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.

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.

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.

Production of a selective antibacterial fatty acid against Staphylococcus aureus by Bifidobacterium strains.

Aims: C16 monounsaturated fatty acid (C16:1) show antibacterial activity against Staphylococcus aureus, a pathogen associated with various diseases such as atopic dermatitis and bacteremia, while the compound does not exhibit antibacterial activity against Staphylococcus epidermidis, an epidermal commensal that inhibits the growth of S. aureus. In this study, we aimed to find bifidobacterial strains with the ability to produce C16:1 and to find a practical manner to utilize C16:1-producing strains in industry. Methods: Various Bifidobacterium strains were screened for their content of C16:1. The chemical identity of C16:1 produced by a selected strain was analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Medium components that affect the C16:1 content of the selected strain were investigated. Antibacterial activity against staphylococci was compared between the authentic C16:1 isomers and total fatty acids (TFA) extracted from the selected strain. Results: B. adolescentis 12451, B. adolescentis 12-111, B. boum JCM 1211, and Bifidobacterium sp. JCM 7042 showed high C16:1 content among the tested strains. TFA extracted from Bifidobacterium sp. JCM 7042 contained C16:1 at 2.3% as the fatty acid constituent (2.4 mg/L of broth). Through GC-MS and LC-MS analyses, the C16:1 synthesized by Bifidobacterium sp. JCM 7042 was identified as 7-cis-hexadecenoic acid (7-cis-C16:1). The authentic 7-cis-C16:1 showed strong and selective antibacterial activity against S. aureus, similar to 6-cis-C16:1, with a minimum inhibitory concentration (MIC) of < 10 µg/mL. Components that increase C16:1 productivity were not found in the MRS and TOS media; however, Tween 80 was shown to considerably reduce the C16:1 ratio in TFA. Antibacterial activity against S. aureus was observed when the TFA extracted from Bifidobacterium sp. JCM 7042 contained high level of 7-cis-C16:1 (6.1% in TFA) but not when it contained low level of 7-cis-C16:1 (0.1% in TFA). Conclusion: The fatty acid, 7-cis-C16:1, which can selectively inhibit the S. aureus growth, is accumulated in TFA of several bifidobacteria. The TFA extracted from cultured cells of Bifidobacterium sp. JCM 7042 demonstrated antibacterial activity. From a practical viewpoint, our findings are important for developing an efficient method to produce novel skin care cosmetics, functional dairy foods, and other commodities.

Mead acid production by disruption of Δ12-desaturase gene in Mortierella alpina 1S-4.

Mead acid (MA; 20:3ω9) is one of the ω9 series of polyunsaturated fatty acids (PUFAs). MA is used to inhibit the inflammation of joints and is applied to the medicinal or health food field. We aimed to construct MA-producing strains with disruption of the Δ12-desaturase gene (Δ12ds) via an efficient gene-targeting system using the lig4-disrupted strain of Mortierella alpina 1S-4 as the host. The transformants showed a unique fatty acid composition that only comprised ω9-PUFAs and saturated fatty acids, while ω6-and ω3-PUFAs were not detected, and the total composition of ω9-PUFAs, including oleic acid (18:1ω9), 18:2ω9, 20:1ω9, 20:2ω9, and MA, was up to 68.4% of the total fatty acids. The MA production in the Δ12ds-disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ12ds-defective mutant JT-180.

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.

Metabolic engineering of the L-serine biosynthetic pathway improves glutathione production in Saccharomyces cerevisiae.

BACKGROUND: Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast Saccharomyces cerevisiae, and is widely used in the pharmaceutical, food, and cosmetic industries. It has been reported that addition of L-serine (L-Ser) is effective at increasing the intracellular glutathione content because L-Ser is the common precursor of L-cysteine (L-Cys) and glycine (Gly) which are substrates for glutathione biosynthesis. Therefore, we tried to enhance the L-Ser biosynthetic pathway in S. cerevisiae for improved glutathione production. RESULTS: The volumetric glutathione production of recombinant strains individually overexpressing SER2, SER1, SER3, and SER33 involved in L-Ser biosynthesis at 48 h cultivation was increased 1.3, 1.4, 1.9, and 1.9-fold, respectively, compared with that of the host GCI strain, which overexpresses genes involved in glutathione biosynthesis. We further examined simultaneous overexpression of SHM2 and/or CYS4 genes involved in Gly and L-Cys biosynthesis, respectively, using recombinant GCI strain overexpressing SER3 and SER33 as hosts. As a result, GCI overexpressing SER3, SHM2, and CYS4 showed the highest volumetric glutathione production (64.0 ± 4.9 mg/L) at 48 h cultivation, and this value is about 2.5-fold higher than that of the control strain. CONCLUSIONS: This study first revealed that engineering of L-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase.

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.

Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson's disease.

Mitochondrial degeneration is considered one of the major causes of Parkinson's disease (PD). Improved mitochondrial functions are expected to be a promising therapeutic strategy for PD. In this study, we introduced a light-driven proton transporter, Delta-rhodopsin (dR), to Drosophila mitochondria, where the mitochondrial proton-motive force (Δp) and mitochondrial membrane potential are maintained in a light-dependent manner. The loss of the PD-associated mitochondrial gene CHCHD2 resulted in reduced ATP production, enhanced mitochondrial peroxide production and lower Ca2+-buffering activity in dopaminergic (DA) terminals in flies. These cellular defects were improved by the light-dependent activation of mitochondrion-targeted dR (mito-dR). Moreover, mito-dR reversed the pathology caused by the CHCHD2 deficiency to suppress α-synuclein aggregation, DA neuronal loss, and elevated lipid peroxidation in brain tissue, improving motor behaviors. This study suggests the enhancement of Δp by mito-dR as a therapeutic mechanism that ameliorates neurodegeneration by protecting mitochondrial functions.

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.

Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose.

BACKGROUND: L-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to D-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as D-glucose, leading to a preferential utilization of D-glucose over pentoses. In order to simultaneously utilize both D-glucose and L-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. RESULTS: Corynebacterium glutamicum ATCC 31831 utilized D-glucose and L-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized D-glucose over L-arabinose at a high concentration (15 g/L each), although L-arabinose and D-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for D-glucose and L-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of L-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of D-glucose and L-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized D-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of L-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of D-glucose and L-arabinose. CONCLUSIONS: Metabolome analysis identified potential bottlenecks in D-glucose and L-arabinose metabolism and was then applied to the following rational metabolic engineering. Manipulation of only two genes enabled simultaneous utilization of D-glucose and L-arabinose at the same rate in metabolically engineered C. glutamicum. This is the first report of rational metabolic design and engineering for simultaneous hexose and pentose utilization without inactivating the phosphotransferase system.

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.

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae.

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, ß-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

Enzymatic improvement of mitochondrial thiol oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae.

BACKGROUND: Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes. RESULTS: We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. CONCLUSIONS: This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.