Mutational analysis in Corynebacterium stationis MFS transporters for improving nucleotide bioproduction.

Product secretion from an engineered cell can be advantageous for microbial cell factories. Extensive work on nucleotide manufacturing, one of the most successful microbial fermentation processes, has enabled Corynebacterium stationis to transport nucleotides outside the cell by random mutagenesis; however, the underlying mechanism has not been elucidated, hindering its applications in transporter engineering. Herein, we report the nucleotide-exporting major facilitator superfamily (MFS) transporter from the C. stationis genome and its hyperactive mutation at the G64 residue. Structural estimation and molecular dynamics simulations suggested that the activity of this transporter improved via two mechanisms: (1) enhancing interactions between transmembrane helices through the conserved "RxxQG" motif along with substrate binding and (2) trapping substrate-interacting residue for easier release from the cavity. Our results provide novel insights into how MFS transporters change their conformation from inward- to outward-facing states upon substrate binding to facilitate efflux and can contribute to the development of rational design approaches for efflux improvements in microbial cell factories. KEYPOINTS: • An MFS transporter from C. stationis genome and its mutation at residue G64 were assessed • It enhanced the transporter activity by strengthening transmembrane helix interactions and trapped substrate-interacting residues • Our results contribute to rational design approach development for efflux improvement.

Expression, purification and crystallization of N-acetyl-(R)-ß-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme.

N-Acetyl-(R)-ß-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-ß-phenylalanine to produce enantiopure (R)-ß-phenylalanine. In previous studies, Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 were isolated as (R)-enantiomer-specific N-acetyl-(R)-ß-phenylalanine acylase-producing organisms and the properties of the native enzyme from Burkholderia sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure-function relationships of the enzymes derived from both organisms. The recombinant N-acetyl-(R)-ß-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the Burkholderia enzyme belonged to space group P41212, with unit-cell parameters a = b = 112.70-112.97, c = 341.50-343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of N,N-dimethylformamidase from Paracoccus sp. strain DMF. The crystals of the Variovorax enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the N-acetyl-(R)-ß-phenylalanine acylases were clarified to be dimeric in solution.

Cryo-EM structures of thylakoid-located voltage-dependent chloride channel VCCN1.

In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca2+-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca2+-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.

Effusibacillus dendaii sp. nov. isolated from farm soil.

A Gram-positive, rod-shaped, spore-forming, thermophilic, and acidophilic bacterium, designated as strain skT53T, was isolated from farm soil in Tokyo, Japan. Under aerobic conditions, the strain grew at 35-55 °C (optimum temperature 44-55 °C) and pH 4.0-6.0 (optimum pH 5.0). Phylogenetic analysis of the 16S rRNA gene sequence showed that the isolate was moderately related to the type strain of Effusibacillus consociatus (94.3% similarity). The G + C content of the genomic DNA was 48.2 mol%, and MK-7 was the predominant respiratory quinone in the strain. The major fatty acids were anteiso-C15:0, iso-C15:0, and iso-C16:0. Based on the phenotypic and chemotaxonomic characteristics, as well as 16S rRNA gene sequence similarity and whole genome analyses, strain skT53T represents a novel species in the genus Effusibacillus, for which the name Effusibacillus dendaii sp. nov. has been proposed. The type strain is skT53T (= NBRC 114101 T = TBRC 11241 T).

Complete Genome Sequence of Effusibacillus sp. Strain skT53, Isolated from Farm Soil.

This study reports the complete genome sequence of Effusibacillus sp. strain skT53. The genome is 3,454,394 bp in length and has a G+C content of 48.22 mol%.

Characterization of lysine acetylation in the peripheral subunit-binding domain of the E2 subunit of the pyruvate dehydrogenase-2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

In Corynebacterium glutamicum, pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) form a unique hybrid complex in which CgE1p and CgE1o are associated with the CgE2-CgE3 subcomplex. We analyzed the role of a lysine acetylation site in the peripheral subunit-binding domain of CgE2 in PDH and ODH functions. Acetylation-mimic substitution at Lys391 of CgE2 severely reduced the interaction of CgE2 with CgE1p and CgE3, but not with CgE1o, indicating the critical role of this residue in the assembly of CgE1p and CgE3 into the complex. It also suggested that Lys391 acetylation inhibited the binding of CgE1p and CgE3 to CgE2, thereby affecting PDH and ODH activities. Interestingly, the CgE2-K391R variant strain showed increased l-glutamate production and reduced pyruvate accumulation. Kinetic analysis suggested that the increased affinity of the K391R variant toward pyruvate might be advantageous for l-glutamate production.

Mechanosensitive channels of Corynebacterium glutamicum functioning as exporters of l-glutamate and other valuable metabolites.

In the industrial l-glutamate production established on the use of Corynebacterium glutamicum, l-glutamate synthesized intracellularly is exported through mechanosensitive transmembrane channel proteins (MscCG and MscCG2) activated by the force-from-lipids. The involvement of MscCG2 in l-glutamate export by C. glutamicum was demonstrated in 2018; however, MscCG was previously found to be the major exporter of l-glutamate. Recent advances in research methods, such as development of the microbial patch clamp, revealed unique characteristics of MscCG, including its conductance, opening and closing thresholds, and gating hysteresis, as well as the significant effect of membrane lipids on the channel properties. In addition, the cryoelectron microscopic structure of Escherichia coli MscS, the canonical representative of the mechanosensitive channel family to which MscCG and MscCG2 belong, revealed its new membrane-interacting region, new position within the lipid bilayer, and hook lipids in a newly defined cavity between subunits. In this short review, the applications of bacterial mechanosensitive channels in the development of effective microbial cell factories, which will contribute to sustainable development, are discussed.

"Force-From-Lipids" mechanosensation in Corynebacterium glutamicum.

Since the mechanosensitive channel MscCG has been identified as the major glutamate efflux system in Corynebacterium glutamicum, studies of mechanotransduction processes in this bacterium have helped to unpuzzle a long-unresolved mystery of glutamate efflux that has been utilised for industrial monosodium glutamate production. The patch clamp recording from C. glutamicum giant spheroplasts revealed the existence of three types of mechanosensitive (MS) channels in the cell membrane of this bacterium. The experiments demonstrated that the MS channels could be activated by membrane tension, indicating that the channel gating by mechanical force followed the "Force-From-Lipids (FFL)" principle characteristic of ion channels inherently sensitive to transbilayer pressure profile changes in the mechanically stressed membrane bilayer. Mechanical properties of the C. glutamicum membrane are characteristics of very soft membranes, which in the C. glutamicum membrane are due to negatively charged lipids as its exclusive constituents. Given that membrane lipids are significantly altered during the fermentation process in the monosodium glutamate production, MS channels seem to respond to changes in force transmission through the membrane bilayer due to membrane lipid dynamics. In this review, we describe the recent results describing corynebacterial FFL-dependent mechanosensation originating from the particular lipid composition of the C. glutamicum membrane and unique structure of MscCG-type channels.

Corynebacterium glutamicum mechanosensitive channels: towards unpuzzling "glutamate efflux" for amino acid production.

Corynebacterium glutamicum has been utilized for industrial amino acid production, especially for monosodium glutamate (MSG), the food-additive for the "UMAMI" category of taste sensation, which is one of the five human basic tastes. Glutamate export from these cells is facilitated by the opening of mechanosensitive channels in the cell membrane within the bacterial cell envelope following specific treatments, such as biotin limitation, addition of Tween 40 or penicillin. A long-unsolved puzzle still remains how and why C. glutamicum mechanosensitive channels are activated by these treatments to export glutamate. Unlike mechanosensitive channels in other bacteria, these channels are not simply osmotic safety valves that prevent these bacteria from bursting upon a hypo-osmotic shock. They also function as metabolic valves to continuously release glutamate as components of a pump-and-leak mechanism regulating the cellular turgor pressure. Recent studies have demonstrated that the opening of the mechanosensitive channel, MscCG, mainly facilitates the efflux of glutamate and not of other amino acids and that the "force-from-lipids" gating mechanism of channels also applies to the MscCG channel. The bacterial types of mechanosensitive channels are found in cell-walled organisms from bacteria to land plants, where their physiological functions have been specialized beyond their basic function in bacterial osmoregulation. In the case of the C. glutamicum MscCG channels, they have evolved to function as specialized glutamate exporters.

Evolutionary specialization of MscCG, an MscS-like mechanosensitive channel, in amino acid transport in Corynebacterium glutamicum.

MscCG, a mechanosensitive channel of Corynebacterium glutamicum provides a major export mechanism for glutamate in this Gram-positive bacterium, which has for many years been used for industrial production of glutamate and other amino acids. The functional characterization of MscCG is therefore, of great significance to understand its conductive properties for different amino acids. Here we report the first successful giant spheroplast preparation of C. glutamicum amenable to the patch clamp technique, which enabled us to investigate mechanosensitive channel activities of MscCG in the native membrane of this bacterium. Single channel recordings from these spheroplasts revealed the presence of three types of mechanosensitive channels, MscCG, MscCG2, and CgMscL, which differ largely from each other in their conductance and mechanosensitivity. MscCG has a relatively small conductance of ~340 pS followed by an intermediate MscCG2 conductance of ~1.0 nS and comparably very large conductance of 3.7 nS exhibited by CgMscL. By applying Laplace's law, we determined that very moderate membrane tension of ~5.5 mN/m was required for half activation of MscCG compared to ~12 mN/m required for half activation of both MscCG2 and CgMscL. Furthermore, by combining the micropipette aspiration technique with molecular dynamics simulations we measured mechanical properties of the C. glutamicum membrane, whose area elasticity module of KA ≈ 15 mN/m is characteristic of a very soft membrane compared to the three times larger area expansion modulus of KA ≈ 44 mN/m of the more elastic E. coli membrane. Moreover, we demonstrate that the "soft" properties of the C. glutamicum membrane have a significant impact on the MscCG gating characterized by a strong voltage-dependent hysteresis in the membrane of C. glutamicum compared to a complete absence of the hysteresis in the E. coli cell membrane. We thus propose that MscCG has evolved and adapted as an MscS-like channel to the mechanical properties of the C. glutamicum membrane enabling the channel to specialize in transport of amino acids such as glutamate, which are major osmolytes helping the bacterial cells survive extreme osmotic stress.

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor, OdhI, involved in glutamate production in Corynebacterium glutamicum.

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.

ATP-dependent modulation of MgtE in Mg2+ homeostasis.

Magnesium is an essential ion for numerous physiological processes. MgtE is a Mg2+ selective channel involved in the maintenance of intracellular Mg2+ homeostasis, whose gating is regulated by intracellular Mg2+ levels. Here, we report that ATP binds to MgtE, regulating its Mg2+-dependent gating. Crystal structures of MgtE-ATP complex show that ATP binds to the intracellular CBS domain of MgtE. Functional studies support that ATP binding to MgtE enhances the intracellular domain affinity for Mg2+ within physiological concentrations of this divalent cation, enabling MgtE to function as an in vivo Mg2+ sensor. ATP dissociation from MgtE upregulates Mg2+ influx at both high and low intracellular Mg2+ concentrations. Using site-directed mutagenesis and structure based-electrophysiological and biochemical analyses, we identify key residues and main structural changes involved in the process. This work provides the molecular basis of ATP-dependent modulation of MgtE in Mg2+ homeostasis.MgtE is an Mg2+ transporter involved in Mg2+ homeostasis. Here, the authors report that ATP regulates the Mg+2-dependent gating of MgtE and use X-ray crystallography combined with functional studies to propose the molecular mechanisms involved in this process.

Microbial innovations in the world of food.

Technological developments in Japan based on the results of microbial research were a major pillar supporting the postwar industrial revolution. The wellspring of these advancements was the sophisticated technology used in traditional brewing, a foundation of the characteristic Japanese food culture. In this manuscript, we will describe the fermentative production of amino acids and nucleic acids following the discovery of the umami component so distinct in Japanese cuisine, which finally revealed the true power of microbial production. Thereafter, we will describe acetic acid production stemming from brewed vinegar production and the fermentative production of some other organic acids. Finally, we will delve into the massive scale of innovations achieved by the discovery of valuable micro-organisms and how they have affected the field of food.

Concise SAR Exploration Based on the "Head-to-Tail" Approach: Discovery of PI4KIIIα Inhibitors Bearing Diverse Scaffolds.

In typical kinase inhibitor programs, a hinge binder showing best potency with preferential specificity is initially selected, followed by fine-tuning of the accompanying substituents on its core module. A shortcoming of this approach is that the exclusive focus on a single chemotype can endanger all the analogues in the series if a critical shortcoming is revealed. Thus, an early evaluation of structure-activity relationships (SARs) can mitigate unforeseen outcomes within a series of multiple compounds, although there have been very few examples to follow such a policy. PI4KIIIα is one of four mammalian phosphatidylinositol-4 kinases and has recently drawn significant attention as an emerging target for hepatitis C virus (HCV) treatment. In this letter, a novel "head-to-tail" approach to discover a diverse set of PI4KIIIα inhibitors is reported. We believe this method will generate distinct core scaffolds, a rational strategy to circumvent potential risks in general kinase programs.

Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more easily than acetylation in such conditions where the substrates for both acetylation and succinylation are limited. This is the first study investigating the acetylome and succinylome of C. glutamicum, and it provides new insight into the roles of acyl modifications in C. glutamicum biology.

Purification and characterization of enantioselective N-acetyl-ß-Phe acylases from Burkholderia sp. AJ110349.

For the production of enantiopure ß-amino acids, enantioselective resolution of N-acyl ß-amino acids using acylases, especially those recognizing N-acetyl-ß-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-ß-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-ß-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-ß-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-ß-Phe (>99% ee) and (S)-ß-Phe (>99% ee) were obtained from the racemic substrate.

The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

Triggered J-aggregation in mixed Langmuir-Blodgett films of amphiphilic spiropyran having a methoxy group at the 5' position and an azobenzene derivative.

Here, we describe the formation of J-aggregates triggered by isomerization of an azobenzene derivative, N-[p-[(p-dodecylphenylazo)phenyloxy]dodecylpyridinium bromide (AzP), in mixed Langmuir-Blodgett (LB) films that contain an amphiphilic spiropyran with a methoxy group at the 5' position, MeO-SP1822. Pure LB films of MeO-SP1822 consist of multilayer domains embedded in a monolayer. UV irradiation of the films causes the isomerization of MeO-SP1822 to its merocyanine form, MeO-MC1822. Pure LB films of AzP comprise finger-like domains and granular domains. Irradiating mixed films of MeO-SP1822 and AzP with alternating UV and visible light causes J-aggregation of MeO-MC1822, with the amount of J-aggregates reaching a maximum at a 1:1 molar ratio. J-aggregation occurs in flat finger-like structures originating in the AzP-rich granular domains that are located on top of the MeO-MC1822-rich multilayer domains. J-aggregates are also present under the AzP-rich granular domains, though these domains do not serve as nucleation sites for the finger-like structures. We propose that granular domains serving as nucleation sites are partially buried in the multilayer domains, whereas those triggering the J-aggregation of MeO-MC1822 under the granular domains are situated on top of the multilayer domains.

L-Glutamate secretion by the N-terminal domain of the Corynebacterium glutamicum NCgl1221 mechanosensitive channel.

The Corynebacterium glutamicum NCgl1221 mechanosensitive channel mediates L-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. The NCgl1221 protein has an N-terminal domain (1-286 a.a.) homologous to the Escherichia coli MscS and a long C-terminal domain (287-533 a.a.) of unknown function. In order to investigate the role of the C-terminal domain in L-glutamate secretion, we constructed a series of C-terminally truncated mutants of NCgl1221. We found that the N-terminal domain, homologous to E. coli MscS, retained the ability to cause L-glutamate secretion in response to the treatment. Electrophysiological analysis confirmed that the N-terminal domain mediated L-glutamate secretion. 3D homology modeling has suggested that the N-terminal domain of NCgl1221 has an extra loop structure (221-232 a.a.) that is not found in most other MscS proteins. The mutant NCgl1221, deleted for this loop structure, lost the ability to secrete L-glutamate. In addition, we found that mutant NCgl1221 lacking the C-terminal extracytoplasmic domain (420-533 a.a.) produced L-glutamate without any inducing treatment. These results suggest that the N-terminal domain is necessary and sufficient for the excretion of L-glutamate in response to inducing treatment, and that the C-terminal extracytoplasmic domain has a negative regulatory role in L-glutamate production.

Isolation of new polyketide metabolites, linearolides A and B, from Streptomyces sp. RK95-74.

Although all Streptomyces strains are now thought to have 20-30 gene clusters for secondary metabolite biosynthesis, we cannot actually identify so many kinds of metabolites from one strain by conventional methods. Using Streptomyces sp. RK95-74, previously found as a cytotrienin producer, we searched new metabolites other than cytotrienin derivatives. Following the cultivation with new media and the peak-guided fractionation, we have found new compounds with new polyketide scaffold, named linearolides A and B.