthin. The recombinant crustacyanin of European lobster spontaneously formed an inclusion body when it was over-expressed in Escherichia coli. In this study, fusing the NusA-tag to the crustacyanin subunits made it possible to express in a soluble fraction and solubilize astaxanthin in aqueous solution. By cutting off the NusA-tag, the crustacyanin subunits generated the pure insoluble form, and captured and concentrated astaxanthin. Overall, the attaching and releasing NusA-tag method has the potential to supply solubilized carotenoids in aqueous solution and concentrated carotenoids, respectively.

Carotenoid Nostoxanthin Production by Sphingomonas sp. SG73 Isolated from Deep Sea Sediment.

Carotenoids are used commercially for dietary supplements, cosmetics, and pharmaceuticals because of their antioxidant activity. In this study, colored microorganisms were isolated from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan. One strain was found to be a pure yellow carotenoid producer, and the strain was identified as Sphingomonas sp. (Proteobacteria) by 16S rRNA gene sequence analysis; members of this genus are commonly isolated from air, the human body, and marine environments. The carotenoid was identified as nostoxanthin ((2,3,2',3')-ß,ß-carotene-2,3,2',3'-tetrol) by mass spectrometry (MS), MS/MS, and ultraviolet-visible absorption spectroscopy (UV-Vis). Nostoxanthin is a poly-hydroxy yellow carotenoid isolated from some photosynthetic bacteria, including some species of Cyanobacteria. The strain Sphingomonas sp. SG73 produced highly pure nostoxanthin of approximately 97% (area%) of the total carotenoid production, and the strain was halophilic and tolerant to 1.5-fold higher salt concentration as compared with seawater. When grown in 1.8% artificial sea salt, nostoxanthin production increased by 2.5-fold as compared with production without artificial sea salt. These results indicate that Sphingomonas sp. SG73 is an efficient producer of nostoxanthin, and the strain is ideal for carotenoid production using marine water because of its compatibility with sea salt.

Production of transglutaminase in glutathione-producing recombinant Saccharomyces cerevisiae.

Transglutaminase (TG) catalyzes the formation of cross-links between proteins. TG from Streptoverticillium mobaraense (SmTG) is used widely in food, cosmetic, biomaterial and medical industries. SmTG is occasionally supplied as a mixture with the activator peptide glutathione. Currently, glutathione is industrially produced using a budding yeast, Saccharomyces cerevisiae, because of its intracellular high content of glutathione. In this study, active SmTG was produced together with glutathione in S. cerevisiae. SmTG extracted from S. cerevisiae expressing SmTG showed cross-linking activity when BSA and sodium caseinate were substrates. The cross-linking activity of SmTG increased proportionally as the concentration of added glutathione increased. Furthermore, SmTG was prepared by extracting SmTG from an engineered S. cerevisiae whose glutathione synthetic pathway was enhanced. The SmTG solution showed higher activity when compared with a SmTG solution prepared from a S. cerevisiae strain without enhanced glutathione production. This result indicates that a high content of intracellular glutathione further enhances active SmTG production in S. cerevisiae. S. cerevisiae co-producing SmTG and a higher content of glutathione has the potential to supply a ready-to-use industrial active TG solution.

Development of astaxanthin production from citrus peel extract using Xanthophyllomyces dendrorhous.

Developing a use for the inedible parts of citrus, mainly peel, would have great environmental and economic benefits worldwide. Astaxanthin is a value-added fine chemical that affects fish pigmentation and has recently been used in healthcare products for humans, resulting in an increased demand. This study aimed to produce astaxanthin from a citrus, ponkan, peel extract using the yeast Xanthophyllomyces dendrorhous, which has the ability to use both pentose and hexose. Feeding on only ponkan peel extract enhanced X. dendrorhous growth and the concomitant astaxanthin production. Additionally, we determined that pectin and its arabinose content were the main substrate and sole carbon source, respectively, for X. dendrorhous growth and astaxanthin production. Thus, ponkan peel extract could become a valuable resource for X. dendrorhous-based astaxanthin production. Using citrus peel extract for microbial fermentation will allow the development of processes that produce value-added chemicals from agricultural byproducts.

5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae.

BACKGROUND: 5'-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. RESULTS: We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. CONCLUSIONS: In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae.

Transporter engineering in biomass utilization by yeast.

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.

Inhibition of thermophilic F1-ATPase by the ε subunit takes different path from the ADP-Mg inhibition.

The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3ß3γδε. The γ and ε subunits rotate relative to the α3ß3δ sub-complex on ATP hydrolysis by the ß subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3ß3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation.

F(1)-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the gamma-subunit against the alpha(3)beta(3) ring. The crystal structures of F(1), which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F(1) has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F(1), it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at betaE391 and gammaR84 of F(1) from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F(1), the corresponding residues in the ADP-bound beta (beta(DP)) and gamma were in direct contact. The betaE190D mutation was additionally introduced into the beta to slow ATP hydrolysis. By incorporating a single copy of the mutant beta-subunit, the chimera F(1), alpha(3)beta(2)beta(E190D/E391C)gamma(R84C), was prepared. In single-molecule rotation assay, chimera F(1) showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of beta(E190D/E391C). When the mutant beta and gamma were cross-linked through a disulfide bond between betaE391C and gammaR84C, F(1) paused the rotation at the catalytic dwell angle of beta(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that beta(DP) is the catalytically active form. The former point was again confirmed in experiments where F(1) rotation was inhibited by adenosine-5'-(beta,gamma-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F(1).

Activation of pausing F1 motor by external force.

A rotary motor F(1), a catalytic part of ATP synthase, makes a 120 degrees step rotation driven by hydrolysis of one ATP, which consists of 80 degrees and 40 degrees substeps initiated by ATP binding and probably by ADP and/or P(i) dissociation, respectively. During active rotations, F(1) spontaneously fails in ADP release and pauses after a 80 degrees substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >+40 degrees with magnetic tweezers, the pausing F(1) resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F(1) weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an F(o) motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.