A diverse proteome is present and enzymatically active in metabolite extracts.

Metabolite extraction is the critical first-step in metabolomics experiments, where it is generally regarded to inactivate and remove proteins. Here, arising from efforts to improve extraction conditions for polar metabolomics, we discover a proteomic landscape of over 1000 proteins within metabolite extracts. This is a ubiquitous feature across several common extraction and sample types. By combining post-resuspension stable isotope addition and enzyme inhibitors, we demonstrate in-extract metabolite interconversions due to residual transaminase activity. We extend these findings with untargeted metabolomics where we observe extensive protein-mediated metabolite changes, including in-extract formation of glutamate dipeptide and depletion of total glutathione. Finally, we present a simple extraction workflow that integrates 3 kDa filtration for protein removal as a superior method for polar metabolomics. In this work, we uncover a previously unrecognized, protein-mediated source of observer effects in metabolomics experiments with broad-reaching implications across all research fields using metabolomics and molecular metabolism.

Enzymatic synthesis and sensory evaluation of the novel kokumi compound N-butyryl phenylalanine.

In this study, food-grade glutamine transaminase (TGase) was utilized for the green-catalyzed preparation of N-butyryl amino acids. For improving the reusability of the enzyme preparation, immobilized TG enzyme (94.23% immobilization rate) was prepared. Furthermore, the yield of N-butyryl phenylalanine (BP) synthesized by TGase was obtained as 20.73% by one-factor experiment. The BP synthesis yield of immobilized TGase was 95.03% of that of TGase and remained above 60% of the initial enzyme activity after five runs. The sensory evaluation and E-tongue results showed that the addition of BP significantly increased the umami, saltiness, and richness intensities of the samples, and decreased the intensities of sourness, bitterness, and aftertaste-B. The molecular docking results indicated that hydrogen bonding dominated the binding of BP to taste receptors in the taste presentation mechanism of BP. These results confirmed the potential of BP as a flavor enhancer with promising applications in the food industry.

Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

[Enzymatic production of 1,4-cyclohexanedimethylamine].

1,4-cyclohexanedimethylamine (1,4-BAC) is an important monomer for bio-based materials, it finds wide applications in various fields including organic synthesis, medicine, chemical industry, and materials. At present, its synthesis primarily relies on chemical method, which suffer from issues such as expensive metal catalyst, harsh reaction conditions, and safety risks. Therefore, it is necessary to explore greener alternatives for its synthesis. In this study, a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. This pathway used Escherichia coli derived aminotransferase (EcTA), Saccharomyces cerevisiae derived glutamate dehydrogenase (ScGlu-DH), and Candida boidinii derived formate dehydrogenase (CbFDH). Through structure-guided protein engineering, a beneficial mutant, EcTAF91Y, was obtained, exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type. By constructing recombinant strains and optimizing reaction conditions, it was found that under the optimal conditions, a substrate concentration of 40 g/L could produce (27.4±0.9) g/L of the product, corresponding to a molar conversion rate of 67.5%±2.1%.

Multisubstrate specificity shaped the complex evolution of the aminotransferase family across the tree of life.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

One-Pot Biocatalytic Route from Alkanes to α,ω-Diamines by Whole-Cell Consortia of Engineered Yarrowia lipolytica and Escherichia coli.

Metabolically engineered microbial consortia can contribute as a promising production platform for the supply of polyamide monomers. To date, the biosynthesis of long-chain α,ω-diamines from n-alkanes is challenging because of the inert nature of n-alkanes and the complexity of the overall synthesis pathway. We combined an engineered Yarrowia lipolytica module with Escherichia coli modules to obtain a mixed strain microbial consortium that could catalyze an efficient biotransformation of n-alkanes into corresponding α,ω-diamines. The engineered Y. lipolytica strain was constructed (YALI10) wherein the two genes responsible for ß-oxidation and the five genes responsible for the overoxidation of fatty aldehydes were deleted. This newly constructed YALI10 strain expressing transaminase (TA) could produce 0.2 mM 1,12-dodecanediamine (40.1 mg/L) from 10 mM n-dodecane. The microbial consortia comprising engineered Y. lipolytica strains for the oxidation of n-alkanes (OM) and an E. coli amination module (AM) expressing an aldehyde reductase (AHR) and transaminase (TA) improved the production of 1,12-diamine up to 1.95 mM (391 mg/L) from 10 mM n-dodecane. Finally, combining the E. coli reduction module (RM) expressing a carboxylic acid reductase (CAR) and an sfp phosphopantetheinyl transferase with OM and AM further improved the production of 1,12-diamine by catalyzing the reduction of undesired 1,12-diacids into 1,12-diols, which further undergo amination to give 1,12-diamine as the target product. This newly constructed mixed strain consortium comprising three modules in one pot gave 4.1 mM (41%; 816 mg/L) 1,12-diaminododecane from 10 mM n-dodecane. The whole-cell consortia reported herein present an elegant "greener" alternative for the biosynthesis of various α,ω-diamines (C8, C10, C12, and C14) from corresponding n-alkanes.

Evolving ω-amine transaminase AtATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates.

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus (AtATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate (R)-(+)-1-(1-naphthyl)ethylamine [(R)-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 (AtATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to AtATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing (R)-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the AtATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level.

Sequence-Guided Redesign of an Omega-Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

Mycobacterium tuberculosis produces D-serine under hypoxia to limit CD8+ T cell-dependent immunity in mice.

Adaptation to hypoxia is a major challenge for the survival of Mycobacterium tuberculosis (Mtb) in vivo. Interferon (IFN)-γ-producing CD8+ T cells contribute to control of Mtb infection, in part by promoting antimicrobial activities of macrophages. Whether Mtb counters these responses, particularly during hypoxic conditions, remains unknown. Using metabolomic, proteomic and genetic approaches, here we show that Mtb induced Rv0884c (SerC), an Mtb phosphoserine aminotransferase, to produce D-serine. This activity increased Mtb pathogenesis in mice but did not directly affect intramacrophage Mtb survival. Instead, D-serine inhibited IFN-γ production by CD8+ T cells, which indirectly reduced the ability of macrophages to restrict Mtb upon co-culture. Mechanistically, D-serine interacted with WDR24 and inhibited mTORC1 activation in CD8+ T cells. This decreased T-bet expression and reduced IFN-γ production by CD8+ T cells. Our findings suggest an Mtb evasion mechanism where pathogen metabolic adaptation to hypoxia leads to amino acid-dependent suppression of adaptive anti-TB immunity.

Transaminase-Catalyzed Synthesis of ß-Branched Noncanonical Amino Acids Driven by a Lysine Amine Donor.

Transaminases are choice biocatalysts for the synthesis of chiral primary amines, including amino acids bearing contiguous stereocenters. In this study, we employ lysine as a "smart" amine donor in transaminase-catalyzed dynamic kinetic resolution reactions to access ß-branched noncanonical arylalanines. Our mechanistic investigation demonstrates that, upon transamination, the lysine-derived ketone byproduct readily cyclizes to a six-membered imine, driving the equilibrium in the desired direction and thus alleviating the need to load superstoichiometric quantities of the amine donor or deploy a multienzyme cascade. Lysine also shows good overall compatibility with a panel of wild-type transaminases, a promising hint of its application as a smart donor more broadly. Indeed, by this approach, we furnished a broad scope of ß-branched arylalanines, including some bearing hitherto intractable cyclopropyl and isopropyl substituents, with high yields and excellent selectivities.

Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

An integrated cofactor and co-substrate recycling pathway for the biosynthesis of 1,5-pentanediol.

BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.

Bcat2-Mediated Branched-Chain Amino Acid Catabolism Is Linked to the Aggravated Inflammation in Obese with Psoriasis Mice.

SCOPE: The global prevalence of obesity has significantly increased, presenting a major health challenge. High-fat diet (HFD)-induced obesity is closely related to the disease severity of psoriasis, but the mechanism is not fully understood. METHODS AND RESULTS: The study utilizes the HFD-induced obesity model along with an imiquimod (IMQ)-induced psoriasis-like mouse model (HFD-IMQ) to conduct transcriptomics and metabolomic analyses. HFD-induced obese mice exhibits more severe psoriasis-like lesions compared to normal diet (ND)-IMQ mice. The expression of genes of the IL-17 signaling pathway (IL-17A, IL-17F, S100A9, CCL20, CXCL1) is significantly upregulated, leading to an accumulation of T cells and neutrophils in the skin. Moreover, the study finds that there is an inhibition of the branched-chain amino acids (BCAAs) catabolism pathway, and the key gene branched-chain amino transferase 2 (Bcat2) is significantly downregulated, and the levels of leucine, isoleucine, and valine are elevated in the HFD-IMQ mice. Furthermore, the study finds that the peroxisome proliferator-activated receptor gamma (PPAR γ) is inhibited, while STAT3 activity is promoted in HFD-IMQ mice. CONCLUSION: HFD-induced obesity significantly amplifies IL-17 signaling and exacerbates psoriasis, with a potential role played by Bcat2-mediated BCAAs metabolism. The study suggests that BCAA catabolism and PPAR γ-STAT3 exacerbate inflammation in psoriasis with obesity.

Multifunctionality of arginine residues in the active sites of non-canonical d-amino acid transaminases.

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications.

Expanding the Genetic Spectrum of AGXT Gene Variants in Egyptian Patients with Primary Hyperoxaluria Type I.

Introduction: Approximately 80% of primary hyperoxaluria cases are caused by primary hyperoxaluria type 1 (PH1, OMIM# 259900), which is characterized by pathogenic variants in the AGXT gene, resulting in deficiency of the liver-specific enzyme alanine-glyoxylate aminotransferase (AGT). This leads to increased production of oxalate, which cannot be effectively eliminated from the body, resulting in its accumulation primarily in the kidneys and other organs. Subjects and Methods: This study included 17 PH1 Egyptian patients from 12 unrelated families, recruited from the Inherited Kidney Disease Outpatient Clinic and the Dialysis Units, Cairo University Hospitals, during the period from January 2018 to December 2019, aiming to identify the pathogenic variants in the AGXT gene. Results: Six different variants were detected. These included three frameshift and three missense variants, all found in homozygosity within the respective families. The most common variant was c.121G>A;p.(Gly41Arg) detected in four families, followed by c.725dup;p.(Asp243GlyfsTer12) in three families, c.33dup;p.(Lys12Glnfs156) in two families, and c.731T >C;p.(Ile244Thr), c.33delC;p.(Lys12Argfs34), and c.568G>A;p.(Gly190Arg) detected in one family each. Conclusion: Consanguineous Egyptian families with history of renal stones or renal disease suspicious of primary hyperoxaluria should undergo AGXT genetic sequencing, specifically targeting exons 1 and 7, as variants in these two exons account for >75% of disease-causing variants in Egyptian patients with confirmed PH1.

Leucine zipper as a bridge for transaminase self-assembly: A fusion enzyme for efficient chiral conversion of d-phenylglycine.

Amino acid transferase is a family of enzymes used to catalyze and separate chiral amino acids. However, due to the low efficiency, by-products and reverse reactions occur in cascade reactions. Therefore, in the research, phenylglycine aminotransferase and aspartate aminotransferase were self-assembled in vitro by leucine zipper. The self-assembled enzyme system with d-phenylglycine and α-ketoglutarate as substrates were used for the chiral transformation reaction. By studying the enzyme combination, kinetic reaction stability and catalytic efficiency, it was found that the self-assembled enzyme showed improved stability and better affinity to the substrate than the control and achieved only ee value of 17.86% for the control at the substrate ratio was 1:2. In contrast, the self-assembled enzyme basically catalyzed the complete conversion of d-Phg to l-Phg, with the ee value as 99%. These results demonstrated the feasibility of the leucine zipper and the conversion of d-phenylglycine to the l-type by fusion enzyme.

Study on anti-BmNPV mechanism of branched-chain amino acid aminotransferases in silkworm.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV.

Multifunctional enzymes related to amino acid metabolism in bacteria.

In bacteria, d-amino acids are primarily synthesized from l-amino acids by amino acid racemases, but some bacteria use d-amino acid aminotransferases to synthesize d-amino acids. d-Amino acids are peptidoglycan components in the cell wall involved in several physiological processes, such as bacterial growth, biofilm dispersal, and peptidoglycan metabolism. Therefore, their metabolism and physiological roles have attracted increasing attention. Recently, we identified novel bacterial d-amino acid metabolic pathways, which involve amino acid racemases, with broad substrate specificity, as well as multifunctional enzymes with d-amino acid-metabolizing activity. Here, I review these multifunctional enzymes and their related d- and l-amino acid metabolic pathways in Escherichia coli and the hyperthermophile Thermotoga maritima.

Raman spectroscopy and one-dimensional convolutional neural network modeling as a real-time monitoring tool for in vitro transaminase-catalyzed synthesis of a pharmaceutically relevant amine precursor.

Raman spectroscopy has been used to measure the concentration of a pharmaceutically relevant model amine intermediate for positive allosteric modulators of nicotinic acetylcholine receptor in a ω-transaminase-catalyzed conversion. A model based on a one-dimensional convolutional neural network was developed to translate raw data augmented Raman spectra directly into substrate concentrations, with which the conversion from ketone to amine by ω-transaminase could be determined over time. The model showed very good predictive capabilities, with R2 values higher than 0.99 for the spectra included in the modeling and 0.964 for an independent dataset. However, the model could not extrapolate outside the concentrations specified by the model. The presented work shows the potential of Raman spectroscopy as a real-time monitoring tool for biocatalytic reactions.