Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

An artificial pathway for trans-4-hydroxy-L-pipecolic acid production from L-lysine in Escherichia coli.

Trans-4-hydroxy-L-pipecolic acid (trans-4-HyPip) is a hydroxylated product of L-pipecolic acid, which is widely used in the pharmaceutical and chemical industries. Here, a trans-4-HyPip biosynthesis module was designed and constructed in Escherichia coli by overexpressing lysine α-oxidase, Δ1-piperideine-2-carboxylase reductase, glucose dehydrogenase, lysine permease, catalase and L-pipecolic acid trans-4-hydroxylase for expanding the lysine catabolism pathway. A total of 4.89 g/L of trans-4-HyPip was generated in shake flasks from 8 g/L of L-pipecolic acid. By this approach, 14.86 g/L of trans-4-HyPip was produced from lysine after 48 h in a 5 L bioreactor. As far as we know, this is the first multi-enzyme cascade catalytic system for the production of trans-4-HyPip using E. coli from L-lysine. Therefore, it can be considered as a potential candidate for the industrial production of trans-4-HyPip in microorganisms.

Analysis of the differences in the chemical composition of monascus rice and highland barley monascus.

Monascus rice (MR) and highland barley monascus (HBM), the monascus fermented products, are applied in food and medicine to reduce cholesterol and promote digestion. Due to the fermentation substrates, their compositions are different. However, the exact differences have not been reported to date. By UPLC-Q-Orbitrap HRMS analysis, multiple components of twenty batches of MR and HBM samples were identified. In total, 100 components were confirmed (e.g., monacolins, pigments, decalin derivatives, amino acids). Then, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to filter the components of MR and HBM. In the PCA model, 88.1% of the total variance was uncovered. The OPLS-DA model showed better discrimination between MR and HBM, and the values of R2X, R2Y, and Q2 were 0.837, 0.996, and 0.956, respectively. Based on the value of the variable importance in projection (VIP) and the result of the t-test, 424 components (VIP > 1, p < 0.05) were acquired. Finally, 11 differential components were selected as the characteristic compounds to discriminate between MR and HBM: the content of 9 monacolins (3-hydroxy-3,5-dihydrodehydromonacolin K, monacolin K, dehydromonacolin K, dehydromonacolin J hydroxy acid, monacophenyl, dihydromonacolin J hydroxy acid, monacolin L, dehydromonacolin J, and monacolin R) in HBM was higher than the content in MR, but the content of 2 pigments (ankaflavin and monascin) was lower in HBM and higher in MR. The findings revealed the similarities and differences in the chemical composition between MR and HBM, which is expected to provide a basis for quality control of HBM.

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids.

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4-7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80-90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.

Impact of drinking water supplemented 2-hydroxy-4-methylthiobutyric acid in combination with acidifier on performance, intestinal development, and microflora in broilers.

In addition to offering methionine, 2-hydroxy-4-methylthiobutyric acid (HMTBa) is also an organic acid and shows excellent bacteriostasis. Therefore, 3 experiments were conducted to determine the influence of drinking water supplemented HMTBa in combination with acidifier on performance, intestinal development, and microflora in broilers. The addition of different concentration (0.02-0.20%) of the blend of HMTBa and other acids significantly reduced the pH of water and exerted antimicrobial activity in dose-dependent manner in vitro. The outcomes from animal trial consisting of the drinking water with blended acidifier at 0.00, 0.05, 0.10, 0.15, and 0.20% indicated that the water with 0.15 or 0.20% acidifier resulted in linear and quadratic higher body weight at 42 d, gain and water consumption during 1 to 42 d (P < 0.05). In experiment 3, responding to graded blended acidifier in drinking water, birds receiving 0.10, 0.15, and 0.20% acidifier decreased the internal pH of gastrointestinal tract and muscle, and exhibited increased duodenal weight, length, villus high, and the ratio of villus high to crypt depth. Drinking water with 0.2% blended acidifier increased the abundance of probiotics (Bacteroidaceae, Ruminococcaceae, and Lachnospiraceae) and decreased the account of pathogenic bacteria such as Desulfovibrionaceae. Alternations in gut microflora were closely related to the metabolism of carbohydrate, amino acid, and vitamins. These findings, therefore, suggest that drinking water with 0.10 to 0.13% the combination HMTBa with acidifier might benefit to intestinal development and gut microbiota, and the subsequent produce a positive effect on the performance of broilers.

The role of oxidative stress in the susceptibility of noise-impaired cochleae to synaptic loss induced by intracochlear electrical stimulation.

Intracochlear electrical stimulation (ES) generated by cochlear implants (CIs) is used to activate auditory nerves to restore hearing perception in deaf subjects and those with residual hearing who use electroacoustic stimulation (EAS) technology. Approximately 1/3 of EAS recipients experience loss of residual hearing a few months after ES activation, but the underlying mechanism is unknown. Clinical evidence indicates that the loss is related to the previous history of noise-induced hearing loss (NIHL). In this report, we investigated the impact of intracochlear ES on oxidative stress levels and synaptic counts in inner hair cells (IHCs) of the apical, middle and basal regions of guinea pigs with normal hearing (NH) and NIHL. Our results demonstrated that intracochlear ES with an intensity of 6 dB above the thresholds of electrically evoked compound action potentials (ECAPs) could induce the elevation of oxidative stress levels, resulting in a loss of IHC synapses near the electrodes in the basal and middle regions of the NH cochleae. Furthermore, the apical region of cochleae with NIHL were more susceptible to synaptic loss induced by relatively low-intensity ES than that of NH cochleae, resulting from the additional elevation of oxidative stress levels and the reduced antioxidant capability throughout the whole cochlea.

Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis.

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.

Production of 7,10,12-trihydroxy-8(E)-octadecenoic acid from ricinoleic acid by Pseudomonas aeruginosa KNU-2B.

Microbial production of hydroxy fatty acids (HFAs) was widely studied because of important biological properties of HFAs. Among microorganisms producing HFAs, Pseudomonas aeruginosa PR3 was well known to produce various HFAs from different unsaturated fatty acids. Recently, a new variant species of P. aeruginosa PR3 was isolated and characterized, showing improved efficiency for producing 7,10-dihydroxy-8(E)-octadecenoic acid from oleic acid. In this study, we report the production of 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) from ricinoleic acid by the newly isolated P. aeruginosa KNU-2B. TOD was efficiently produced from ricinoleic acid by KNU-2B with the maximum conversion yield of 56.7% under the optimum reaction conditions of pH 8.0 and 48-h incubation at 27 °C, 150 rpm. Under optimized reaction conditions, maximum TOD production reached 340.3 mg/100 mL of the culture. However, requirement of nutritional factors by KNU-2B for production of TOD were considerably different from those by PR3 strain.

Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon Extension of Aldehydes to Chiral α-Hydroxy Acids.

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine-diphosphate-dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl-CoA thioester substrates through the release of the C1 -unit formyl-CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C1 -extensions of various aldehydes to yield the corresponding 2-hydroxyacyl-CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes-a specific oxalyl-CoA synthetase and a 2-hydroxyacyl-CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)-α-hydroxy acids with enantiomeric excess up to 99 %.

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase.

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD-containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2-(2-hydroxyethoxy)acetic acid. Such a facile cofactor-independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks.

Eosinophils synthesize trihydroxyoctadecenoic acids (TriHOMEs) via a 15-lipoxygenase dependent process.

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived lipid mediators reported to be dysregulated in obstructive lung disease. In contrast to many other oxylipins, TriHOME biosynthesis in humans is still poorly understood. The association of TriHOMEs with inflammation prompted the current investigation into the ability of human granulocytes to synthesize the 16 different 9,10,13-TriHOME and 9,12,13-TriHOME isomers and of the TriHOME biosynthetic pathway. Following incubation with linoleic acid, eosinophils and (to a lesser extent) the mast cell line LAD2, but not neutrophils, formed TriHOMEs. Stereochemical analysis revealed that TriHOMEs produced by eosinophils predominantly evidenced the 13(S) configuration, suggesting 15-lipoxygenase (15-LOX)-mediated synthesis. TriHOME formation was blocked following incubation with the 15-LOX inhibitor BLX-3887 and was shown to be largely independent of soluble epoxide hydrolase and cytochrome P450 activities. TriHOME synthesis was abolished when linoleic acid was replaced with 13-HODE, but increased in incubations with 13-HpODE, indicating the intermediary role of epoxy alcohols in TriHOME formation. In contrast to eosinophils, LAD2 cells formed TriHOMEs having predominantly the 13(R) configuration, demonstrating that there are multiple synthetic routes for TriHOME formation. These findings provide for the first-time insight into the synthetic route of TriHOMEs in humans and expand our understanding of their formation in inflammatory diseases.

One-pot lipase-catalyzed esterification of ε-caprolactone with methyl-d-glucopyranoside and its elongation with free 6-hydroxyhexanoate monomer units.

One-pot synthesis of sugar-functionalized oligomeric caprolactone was carried out by lipase-catalyzed esterification of ε-caprolactone (ECL) with methyl-d-glucopyranoside (MGP) followed by the elongation of functionalized oligomer chain. Functionalization was performed in a custom-fabricated glass reactor equipped with Rushton turbine impeller and controlled temperature at 60 °C using tert-butanol as reaction medium. The overall reaction steps include MGP esterification of ECL monomer and its subsequent elongation by free 6-hydroxyhexanoate monomer units. A ping-pong bi-bi mechanism without ternary complex was proposed for esterification of ECL and MGP with apparent values of kinetic constant, namely maximal velocity (Vmax ), Michaelis constant for MGP (KmMGP ), and Michaelis constant for ECL (KmECL ) at 3.848 × 10-3  M H-1 , 8.189 × 10-2  M, and 6.050 M, respectively. Chain propagation step of MGP-functionalized ECL oligomer exhibits the properties of living polymerization mechanism. Linear relationship between conversion (%) and number average molecular weight, Mn (g mol-1 ), of functionalized oligomer was observed. Synthesized functionalized oligomer showed narrow range of molecular weight from 1,400 to 1,600 g mol-1 with more than 90% conversion achieved. Structural analysis confirmed the presence of covalent bond between the hydroxyl group in MGP with carboxyl end group of ECL oligomer.

New Hydroxydecanoic Acid Derivatives Produced by an Dndophytic Yeast Aureobasidium pullulans AJF1 from Flowers of Aconitum carmichaeli.

Endophytes have been recognized as a source for structurally novel and biologically active secondary metabolites. Among the host plants for endophytes, some medicinal plants that produce pharmaceuticals have been reported to carry endophytes, which could also produce bioactive secondary metabolites. In this study, the medicinal plant Aconitum carmichaeli was selected as a potential source for endophytes. An endophytic microorganism, Aureobasidium pullulans AJF1, harbored in the flower of Aconitum carmichaeli, was cultured on a large scale and extracted with an organic solvent. Extensive chemical investigation of the extracts resulted in isolation of three lipid type compounds (1-3), which were identified to be (3R,5R)-3,5-dihydroxydecanoic acid (1), (3R,5R)-3-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-5-hydroxydecanoic acid (2), and (3R,5R)-3-(((3R,5R)-5-(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-3-hydroxydecanoyl)oxy)-5-hydroxydecanoic acid (3) by chemical methods in combination with spectral analysis. Compounds 2 and 3 had new structures. Absolute configurations of the isolated compounds (1-3) were established using modified Mosher's method together with analysis of NMR data for their acetonide derivatives. All the isolates (1-3) were evaluated for antibiotic activities against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and their cytotoxicities against MCF-7 cancer cells. Unfortunately, they showed low antibiotic activities and cytotoxic activities.

The Transcriptome and Flux Profiling of Crabtree-Negative Hydroxy Acid-Producing Strains of Saccharomyces cerevisiae Reveals Changes in the Central Carbon Metabolism.

Saccharomyces cerevisiae (S. cerevisiae) is the yeast cell factory of choice for the production of many biobased chemicals. However, it is a Crabtree-positive yeast and so shuttles a large portion of carbon into ethanol. Ethanol formation can be eliminated by deleting pyruvate decarboxylase (PDC) activity. It is not yet well understood how PDC-negative yeasts are affected when engineered to produce other products than ethanol. In this study, pathways are introduced for the production of three hydroxy acids (lactic, malic, or 3-hydroxypropionic acid [3HP]) into an evolved PDC-negative strain. These strains are characterized via transcriptome and flux profiling to elucidate the effects that the production of these hydroxy acids has on the host strain. Expression of lactic and malic acid biosynthesis pathways improved the maximum specific growth rate (µmax ) of the strain by 64% and 20%, respectively, presumably due to nicotinamide adenine dinucleotide regeneration. All strains show a very high flux ( > 90% of glucose uptake) into the oxidative pentose phosphate pathway under batch fermentation conditions. The study, for the first time, directly compares the flux and transcriptome profiles of several hydroxy acid-producing strains of an evolved PDC-negative S. cerevisiae and suggests directions for future metabolic engineering.

Re-examining the role of the gut microbiota in the conversion of the lipid-lowering statin monacolin K (lovastatin) into its active ß-hydroxy acid metabolite.

Monacolin K (MK, lovastatin), a naturally occurring statin, only exerts lipid-lowering effects in its active ß-hydroxy acid form (MKA). This activation was thought to be mediated by the gut microbiota (GM). We report here for the first time that the GM does not convert MK into MKA (a spontaneous pH-dependent conversion) but catabolises MKA. The GM might hamper the lipid-lowering effects by degrading the active metabolite MKA.

A novel d-2-hydroxy acid dehydrogenase with high substrate preference for phenylpyruvate originating from lactic acid bacteria: Structural analysis on the substrate specificity.

2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. d-lactate dehydrogenases (d-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C3 phenyl group. The 2-HADH from P. claussenii was found to have the highest kcat/Km on PPA with 1,348.03 s-1 mM-1 among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the d-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.

Feeding broiler chicks diets containing keto- and hydroxy-acids: performance and carcass weight.

This study evaluated reduced dietary CP and supplementing amino acid analogs to sustain growth and carcass weight in 0- to 21-day-old Cobb × Avian-48 male broiler chicks. A total of 6 diets with 3 levels of CP (22.5, 19.5, and 16.5%) and 2 sources of AA analogs, either synthetic amino acids (SA) or keto-/hydroxy-acids (KA), were assigned randomly to 36 cages (8 chicks/cage) in a 3 × 2 factorial design. For SA diets, DL-Met, DL-Met + L-Ile, and D-Met + L-Ile + L-Val were used to supplement 22.5, 19.5, and 16.5% CP diets, respectively, and for corresponding KA diets, DL-Met was replaced with methionine hydroxy analog (MHA), L-Ile was replaced with keto-Ile, and L-Val was replaced with keto-Val. Water and all isocaloric diets (3,050 kcal ME/kg) were given ad libitum. Lowering dietary CP to 16.5% reduced BW at 7, 14, and 21 D (P ≤ 0.0001) and feed intake at 8 to 14, 15 to 21, and 0 to 21 D (P ≤ 0.001). Body weight gain (BWG) was reduced and feed-to-gain ratio (FGR) was increased (P ≤ 0.003 to 0.0001) at all times for chicks fed 16.5% CP; however, chicks fed 22.5 and 19.5% CP had comparable performance. Differences in 0 to 7 D BWG (SA, 122.9 vs. KA, 113.9 g/bird; P ≤ 0.04), a 0 to 21 D FGR cumulative effect (1.45 vs. 1.51; P ≤ 0.02), and a 15 to 21 D (P ≤ 0.04) and 0 to 21 D (P ≤ 0.05) CP × AA interaction were also observed. Greater liver weight among 16.5 vs. 19.5 or 22.5% CP fed chicks was found at 14 and 21 D (P ≤ 0.0001 and P = 0.06, respectively). Lower dietary CP reduced spleen weight on day 21 birds (P ≤ 0.0005) with lighter spleens among 16.5 and 19.5% vs. the 22.5% CP fed group (0.090, 0.095, 0.119 g/100 g BW, respectively). Breast weight at 21 D was significantly less for 16.5 vs. 22.5% CP fed chicks. Fat pad weight on day 21 was heaviest among 16.5% chicks (P ≤ 0.0004). Overall, lowering dietary CP to 16.5% had a negative effect, but keto-acid supplementation supported 0 to 21 D broiler growth compared to SA; however, transamination efficiency of KA may be lower for 0 to 7D old chicks compared to older birds.

Mechanism of a Standalone ß-Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme.

Enzyme-catalyzed ß-lactone formation from ß-hydroxy acids is a crucial step in bacterial biosynthesis of ß-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for ß-lactone synthetase activity using synthetic ß-hydroxy acid substrates with alkene or alkyne moieties. ß-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain ß-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the ß-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the ß-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.

A multi-enzyme cascade reaction for the production of 6-hydroxyhexanoic acid.

Multi-enzyme cascade reactions capture the essence of nature's efficiency by increasing the productivity of a process. Here we describe one such three-enzyme cascade for the synthesis of 6-hydroxyhexanoic acid. Whole cells of Escherichia coli co-expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase (CHMO) for internal cofactor regeneration were used without the supply of external NADPH or NADP+. The product inhibition caused by the ε-caprolactone formed by the CHMO was overcome by the use of lipase CAL-B for in situ conversion into 6-hydroxyhexanoic acid. A stirred tank reactor under fed-batch mode was chosen for efficient catalysis. By using this setup, a product titre of >20 g L-1 was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.

Highly Efficient Deracemization of Racemic 2-Hydroxy Acids in a Three-Enzyme Co-Expression System Using a Novel Ketoacid Reductase.

Enantiopure 2-hydroxy acids (2-HAs) are important intermediates for the synthesis of pharmaceuticals and fine chemicals. Deracemization of racemic 2-HAs into the corresponding single enantiomers represents an economical and highly efficient approach for synthesizing chiral 2-HAs in industry. In this work, a novel ketoacid reductase from Leuconostoc lactis (LlKAR) with higher activity and substrate tolerance towards aromatic α-ketoacids was discovered by genome mining, and then its enzymatic properties were characterized. Accordingly, an engineered Escherichia coli (HADH-LlKAR-GDH) co-expressing 2-hydroxyacid dehydrogenase, LlKAR, and glucose dehydrogenase was constructed for efficient deracemization of racemic 2-HAs. Most of the racemic 2-HAs were deracemized to their (R)-isomers at high yields and enantiomeric purity. In the case of racemic 2-chloromandelic acid, as much as 300 mM of substrate was completely transformed into the optically pure (R)-2-chloromandelic acid (> 99% enantiomeric excess) with a high productivity of 83.8 g L-1 day-1 without addition of exogenous cofactor, which make this novel whole-cell biocatalyst more promising and competitive in practical application.