Large-scale preparation of yeast strains expressing condensates derived from a glycolytic enzyme via controlled dissolved oxygen levels under hypoxia.

Under hypoxia, Saccharomyces cerevisiae forms cytoplasmic condensates composed of proteins, including glycolytic enzymes, that are thought to regulate cellular metabolism. However, the hypoxic conditions required for condensate formation remain unclear. In this study, we developed a 300-mL-scale culture method to produce condensate-forming cells by precisely controlling the dissolved oxygen (DO) level in the media. Using enolase as a model, a foci formation rate of more than 50% was achieved at ∼0.1% DO, and the results showed that the DO level affected the foci formation rate. The foci formation rates of the previously reported foci-deficient strains and strains with single amino acid substitutions in the endogenous enolase were examined, and the effect of these amino acid substitutions on glucose consumption and ethanol and glycerol production under hypoxia was evaluated. The results of this study contribute to the investigation of the mechanisms that regulate biomacromolecular condensates under hypoxia.

Small-scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae.

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small-scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small-scale hypoxic culture in a bench-top multi-gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small-scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small-scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes.

Design of novel primer sets for easy detection of Ruegeria species from seawater.

Some coral-associated bacteria show protective roles for corals against pathogens. However, the distribution of coral-protecting bacteria in seawater is not well known. In addition, compared with the methods for investigating coral pathogens, few methods have been developed to detect coral-protecting bacteria. Here we prepared a simple method for detecting Ruegeria spp., some strains of which inhibit growth of the coral pathogen Vibrio coralliilyticus. We successfully obtained two Ruegeria-targeting primer sets through in silico and in vitro screening. The primer sets r38F-r30R and r445F-r446R, in addition to the newly designed universal primer set U357'F-U515'R, were evaluated in vitro using environmental DNA extracted from seawater collected in Osaka. These methods and primers should contribute to revealing the distribution of Ruegeria spp. in marine environments.

Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds.

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.

Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use.

Biocatalysts (enzymes) have many advantages as catalysts for the production of useful compounds as compared to chemical catalysts. The stereoselectivity of the enzymes is one advantage, and thus the stereoselective production of chiral compounds using enzymes is a promising approach. Importantly, industrial application of the enzymes for chiral compound production requires the discovery of a novel useful enzyme or enzyme function; furthermore, improving the enzyme properties through protein engineering and directed evolution approaches is significant. In this review, the significance of several enzymes showing stereoselectivity (quinuclidinone reductase, aminoalcohol dehydrogenase, old yellow enzyme, and threonine aldolase) in chiral compound production is described, and the improvement of these enzymes using protein engineering and directed evolution approaches for further usability is discussed. Currently, enzymes are widely used as catalysts for the production of chiral compounds; however, for further use of enzymes in chiral compound production, improvement of enzymes should be more essential, as well as discovery of novel enzymes and enzyme functions.

An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.

(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.

Fermentative production of 1-propanol from sugars using wild-type and recombinant Shimwellia blattae.

Shimwellia blattae is an enteric bacterium and produces endogenous enzymes that convert 1,2-propanediol (1,2-PD) to 1-propanol, which is expected to be used as a fuel substitute and a precursor of polypropylene. Therefore, if S. blattae could be induced to generate its own 1,2-PD from sugars, it might be possible to produce 1-propanol from sugars with this microorganism. Here, two 1,2-PD production pathways were constructed in S. blattae, resulting in two methods for 1-propanol production with the bacterium. One method employed the L-rhamnose utilization pathway, in which L-rhamnose is split into dihydroxyacetone phosphate and 1,2-PD. When wild-type S. blattae was cultured with L-rhamnose, an accumulation of 1,2-PD was observed. The other method for producing 1,2-PD was to introduce an engineered 1,2-PD production pathway from glucose into S. blattae. In both cases, the produced 1,2-PD was then converted to 1-propanol by 1,2-PD converting enzymes, whose production was induced by the addition of glycerol.

Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation.

L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity.

L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Šresolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Šoutwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR.

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily.

Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies.

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27-42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.

LplR, a repressor belonging to the TetR family, regulates expression of the L-pantoyl lactone dehydrogenase gene in Rhodococcus erythropolis.

The L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) gene (lpldh) has been cloned from Rhodococcus erythropolis AKU2103, and addition of 1,2-propanediol (1,2-PD) was shown to be required for lpldh expression in this strain. In this study, based on an exploration of the nucleotide sequence around lpldh, a TetR-like regulator gene, which we designated lplR, was found upstream of lpldh, and three putative open reading frames existed between the two genes. Disruption of lplR led to 22.8 times higher lpldh expression, even without 1,2-PD induction, than that in wild-type R. erythropolis AKU2103 without 1,2-PD addition. Introduction of a multicopy vector carrying lplR (multi-lplR) into the wild-type and ΔlplR strains led to no detectable LPLDH activity even in the presence of 1,2-PD. The results of an electrophoretic mobility shift assay revealed that purified LplR bound to a 6-bp inverted-repeat sequence located in the promoter/operator region of the operon containing lpldh. These results indicated that LplR is a negative regulator in lpldh expression. Based on the clarification of the expression mechanism of lpldh, recombinant cells showing high LPLDH activity were constructed and used as a catalyst for the conversion of L-PL to ketopantoyl lactone. Finally, a promising production process of D-PL from DL-PL was constructed.

Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens.

(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Šresolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2(1), with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, ß = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V(M) = 2.08 Å(3) Da(-1)).

Expression, purification, crystallization and preliminary X-ray analysis of carbonyl reductase S1 from Candida magnoliae.

The NADPH-dependent carbonyl reductase S1 from Candida magnoliae stereoselectively catalyzes the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), which is a chiral compound valuable as a building block for pharmaceuticals. Carbonyl reductase S1 was expressed in Escherichia coli and purified by Ni-affinity, ion-exchange and size-exclusion chromatography. Crystals of carbonyl reductase S1 were obtained by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. X-ray diffraction data were collected to 1.90 Šresolution using a synchrotron-radiation source. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.7, c = 307.5 Å. The asymmetric unit contained two molecules of the protein, with a solvent content of 44.2%.

Cloning and overexpression of ketopantoic acid reductase gene from Stenotrophomonas maltophilia and its application to stereospecific production of D-pantoic acid.

Ketopantoic acid (KPA) reductase catalyzes the stereospecific reduction of ketopantoic acid to D-pantoic acid. Based on the N-terminal amino acid sequence of KPA reductase from Stenotrophomonas maltophilia 845, the KPA reductase gene was cloned from S. maltophilia NBRC14161 and sequenced. This gene contains an open reading frame of 777 bp encoding 258 amino acid residues, and the deduced amino acid sequence showed high similarity to the SDR superfamily proteins. An expression vector, pETSmKPR, containing the full KPA reductase gene was constructed and introduced into Escherichia coli BL21 (DE3) to overexpress the enzyme. Bioreduction of KPA using E. coli transformant cells coexpressing KPA reductase together with cofactor regeneration enzyme gene was also performed. The conversion yield of KPA to D-pantoic acid reached over 88% with a substrate concentration up to 1.17 M.

L-pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of L-pantoyl lactone.

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.

Genetic analysis around aminoalcohol dehydrogenase gene of Rhodococcus erythropolis MAK154: a putative GntR transcription factor in transcriptional regulation.

NADP(+)-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 catalyzes the reduction of (S)-1-phenyl-1-keto-2-methylaminopropane ((S)-MAK) to d-pseudoephedrine, which is used as a pharmaceutical. AADH is suggested to participate in aminoalcohol or aminoketone metabolism in this organism because it is induced by the addition of several aminoalcohols, such as 1-amino-2-propanol. Genetic analysis of around the aadh gene showed that some open reading frames (ORFs) are involved in this metabolic pathway. Four of these ORFs might form a carboxysome-like polyhedral organelle, and others are predicted to encode aminotransferase, aldehyde dehydrogenase, phosphotransferase, and regulator protein. OrfE, a homologous ORF of the FadR subfamily of GntR transcriptional regulators, lies downstream from aadh. To investigate whether or not orfE plays a role in the regulation of aadh expression, the gene disruption mutant of R. erythropolis MAK154 was constructed. The ΔorfE strain showed higher AADH activity than wild-type strain. In addition, a transformed strain, which harbored multi-orfE, showed no AADH activity even in the induced condition with 1-amino-2-propanol. These results suggest that OrfE is a negative regulator that represses aadh expression in the absence of 1-amino-2-propanol.

Specific Detection of Coral-Associated Ruegeria, a Potential Probiotic Bacterium, in Corals and Subtropical Seawater.

Coral microbial flora has been attracting attention because of their potential to protect corals from environmental stresses or pathogens. Although coral-associated bacteria are considered to be acquired from seawater, little is known about the relationships between microbial composition in corals and its surrounding seawater. Here, we tested several methods to identify coral-associated bacteria in coral and its surrounding seawater to detect specific types of Ruegeria species, some of which exhibit growth inhibition activities against the coral pathogen Vibrio coralliilyticus. We first isolated coral-associated bacteria from the reef-building coral Galaxea fascicularis collected at Sesoko Island, Okinawa, Japan, via random colony picking, which showed the existence of varieties of bacteria including Ruegeria species. Using newly constructed primers for colony PCR, several Ruegeria species were successfully isolated from G. fascicularis and seawater. We further investigated the seawater microbiome in association with the distance from coral reefs. By seasonal sampling, it was suggested that the seawater microbiome is more affected by seasonality than the distance from coral reefs. These methods and results may contribute to investigating and understanding the relationships between the presence of corals and microbial diversity in seawater, in addition to the efficient isolation of specific bacterial species from coral or its surrounding seawater.