Rational Design of the Soluble Variant of l-Pipecolic Acid Hydroxylase using the α-Helix Rule and the Hydropathy Contradiction Rule.

The production of recombinant proteins in Escherichia coli is an important application of biotechnology. 2-Oxoglutarate-dependent l-pipecolic acid hydroxylase derived from Xenorhabdus doucetiae (XdPH) is an excellent biocatalyst that catalyzes the hydroxylation of l-pipecolic acid to produce cis-5-hydroxy-l-pipecolic acid. However, the enzyme tends to form aggregates in the E. coli expression system. Our group established two rules, namely, the "α-helix rule" and the "hydropathy contradiction rule," to select residues to be altered for improving the heterologous recombinant production of proteins, by analyzing their primary structure. We rationally designed XdPH variants that are expressed in highly soluble and active forms in the E. coli expression system using these hotspot prediction methods, and the L142R variant showed a remarkably high soluble expression level compared to the wild-type XdPH. Further mutations were introduced into the L142R gene by site-directed mutagenesis. Moreover, the I28P/L142R and C76Y/L142R double variants displayed improved soluble expression levels compared to the single variants. These variants were also more thermostable than the wild-type XdPH. To analyze the effect of the alteration on one of the hotspots, L142 was replaced with various hydrophilic and positively charged residues. The remarkable increase in soluble protein expression caused by the alterations suggests that the decrease in the hydrophobicity of the protein surface and the enhancement of the interaction between nearby residues are important factors determining the solubility of the protein. Overall, this study demonstrated the effectiveness of our protocol in identifying aggregation hotspots for recombinant protein production and in basic biochemical research.

Transporter-Driven Engineering of a Genetic Biosensor for the Detection and Production of Short-Branched Chain Fatty Acids in Saccharomyces cerevisiae.

Biosensors can be used for real-time monitoring of metabolites and high-throughput screening of producer strains. Use of biosensors has facilitated strain engineering to efficiently produce value-added compounds. Following our recent work on the production of short branched-chain fatty acids (SBCFAs) in engineered Saccharomyces cerevisiae, here we harnessed a weak organic acid transporter Pdr12p, engineered a whole-cell biosensor to detect exogenous and intracellular SBCFAs and optimized the biosensor's performance by varying PDR12 expression. We firstly constructed the biosensor and evaluated its response to a range of short-chain carboxylic acids. Next, we optimized its sensitivity and operational range by deletion and overexpression of PDR12. We found that the biosensor responded to exogenous SBCFAs including isovaleric acid, isobutyric acid and 2-methylbutanoic acid. PDR12 deletion enhanced the biosensor's sensitivity to isovaleric acid at a low concentration and PDR12 overexpression shifted the operational range towards a higher concentration. Lastly, the deletion of PDR12 improved the biosensor's sensitivity to the SBCFAs produced in our previously engineered SBCFA-overproducing strain. To our knowledge, our work represents the first study on employing an ATP-binding-cassette transporter to engineer a transcription-factor-based genetic biosensor for sensing SBCFAs in S. cerevisiae. Our findings provide useful insights into SBCFA detection by a genetic biosensor that will facilitate the screening of SBCFA-overproducing strains.

Asymmetric synthesis of intermediate for (1R,2S)-ethyl 1-amino-2-vinylcyclopropanecarboxylate by desymmetrization using engineered esterase from Bacillus subtilis.

(1R,2S)-Ethyl 1-amino-2-vinylcyclopropanecarboxylate (VCPA), is a key intermediate for anti-hepatitis C virus drugs. In this study, we developed an efficient manufacturing method of intermediate for (1R,2S)-VCPA by enzymatic desymmetrization of a malonate diester derivative. In synthesis scheme of VCPA (1S,2S)-1-(ethoxycarbonyl)-2-vinylcyclopropanecarboxylic acid (VCPME) is the monoester intermediate, which is converted from 2-vinylcyclopropane-1,1-dicarboxylate diethyl ester (VCPDE). As a result of esterase screening for producing (1S,2S)-VCPME from VCPDE by enzymatic desymmetrization, p-nitrobenzyl esterase from Bacillus subtilis NBRC3027 (PNBE3027) showed high enantioselectivity (more than 90% e.e.). Based on the homology model of PNBE3027, a library of mutants with the substitution of L70, L270, L273, and L313 in substrate-binding pocket was created for improvement in enantioselectivity. (1S,2S)-VCPME produced by the best variant harboring L70D, L270Q, L273R, and L313M showed 98.9% e.e. of enanthiopurity. Furthermore, preparative scale production of (1S,2S)-VCPME using the quadruple mutant was achieved. Our investigations present a new efficient process for (1R,2S)-VCPA using esterase and diverse to be applied for the industrial scale production.

Genetic Biosensor Design for Natural Product Biosynthesis in Microorganisms.

Low yield and low titer of natural products are common issues in natural product biosynthesis through microbial cell factories. One effective way to resolve such bottlenecks is to design genetic biosensors to monitor and regulate the biosynthesis of target natural products. In this review, we evaluate the most recent advances in the design of genetic biosensors for natural product biosynthesis in microorganisms. In particular, we examine strategies for selection of genetic parts and construction principles for the design and evaluation of genetic biosensors. We also review the latest applications of transcription factor- and riboswitch-based genetic biosensors in natural product biosynthesis. Lastly, we discuss challenges and solutions in designing genetic biosensors for the biosynthesis of natural products in microorganisms.

Expression of engineered carbonyl reductase from Ogataea minuta in Rhodococcus opacus and its application to whole-cell bioconversion in anhydrous solvents.

The carbonyl reductase from the methylotrophic yeast Ogataea minuta can catalyze the regio- and enantio-selective reduction of prochiral ketones to chiral alcohols, and is available for industrial manufacturing of statin drugs. We previously conducted a directed evolution experiment of the enzyme, and obtained a mutant (OCR_V166A) with improved tolerance to organic solvents. This expanded the applicability of the enzyme to the bioconversion of water-insoluble compounds (Honda et al., J. Biosci. Bioeng., 123, 673-678, 2017). In the present study, we expressed OCR_V166A in Rhodococcus opacus cells, which have a highly lipophilic surface structure and are dispersible in anhydrous organic solvents, and developed a whole-cell biocatalyst which can function in an organic-solvent-based reaction medium. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeADH) was employed as an NADPH-regenerating enzyme and co-expressed with OCR_V166A in R. opacus. The whole-cell bioconversion of 2,2,2-trifluoroacetophenone to α-(trifluoromethyl)benzyl alcohol was performed in organic solvents, including isopropanol, isobutanol, and cyclohexanol, which served both as reaction media and as substrates for TeADH. The type of organic solvents markedly affected not only the product titer but also the enantio-purity of the product. When isobutanol was used as the reaction medium, the whole-cell biocatalyst showed higher stability than the isolated enzyme. Consequently, a high concentration (1 M) of the substrate was converted to the product with an overall conversion yield of 81% (mol/mol) in 24 h.

Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction.

Hydroxylation via C-H bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available l-lysine with l-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two l-lysine 3S-hydroxylases and four l-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of l-lysine, yielding (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant l-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S,3S)-3-hydroxylysine was produced from 600 mM l-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S,4R)-4-hydroxylysine was produced from 300 mM l-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies.IMPORTANCE The present study identified six l-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of l-lysine hydroxylases was relatively narrow, we found that (2S,3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S,5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the l-arginine hydroxylase VioC also hydroxylated l-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of l-lysine into (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic l-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of L-Pipecolic Acid.

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward L-pipecolic acid (L-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme L-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of L-Pip, and produces optically pure trans-4-hydroxy-L-pipecolic acid (trans-4-L-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had L-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the L and D forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-L-HyPip was successfully developed. Thus, we report a fungal family of L-Pip hydroxylases and the enzymatic preparation of trans-4-L-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.

Heterologous expression of l-lysine α-oxidase from Scomber japonicus in Pichia pastoris and functional characterization of the recombinant enzyme.

Fish have a complex self-defense mechanism against microbial invasion. Recently, l-lysine α-oxidases have been identified from a number of fish species as a novel type of antibacterial protein in the integument. These enzymes exhibit strict substrate specificity for l-lysine, but the underlying mechanisms and details of their catalytic properties remain unknown. In this study, a synthetic gene coding for Scomber japonicus l-lysine α-oxidase, originally termed AIP (for apoptosis-inducing protein), was expressed in Pichia pastoris, and the recombinant enzyme (rAIP) was purified and characterized. rAIP exhibited essentially the same substrate specificity as the native enzyme, catalyzing the oxidative deamination of l-lysine as an exclusive substrate. rAIP was N-glycosylated and remained active over a wide range of pH, with an optimal pH of 7.5. The enzyme was stable in the pH range from 4.5 to 10.0 and was thermally stable up to 60°C. A molecular modelling of rAIP and a comparative structure/sequence analysis with homologous enzymes indicate that Asp(220) and Asp(320) are the substrate-binding residues that are likely to confer exclusive substrate specificity for l-lysine on the fish enzymes.

Functional expression of L-lysine α-oxidase from Scomber japonicus in Escherichia coli for one-pot synthesis of L-pipecolic acid from DL-lysine.

L-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced from L-lysine by a two-step bioconversion involving L-lysine α-oxidase and ∆(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an L-lysine α-oxidase from Scomber japonicus that was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation of L-pipecolic acid in Escherichia coli. A synthetic gene coding for an AIP was expressed in E. coli, and the recombinant enzyme was purified and characterized. The purified enzyme was determined to be a homodimer with a molecular mass of 133.9 kDa. The enzyme essentially exhibited the same substrate specificity as the native enzyme. Optimal temperature and pH for the enzymatic reaction were 70 °C and 7.4, respectively. The enzyme was stable below 60 °C and at a pH range of 5.5-7.5 but was markedly inhibited by Co(2+). To establish a one-pot fermentation system for the synthesis of optically pure L-pipecolic acid from DL-lysine, an E. coli strain carrying a plasmid encoding AIP, Pip2C reductase from Pseudomonas putida, lysine racemase from P. putida, and glucose dehydrogenase from Bacillus subtilis was constructed. The one-pot process produced 45.1 g/L of L-pipecolic acid (87.4 % yield from DL-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).

Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1.

2,4-Diaminopentanoate dehydrogenase (2,4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R,4S)-2,4-diaminopentanoate (2,4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2,4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2,4-DAP, indicating that the protein is a 2,4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2,4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2,4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2,4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2,4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2,4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2,5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway.

Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host.

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.

Two thimet oligopeptidase-like Pz peptidases produced by a collagen-degrading thermophile, Geobacillus collagenovorans MO-1.

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in G. collagenovorans MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.