Identifying a key spot for electron mediator-interaction to tailor CO dehydrogenase's affinity.

Fe‒S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), employ sophisticated artificial electron mediators like viologens to serve as potent biocatalysts capable of cleaning-up industrial off-gases at stunning reaction rates. Unraveling the interplay between these enzymes and their associated mediators is essential for improving the efficiency of CODHs. Here we show the electron mediator-interaction site on ChCODHs (Ch, Carboxydothermus hydrogenoformans) using a systematic approach that leverages the viologen-reactive characteristics of superficial aromatic residues. By enhancing mediator-interaction (R57G/N59L) near the D-cluster, the strategically tailored variants exhibit a ten-fold increase in ethyl viologen affinity relative to the wild-type without sacrificing the turn-over rate (kcat). Viologen-complexed structures reveal the pivotal positions of surface phenylalanine residues, serving as external conduits for the D-cluster to/from viologen. One variant (R57G/N59L/A559W) can treat a broad spectrum of waste gases (from steel-process and plastic-gasification) containing O2. Decoding mediator interactions will facilitate the development of industrially high-efficient biocatalysts encompassing gas-utilizing enzymes.

Tunnel engineering of gas-converting enzymes for inhibitor retardation and substrate acceleration.

Carbon monoxide dehydrogenase (CODH), formate dehydrogenase (FDH), hydrogenase (H2ase), and nitrogenase (N2ase) are crucial enzymatic catalysts that facilitate the conversion of industrially significant gases such as CO, CO2, H2, and N2. The tunnels in the gas-converting enzymes serve as conduits for these low molecular weight gases to access deeply buried catalytic sites. The identification of the substrate tunnels is imperative for comprehending the substrate selectivity mechanism underlying these gas-converting enzymes. This knowledge also holds substantial value for industrial applications, particularly in addressing the challenges associated with separation and utilization of byproduct gases. In this comprehensive review, we delve into the emerging field of tunnel engineering, presenting a range of approaches and analyses. Additionally, we propose methodologies for the systematic design of enzymes, with the ultimate goal of advancing protein engineering strategies.

The Advent of Domain Adaptation into Artificial Intelligence for Gastrointestinal Endoscopy and Medical Imaging.

Artificial intelligence (AI) is a subfield of computer science that aims to implement computer systems that perform tasks that generally require human learning, reasoning, and perceptual abilities. AI is widely used in the medical field. The interpretation of medical images requires considerable effort, time, and skill. AI-aided interpretations, such as automated abnormal lesion detection and image classification, are promising areas of AI. However, when images with different characteristics are extracted, depending on the manufacturer and imaging environment, a so-called domain shift problem occurs in which the developed AI has a poor versatility. Domain adaptation is used to address this problem. Domain adaptation is a tool that generates a newly converted image which is suitable for other domains. It has also shown promise in reducing the differences in appearance among the images collected from different devices. Domain adaptation is expected to improve the reading accuracy of AI for heterogeneous image distributions in gastrointestinal (GI) endoscopy and medical image analyses. In this paper, we review the history and basic characteristics of domain shift and domain adaptation. We also address their use in gastrointestinal endoscopy and the medical field more generally through published examples, perspectives, and future directions.

Comparative Genomic Analysis and BTEX Degradation Pathways of a Thermotolerant Cupriavidus cauae PHS1.

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

Extra disulfide and ionic salt bridge improves the thermostability of lignin peroxidase H8 under acidic condition.

The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t1/2) of 8.2 days at pH 6) exhibits a marked decline in thermostability under acidic conditions (t1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t1/2 (2.52 h at 25 °C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.

Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli.

After glucose, xylose is the most abundant sugar in lignocellulosic carbon sources. However, wild-type Escherichia coli is unable to simultaneously utilize both sugars due to carbon catabolite repression (CCR). In this paper, we describe GX50, an engineered strain capable of utilizing glucose and xylose simultaneously. This strain was obtained by evolving a mutant from which araC has been deleted, and in which genes required for pentose metabolism are constitutively expressed. The strain acquired four additional mutations during adaptive evolution, including intergenic mutations in the 5'-flanking region of xylA and pyrE, and missense mutations in araE (S91I) and ybjG (D99G). In contrast to wild type E. coli, GX50 rapidly converts xylose to xylitol even if glucose is available. Notably, the strain grows well when cultured on glucose, unlike some well-known CCR-insensitive mutants defective in the glucose phosphotransferase system. Our work will advance efforts to design a metabolically efficient platform strain for potential use in producing chemicals from lignocellulose.

Identification and characterization of 2-keto-3-deoxy-L-rhamnonate dehydrogenase belonging to the MDR superfamily from the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans: implications to L-rhamnose metabolism in archaea.

We identified the non-phosphorylated L-rhamnose metabolic pathway (Rha_NMP) genes that are homologous to those in the thermoacidophilic archaeon Thermoplasma acidophilum in the genome of the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans. However, unlike previously known 2-keto-3-deoxy-L-rhamnonate (L-KDR) dehydrogenase (KDRDH) which belongs to the short chain dehydrogenase/reductase superfamily, the putative KDRDHs in S. thermosulfidooxidans (Sulth_3557) and T. acidophilum (Ta0749) belong to the medium chain dehydrogenase/reductase (MDR) superfamily. We demonstrated that Sulth_3559 and Sulth_3557 proteins from S. thermosulfidooxidans function as L-rhamnose dehydrogenase and KDRDH, respectively. Sulth_3557 protein is an NAD(+)-specific KDRDH with optimal temperature and pH of 50 °C and 9.5, respectively. The K m and V max values for L-KDR were 2.0 mM and 12.8 U/mg, respectively. Sulth_3557 also showed weak 2,3-butanediol dehydrogenase activity. Phylogenetic analysis suggests that Sulth_3557 and its homologs form a new subfamily in the MDR superfamily. The results shown in this study imply that thermoacidophilic archaea metabolize L-rhamnose to pyruvate and L-lactate by using the MDR-family KDRDH similarly to that of the thermoacidophilic bacterium S. thermosulfidooxidans.

A simple and effective method for construction of Escherichia coli strains proficient for genome engineering.

Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.

Sagittula marina sp. nov., isolated from seawater and emended description of the genus Sagittula.

A novel bacterium, designated strain F028-2(T), was isolated from seawater at Damupo beach in Pohang, Korea, and investigated in a taxonomic study using a polyphasic approach. This novel strain was strictly aerobic, non-motile, Gram-stain-negative and rod-shaped, and occasionally formed aggregates. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 6.5-9.0 and 1-7% (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F028-2(T) formed a lineage within the family Rhodobacteraceae of the class Alphaproteobacteria, and was closely related to members of the genera Sagittula and Antarctobacter with 96.3-96.4% sequence similarities. The polar lipid profile of strain F028-2(T) comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, one unidentified phospholipid and six unidentified lipids. The predominant cellular fatty acids were C18:1ω7c and C12:1 3-OH. The genomic DNA G+C content of strain F028-2(T) was 61.6 mol% and the major respiratory quinone was Q-10. Based on phenotypic, phylogenetic and genotypic data, strain F028-2(T) is considered to represent a novel species in the genus Sagittula, for which the name Sagittula marina sp. nov. is proposed. The type strain is F028-2(T) (=KCTC 23543(T)=JCM 17627(T)). An emended description of the genus Sagittula is also proposed.

Psychroserpens damuponensis sp. nov., isolated from seawater.

A novel bacterium, designated strain F051-1(T), isolated from a seawater sample collected from the coast at Damupo beach in Pohang, Korea, was investigated in a polyphasic taxonomic study. Cells were yellow-pigmented, strictly aerobic, Gram-staining-negative and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-30 °C, pH 6.0-9.0 and 1.0-6.0 % (w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F051-1(T) belongs to the genus Psychroserpens in the family Flavobacteriaceae. Its closest relatives were Psychroserpens burtonensis ACAM 188(T) (96.8 % 16S rRNA gene sequence similarity) and Psychroserpens mesophilus KOPRI 13649(T) (95.7 %). The major cellular fatty acids were iso-C(15 : 0), iso-C(15 : 1) G and anteiso-C(15 : 0). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and eight unidentified lipids. The major respiratory quinone was menaquinone-6 and the genomic DNA G+C content of the strain was 33.5 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F051-1(T) represents a novel species within the genus Psychroserpens, for which the name Psychroserpens damuponensis sp. nov. is proposed. The type strain is F051-1(T) ( = KCTC 23539(T)  = JCM 17632(T)).

Winogradskyella damuponensis sp. nov., isolated from seawater.

A novel bacterium, designated F081-2(T), isolated from seawater from Damupo beach in Pohang, Korea, was investigated using a polyphasic taxonomic approach. Cells were yellow-pigmented, strictly aerobic, motile by gliding, Gram-negative-staining and rod-shaped. The temperature, pH and NaCl ranges for growth were 4-35 °C, pH 5.5-9.5 and 1.0-5.0 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain F081-2(T) belonged to a distinct lineage in the genus Winogradskyella of the family Flavobacteriaceae, sharing 93.7-98.1 % similarity with recognized members of the genus. Low levels of DNA-DNA relatedness values were found between strain F081-2(T) and Winogradskyella eximia KCTC 12219(T) (61.1 %), Winogradskyella thalassocola KCTC 12221(T) (47.0 %), Winogradskyella echinorum KCTC 22026(T) (39.3 %), Winogradskyella rapida CCUG 56098(T) (34.3 %) and Winogradskyella arenosi JCM 17633(T) (33.4 %). The major cellular fatty acids were iso-C(15 : 0) (25.3 %), iso-C(15 : 1) G (14.6 %), iso-C(17 : 0) 3-OH (9.3 %), anteiso-C(15 : 0) (7.8 %) and iso-C(15 : 0) 3-OH (7.6 %). The polar lipid profile was composed of phosphatidylethanolamine, one unidentified aminolipid, one unidentified phospholipid, one unidentified aminophospholipid and six unidentified lipids. The major respiratory quinone was menaquinone-6 and the DNA G+C content of the strain was 32.3 mol%. On the basis of phenotypic, phylogenetic and genotypic data, strain F081-2(T) represents a novel species within the genus Winogradskyella, for which the name Winogradskyella damuponensis sp. nov. is proposed. The type strain is F081-2(T) (=KCTC 23552(T) = JCM 17633(T)).

Genome sequence of the unclassified marine Gammaproteobacterium BDW918.

The unclassified marine gammaproteobacterium BDW918, which utilizes volatile fatty acids but not most common carbohydrates and amino acids, was isolated from Dokdo seawater in South Korea. Here we present a draft genome of the strain BDW918, which encodes many putative genes related to fatty acid metabolism and aromatic hydrocarbon degradation.

Characterization of NADP+-specific L-rhamnose dehydrogenase from the thermoacidophilic Archaeon Thermoplasma acidophilum.

Thermoplasma acidophilum utilizes L-rhamnose as a sole carbon source. To determine the metabolic pathway of L-rhamnose in Archaea, we identified and characterized L-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of L-rhamnose to L-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized L-rhamnose, L-lyxose, and L-mannose using only NADP(+) as a cofactor, which is different from NAD(+)/NADP(+)-specific bacterial RhaDs and NAD(+)-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward L-rhamnose at 60 °C and pH 7. The K (m) and k (cat) values were 0.46 mM, 1,341.3 min(-1) for L-rhamnose and 0.1 mM, 1,027.2 min(-1) for NADP(+), respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP(+)-specific RhaD.

Postechiella marina gen. nov., sp. nov., isolated from seawater.

A Gram-staining-negative, strictly aerobic, non-motile, yellow-pigmented bacterium, designated strain M091(T), was isolated from seawater at Damupo beach in Pohang, Republic of Korea, and investigated using a polyphasic taxonomic approach. The novel strain grew optimally at 25 °C, pH 7.0-8.0, and in the presence of 3% (w/v) NaCl. In a phylogenetic analysis based on 16S rRNA gene sequences, strain M091(T) formed a lineage within the family Flavobacteriaceae that was distinct from the most closely related genera of Flaviramulus (95.1% sequence similarity), Algibacter (94.9-93.9%), Mariniflexile (94.8-94.2%), Winogradskyella (94.8-93.2%), Lacinutrix (94.7-93.8%) and Tamlana (94.7-92.9%). The polar lipid profile of the novel strain comprised phosphatidylethanolamine, two unidentified aminolipids, one unidentified phospholipid and seven unidentified lipids. The predominant cellular fatty acids were iso-C(15:0) (20.5%), iso-C(17:0) 3-OH (15.4%), iso-C(15:0) 3-OH (12.4%), C(15:0) (10.9%) and iso-C(15:1) G (9.9%). The genomic DNA G+C content of strain M091(T) was 34.4 mol% and the major respiratory quinone was MK-6. Based on phenotypic and genotypic data, strain M091(T) represents a new genus and novel species in the family Flavobacteriaceae, for which the name Postechiella marina gen. nov., sp. nov. is proposed. The type strain of the type species is M091(T) (=KCTC 23537(T)=JCM 17630(T)).