Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid.

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.

Electrostatic Ratchet for Successive Peptide Synthesis in Nonribosomal Molecular Machine RimK.

A nonribosomal peptide-synthesizing molecular machine, RimK, adds l-glutamic acids to the C-terminus of ribosomal protein S6 (RpsF) in vivo and synthesizes poly-α-glutamates in vitro. However, the mechanism of the successive glutamate addition, which is fueled by ATP, remains unclear. Here, we investigate the successive peptide-synthesizing mechanism of RimK via the molecular dynamics (MD) simulation of glutamate binding. We first show that RimK adopts three stable structural states with respect to the ATP-binding loop and the triphosphate chain of the bound ATP. We then show that a glutamate in solution preferentially binds to a positively charged belt-like region of RimK and the bound glutamate exhibits Brownian motion along the belt. The binding-energy landscape shows that the open-to-closed transition of the ATP-binding loop and the bent-to-straight transition of the triphosphate chain of ATP can function as an electrostatic ratchet that guides the bound glutamate to the active site. We then show the binding site of the second glutamate, which allows us to infer the ligation mechanism. Consistent with MD results, the crystal structure of RimK we obtained in the presence of RpsF presents an electron density that is presumed to correspond to the C-terminus of RpsF. We finally propose a mechanism for the successive peptide synthesis by RimK and discuss its similarity to other molecular machines.

Improving the enzymatic activity of l-amino acid α-ligase for imidazole dipeptide production by site-directed mutagenesis.

Imidazole dipeptides, histidine-containing dipeptides, including carnosine (ß-alanyl-l-histidine), anserine (ß-alanyl-3-methyl-l-histidine), and balenine (ß-alanyl-1-methyl-l-histidine) in animal muscles have physiological functions, such as significant antioxidant and antifatigue effects. They are obtained by extraction from natural raw materials, including chicken and fish meat. However, using natural raw materials entails stable supply and mass production limitations. l-amino acid α-ligase (Lal) catalyzes the formation of various dipeptides from unprotected l-amino acids by conjugating with adenosine 5'-triphosphate (ATP) hydrolysis reaction. In this study, site-directed mutagenesis of Lal was applied to establish an efficient method for producing imidazole dipeptides by the enzymatic process. We significantly improved the conversion rate from substrate amino acids compared with wild-type Lal.

One-pot synthesis of 2,5-diketopiperazine with high titer and versatility using adenylation enzyme.

2,5-Diketopiperazine (DKP) is a cyclic peptide composed of two amino acids and has been recently reported to exhibit various biological activities. DKPs have been synthesized using various methods. In chemical synthesis, a multi-step reaction requiring purification and racemization is problematic. Although enzymatic synthesis can overcome these problems, there has been no example of a general-purpose synthesis of DKPs with high titers. Therefore, we propose a chemoenzymatic method that can synthesize DKPs in a general-purpose manner with high efficiency under mild conditions. The adenylation domain of tyrocidine synthetase A (TycA-A) catalyzes the adenylation reaction of amino acids, and various amides can be synthesized by a nucleophilic substitution reaction with any amine. On the other hand, DKPs can be produced via intramolecular cyclization reactions from dipeptide esters. Based on these observations, we expected a one-pot synthesis of DKPs via dipeptide ester synthesis by TycA-A and cyclization reactions. This method enabled the synthesis of more than 128 types of DKPs without racemization. Importantly, the intramolecular cyclization reaction proceeded largely depending on the pH. In particular, the cyclization reaction proceeded well in the pH range of 6.5-9.5. Based on these results, we constructed a bioreactor with pH-stat for purified enzyme reaction; cyclo(L-Trp-L-Pro) was produced at 4.07 mM by controlling the reaction pH over time using this reactor. The DKPs obtained using this method will provide deeper insights into their structures and functions in future studies. KEY POINTS: • Adenylation enzyme enabled one-pot synthesis of arbitrary 2,5-diketopiperazine. • Little or no racemization occurred during 2,5-diketopiperazine synthesis. • Bioreactor with pH-stat for purified enzymes improved the reaction rate.

Enzymatic Synthesis of l-threo-ß-Hydroxy-α-Amino Acids via Asymmetric Hydroxylation Using 2-Oxoglutarate-Dependent Hydroxylase from Sulfobacillus thermotolerans Strain Y0017.

ß-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of ß-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo-ß-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter-1) l-threo-ß-hydroxy-His and 150 mM l-threo-ß-hydroxy-Gln (24.3 g liter-1) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo-ß-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of ß-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for ß-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln with a titer of over 20 g liter-1 through whole-cell bioconversion using recombinant Escherichia coli cells. As ß-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

Tryptophanase gene deficiency improves the application of dioxygenase to 3-(2-hydroxyethyl)catechol production.

3-(2-Hydroxyethyl)catechol (HEC) is a polyphenol reported to exhibit skin-lightning and antioxidative effects, and hence is expected to be used as cosmetic and food additives and chemical products such as electronic materials. In this study, we established biocatalytic HEC production from 2-phenylethanol using the dioxygenase whose expression was induced by toluene, CumA, and its flanking dehydrogenase, CumB, from an isolated strain, Pseudomonas sp. K17. Escherichia coli cells coexpressing CumA and CumB were stained blue during cultivation in Luria-Bertani medium, and HEC was not produced upon using the cell-free extracts as biocatalysts, likely resulting from the inhibitory effects of the blue dyes. The disruption of the tryptophanase gene of E. coli was found to repress the generation of the blue dyes, and enhanced HEC production. The blue dyes were extracted from the cell-free extracts, and their molecular formula was C16H10N2O3, suggesting they were monooxygenated indigo or its isomers. Although repression of blue dye formation and enhancement of HEC production were observed when cells were cultivated with glucose, the percent yield of HEC was 84% at 20 h, whereas that with tryptophanase disruption strain was 84% at 4 h. It was suggested that tryptophanase gene disruption could contribute to more efficient HEC production.

Screening and characterization of a novel reversible 4-hydroxyisophthalic acid decarboxylase from Cystobasidium slooffiae HTK3.

Owing to carboxylation activity, reversible decarboxylases can use CO2 as a C1-building block to produce useful carboxylic acids. Although many reversible decarboxylases can synthesize aromatic monocarboxylic acids, only a few reversible decarboxylases have been reported to date that catalyze the synthesis of aromatic dicarboxylic acids. In the present study, a reversible 4-hydroxyisophthalic acid decarboxylase was identified in Cystobasidium slooffiae HTK3. Furthermore, recombinant 4-hydroxyisophthalic acid decarboxylase was prepared, characterized, and used for 4-hydroxyisophthalic acid production from 4-hydroxybenzoic acid.

2,5-Furandicarboxylic acid production from furfural by sequential biocatalytic reactions.

2,5-Furandicarboxylic acid (FDCA) is a valuable compound that can be synthesized from biomass-derived hydroxymethylfurfural (HMF), and holds great potential as a promising replacement for petroleum-based terephthalic acid in the production of polyamides, polyesters, and polyurethanes used universally. However, an economical large-scale production strategy for HMF from lignocellulosic biomass is yet to be established. This study aimed to design a synthetic pathway that can yield FDCA from furfural, whose industrial production from lignocellulosic biomass has already been established. This artificial pathway consists of an oxidase and a prenylated flavin mononucleotide (prFMN)-dependent reversible decarboxylase, catalyzing furfural oxidation and carboxylation of 2-furoic acid, respectively. The prFMN-dependent reversible decarboxylase was identified in an isolated strain, Paraburkholderia fungorum KK1, whereas an HMF oxidase from Methylovorus sp. MP688 exhibited furfural oxidation activity and was used as a furfural oxidase. Using Escherichia coli cells coexpressing these proteins, as well as a flavin prenyltransferase, FDCA could be produced from furfural via 2-furoic acid in one pot.

Screening, gene cloning, and characterization of orsellinic acid decarboxylase from Arthrobacter sp. K8 for regio-selective carboxylation of resorcinol derivatives.

Toward a sustainable synthesis of value-added chemicals, the method of CO2 utilization attracts great interest in chemical process engineering. Biotechnological CO2 fixation is a promising technology; however, efficient methods that can fix carbon dioxide are still limited. Instead, some parts of microbial decarboxylases allow the introduction of carboxy group into phenolic compounds using bicarbonate ion as a C1 building block. Here, we identified a unique decarboxylase from Arthrobacter sp. K8 that acts on resorcinol derivatives. A high-throughput colorimetric decarboxylase assay facilitated gene cloning of orsellinic acid decarboxylase from genomic DNA library of strain K8. Sequence analysis revealed that the orsellinic acid decarboxylase belonged to amidohydrolase 2 family, but shared low amino acid sequence identity with those of related decarboxylases. Enzymatic characterization unveiled that the decarboxylase introduces a carboxy group in a highly regio-selective manner. We applied the decarboxylase to enzymatic carboxylation of resorcinol derivatives. Using Escherichia coli expressing the decarboxylase gene as a whole cell biocatalyst, orsellinic acid, 2,4-dihydroxybenzoic acid, and 4-methoxysalicylic acid were produced in the presence of saturated bicarbonate. These findings could provide new insights into the production of useful phenolic acids from resorcinol derivatives.

Efficient and long-term vanillin production from 4-vinylguaiacol using immobilized whole cells expressing Cso2 protein.

Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E. coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E. coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E. coli cells for both continuous and repeated batch vanillin production without any coenzymes.

Enzymatic reactions and microorganisms producing the various isomers of hydroxyproline.

Hydroxyproline is an industrially important compound with applications in the pharmaceutical, nutrition, and cosmetic industries. trans-4-Hydroxy-L-proline is recognized as the most abundant of the eight possible isomers (hydroxy group at C-3 or C-4, cis- or trans-configuration, and L- or D-form). However, little attention has been paid to the rare isomers, probably due to their limited availability. This mini-review provides an overview of recent advances in microbial and enzymatic processes to develop practical production strategies for various hydroxyprolines. Here, we introduce three screening strategies, namely, activity-, sequence-, and metabolite-based approaches, allowing identification of diverse proline-hydroxylating enzymes with different product specificities. All naturally occurring hydroxyproline isomers can be produced by using suitable hydroxylases in a highly regio- and stereo-selective manner. Furthermore, crystal structures of relevant hydroxylases provide much insight into their functional roles. Since hydroxylases acting on free L-proline belong to the 2-oxoglutarate-dependent dioxygenase superfamily, cellular metabolism of Escherichia coli coupled with a hydroxylase is a valuable source of 2-oxoglutarate, which is indispensable as a co-substrate in L-proline hydroxylation. Further, microbial hydroxyproline 2-epimerase may serve as a highly adaptable tool to convert L-hydroxyproline into D-hydroxyproline. KEY POINTS: • Proline hydroxylases serve as powerful tools for selectivel-proline hydroxylation. • Engineered Escherichia coli are a robust platform for hydroxyproline production. • Hydroxyproline epimerase convertsl-hydroxyproline intod-hydroxyproline.

Chemoenzymatic oxygenation method for sesquiterpenoid synthesis based on Fe-chelate and ferric-chelate reductase.

Sesquiterpenoids are one of the most diverse groups in natural compounds with various chemical structures and bioactivities. In our previous work, we developed the chemoenzymatic oxygenation method based on the combination of Fe(II)-EDTA and ferric-chelate reductase that could synthesize (-)-rotundone, a key aroma sesquiterpenoid of black pepper. Fe(II)-EDTA catalyzed the oxygenation of sesquiterpene to sesquiterpenoid, and ferric-chelate reductase catalyzed the supply and regeneration of Fe(II)-EDTA in this system. We then investigated the effect of various Fe2+-chelates on the catalytic oxygenation of sesquiterpene and applied this system to the synthesis of odor sesquiterpenoids. We determined Fe(II)-NTA to be an efficient oxygenation catalyst by the screening approach focusing on ligand structures and coordination atoms of Fe2+-chelates. Valuable odor sesquiterpenoids such as (+)-nootkatone, (-)-isolongifolenone, and (-)-ß-caryophyllene oxide were oxygenatively synthesized from each precursor sesquiterpene by 66%, 82%, and 67% of the molar conversion rate, respectively.Abbreviations: EDTA: ethylenediaminetetraacetate; NTA: nitrilotriacetate; DTPA: diethylenetriaminepentaacetate; phen: o-phenanthroline; cyclam: 1,4,8,11-tetraazacyclotetradecane; TPA: tris(2-pyridylmethyl)amine; GlcDH: glucose dehydrogenase; HP-ß-CD: hydroxypropyl-ß-cyclodextrin.

Development of a synthesis method for odor sesquiterpenoid, (-)-rotundone, using non-heme Fe2+-chelate catalyst and ferric-chelate reductase.

(-)-Rotundone, a sesquiterpenoid that has a characteristic woody and peppery odor, is a key aroma component of spicy foodstuffs, such as black pepper and Australian Shiraz wine. (-)-Rotundone shows the lowest level of odor threshold in natural compounds and remarkably improves the quality of various fruit flavors. To develop a method for the synthesis of (-)-rotundone, we focused on non-heme Fe2+-chelates, which are biomimetic catalysts of the active center of oxygenases and enzymatic supply and regeneration of those catalysts. That is, we constructed a unique combination system composed of the oxidative synthesis of (-)-rotundone using the non-heme Fe2+-chelate catalyst, Fe(II)-EDTA, and the enzymatic supply and regeneration of Fe2+-chelate by ferric-chelate reductase, YqjH, from Escherichia coli. In addition, we improved the yield of (-)-rotundone by the application of cyclodextrin and glucose dehydrogenase to this system, and thus established a platform for efficient (-)-rotundone production.

Ectoine hydroxylase displays selective trans-3-hydroxylation activity towards L-proline.

L-Hydroxyproline (Hyp) is a valuable intermediate for the synthesis of pharmaceuticals; consequently, a practical process for its production has been in high demand. To date, industrial processes have been developed by using L-Pro hydroxylases. However, a process for the synthesis of trans-3-Hyp has not yet been established, because of the lack of highly selective enzymes that can convert L-Pro to trans-3-Hyp. The present study was designed to develop a biocatalytic trans-3-Hyp production process. We speculated that ectoine hydroxylase (EctD), which is involved in the hydroxylation of the known compatible solute ectoine, may possess the ability to hydroxylate L-Pro, since the structures of ectoine and 5-hydroxyectoine resemble those of L-Pro and trans-3-Hyp, respectively. Consequently, we discovered that ectoine hydroxylases from Halomonas elongata, as well as some actinobacteria, catalyzed L-Pro hydroxylation to form trans-3-Hyp. Of these, ectoine hydroxylase from Streptomyces cattleya also utilized 3,4-dehydro-L-Pro, 2-methyl-L-Pro, and L-pipecolic acid as substrates. In the whole-cell bioconversion of L-Pro into trans-3-Hyp using Escherichia coli expressing the ectD gene from S. cattleya, only 12.4 mM trans-3-Hyp was produced from 30 mM L-Pro, suggesting a rapid depletion of 2-oxoglutarate, an essential component of enzyme activity as a cosubstrate, in the host. Therefore, the endogenous 2-oxoglutarate dehydrogenase gene was deleted. Using this deletion mutant as the host, trans-3-Hyp production was enhanced up to 26.8 mM from 30 mM L-Pro, with minimal loss of 2-oxoglutarate. This finding is not only beneficial for trans-3-Hyp production, but also for other E. coli bioconversion processes involving 2-oxoglutarate-utilizing enzymes.

Isolation and characterization of Gram-negative and Gram-positive bacteria capable of producing piceatannol from resveratrol.

Piceatannol is a valuable natural polyphenol with therapeutic potential in cardiovascular and metabolic disease treatment. In this study, we screened for microorganisms capable of producing piceatannol from resveratrol via regioselective hydroxylation. In the first screening, we isolated microorganisms utilizing resveratrol, phenol, or 4-hydroxyphenylacetic acid as a carbon source for growth. In the second screening, we assayed the isolated microorganisms for hydroxylation of resveratrol. Using this screening procedure, a variety of resveratrol-converting microorganisms were obtained. One Gram-negative bacterium, Ensifer sp. KSH1, and one Gram-positive bacterium, Arthrobacter sp. KSH3, utilized 4-hydroxyphenylacetic acid as a carbon source for growth and efficiently hydroxylated resveratrol to piceatannol without producing any detectable by-products. The hydroxylation activity of strains KSH1 and KSH3 was strongly induced by cultivation with 4-hydroxyphenylacetic acid as a carbon source during stationary growth phase. Using the 4-hydroxyphenylacetic acid-induced cells as a biocatalyst under optimal conditions, production of piceatannol by strains KSH1 and KSH3 reached 3.6 mM (0.88 g/L) and 2.6 mM (0.64 g/L), respectively. We also cloned genes homologous to the monooxygenase gene hpaBC from strains KSH1 and KSH3. Introduction of either hpaBC homolog into Escherichia coli endowed the host with resveratrol-hydroxylating activity.

Synthesis of d-Amino Acid-Containing Dipeptides Using the Adenylation Domains of Nonribosomal Peptide Synthetase.

Recent papers have reported dipeptides containing d-amino acids to have novel effects that cannot be observed with ll-dipeptides, and such dipeptides are expected to be novel functional compounds for pharmaceuticals and food additives. Although the functions of d-amino acid-containing dipeptides are gaining more attention, there are few reports on the synthetic enzymes that can accept d-amino acids as substrates, and synthetic methods for d-amino acid-containing dipeptides have not yet been constructed. Previously, we developed a chemoenzymatic system for amide synthesis that comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for d-amino acid-containing-dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis, i.e., an adenylation domain located upstream from the epimerization domain may activate d-amino acid as well as l-amino acid. We successfully synthesized over 40 kinds of d-amino acid-containing dipeptides, including ld-, dl-, and dd-dipeptides, using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward d-amino acid. This paper provides additional information regarding d-amino acid-containing-dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis.IMPORTANCE Because almost all amino acids in nature are l-amino acids, the functioning of d-amino acids has received little attention. Thus, there is little information available on the activity of enzymes toward d-amino acids or synthetic methods for d-amino acid-containing dipeptides. Recently, d-amino acids and d-amino acid-containing peptides have attracted attention as novel functional compounds, and d-amino acid-activating enzymes and synthetic methods are required for the development of the d-amino acid-containing-peptide industry. This study provides additional knowledge regarding d-amino acid-activating enzymes and proposes a unique synthetic method for d-amino acid-containing peptides, including ld-, dl-, and dd-dipeptides.

Structural polymorphism of the Escherichia coli poly-α-L-glutamate synthetase RimK.

Bacterial RimK is an enzyme that catalyzes the polyglutamylation of the C-terminus of ribosomal protein S6 and the synthesis of poly-α-L-glutamate peptides using L-glutamic acid. In the present study, the crystal structure of the Escherichia coli RimK protein complexed with the ATP analogue AMP-PNP was determined at 2.05 Šresolution. Two different conformations of RimK, closed and open forms, were observed in the crystals. The structural polymorphism revealed in this study provided important information to understand the mechanism by which RimK catalyzes the synthesis of poly-α-L-glutamate peptides and the polyglutamylation of ribosomal protein S6.

Efficient monooxygenase-catalyzed piceatannol production: Application of cyclodextrins for reducing product inhibition.

Piceatannol is a rare, costly plant-based stilbene derivative and exhibits various health-enhancing properties. Recently, we demonstrated that piceatannol could be produced from resveratrol through site-selective hydroxylation using Escherichia coli cells expressing the monooxygenase HpaBC. However, piceatannol production ceased at approximately 25 mM, even when sufficient levels of the substrate resveratrol remained in the reaction mixture. In this study, we found that high concentrations (>20-25 mM) of piceatannol significantly inhibited the HpaBC-catalyzed reaction. Cyclodextrins (CDs) reportedly encapsulate various hydrophobic compounds. We found that the addition of ß-CD or γ-CD to the reaction mixture reduced the inhibition caused by the product piceatannol. The effects of ß-CD on piceatannol production were more pronounced than those of γ-CD at high concentrations of the substrate resveratrol and CDs. The production of piceatannol reached 49 mM (12 g L-1) in the presence of ß-CD, a level twice that achieved in the absence of ß-CD. The technique described here might be applicable to the bioproduction of other stilbenes and structurally related compounds.

A chemoenzymatic process for amide bond formation by an adenylating enzyme-mediated mechanism.

Amide bond formation serves as a fundamental reaction in chemistry, and is practically useful for the synthesis of peptides, food additives, and polymers. However, current methods for amide bond formation essentially generate wastes and suffer from poor atom economy under harsh conditions. To solve these issues, we demonstrated an alternative synthesis method for diverse tryptophyl-N-alkylamides by the combination of the first adenylation domain of tyrocidine synthetase 1 with primary or secondary amines as nucleophiles. Moreover, the physiological role of this domain is L-phenylalanine adenylation; however, we revealed that it displayed broad substrate flexibility from mono-substituted tryptophan analogues to even D-tryptophan. To the best of our knowledge, this is the first evidence for an adenylating enzyme-mediated direct amide bond formation via a sequential enzymatic activation of amino acids followed by nucleophilic substitution by general amines. These findings facilitate the design of a promising tool for biocatalytic straightforward amide bond formation with less side products.

Production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class III polyphosphate kinase 2-mediated ATP regeneration.

An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported; however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of l-Trp-l-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM l-Trp-l-Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ-putA-, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of l-Trp-l-Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only l-Trp-l-Pro, but also various other l-Xaa-l-Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.