Polyester degradation by soil bacteria: identification of conserved BHETase enzymes in Streptomyces.

The rising use of plastic results in an appalling amount of waste which is scattered into the environment. One of these plastics is PET which is mainly used for bottles. We have identified and characterized an esterase from Streptomyces, annotated as LipA, which can efficiently degrade the PET-derived oligomer BHET. The Streptomyces coelicolor ScLipA enzyme exhibits varying sequence similarity to several BHETase/PETase enzymes, including IsPETase, TfCut2, LCC, PET40 and PET46. Of 96 Streptomyces strains, 18% were able to degrade BHET via one of three variants of LipA, named ScLipA, S2LipA and S92LipA. SclipA was deleted from S. coelicolor resulting in reduced BHET degradation. Overexpression of all LipA variants significantly enhanced BHET degradation. All variants were expressed in E. coli for purification and biochemical analysis. The optimum conditions were determined as pH 7 and 25 °C for all variants. The activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and indents on the surface of PET films, comparable to the activity of previously described TfCut2 under the same conditions. The abundance of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more polar substrates including these polluting plastics.

The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

Bioprospecting of novel peroxidase from Streptomyces coelicolor strain SPR7 for carcinogenic azo dyes decolorization.

Peroxidase (POX) is a heme-containing oxidoreductase, its voluminous immuno-diagnostic and bioremediatory intuitions have incited optimization and large scale-generation from novel microbial repertoires. Azo dyes are the most detrimental classes of synthetic dyes and they are the common ecotoxic industrial pollutants in wastewater. In addition, azo dyes are refractory to degradation owing to their chemical nature, comprising of azoic linkages, amino moieties with recalcitrant traits. Moreover, they are major carcinogenic and mutagenic on humans and animals, whereby emphasizing the need for decolorization. In the present study, a novel POX from Streptomyces coelicolor strain SPR7 was investigated for the deterioration of ecotoxic dyestuffs. The initial medium component screening for POX production was achieved using, One Factor at a Time and Placket-Burman methodologies with starch, casein and temperature as essential parameters. In auxiliary, Response Surface Methodology (RSM) was recruited and followed by model validation using Back propagation algorithm (BPA). RSM-BPA composite approach prophesied that combination of starch, casein, and temperature at optimal values 2.5%, 0.035% and 35 °C respectively, has resulted in 7 folds enhancement of POX outturn (2.52 U/mL) compared to the unoptimized media (0.36 U/mL). The concentrated enzyme decolorized 75.4% and 90% of the two azo dyes with lignin (10 mM), respectively. Hence, this investigation confirms the potentiality of mangrove actinomycete derived POX for elimination of noxious azo dyes to overcome their carcinogenic, mutagenic and teratogenic effects on humans and aquatic organisms.

Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis.

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.

Substrate Enolate Intermediate and Mimic Captured in the Active Site of Streptomyces coelicolor Methylmalonyl-CoA Epimerase.

Methylmalonyl-CoA epimerase (MMCE) is proposed to use general acid-base catalysis, but the proposed catalytic glutamic acids are highly asymmetrical in the active site unlike many other racemases. To gain insight into the puzzling relationships between catalytic mechanism, structure, and substrate preference, we solved Streptomyces coelicolor MMCE structures with substrate or 2-nitropropionyl-CoA, an intermediate/transition state analogue. Both ligand bound structures have a planar methylmalonate/2-nitropropionyl moiety indicating a deprotonated C2 with ≥4 Šdistances to either catalytic acid. Both glutamates interact with the carboxylate/nitro group, either directly or through other residues. This suggests the proposed catalytic acids sequentially catalyze proton shifts between C2 and carboxylate of the substrate with an enolate intermediate. In addition, our structures provide a platform to design mutations for expanding substrate scope to support combinatorial biosynthesis.

Alanine-scanning mutagenesis of protein mannosyl-transferase from Streptomyces coelicolor reveals strong activity-stability correlation.

In Actinobacteria, protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and ß-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.

SCO6992, a Protein with ß-Glucuronidase Activity, Complements a Mutation at the absR Locus and Promotes Antibiotic Biosynthesis in Streptomyces coelicolor.

Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong ß-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-ß-D-glucuronide and AS-BI-ß-D-glucuronide. The binding between rSCO6992 and Zn2+ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with ß-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported ß-glucuronidases.

Stereoselective synthesis of a 4-⍺-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S.

Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose-response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)-OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)-OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

Poly- and Monoamine Metabolism in Streptomyces coelicolor: The New Role of Glutamine Synthetase-Like Enzymes in the Survival under Environmental Stress.

Soil bacteria from the genus Streptomyces, phylum Actinobacteria, feature a complex metabolism and diverse adaptations to environmental stress. These characteristics are consequences of variable nutrition availability in the soil and allow survival under changing nitrogen conditions. Streptomyces coelicolor is a model organism for Actinobacteria and is able to use nitrogen from a variety of sources including unusual compounds originating from the decomposition of dead plant and animal material, such as polyamines or monoamines (like ethanolamine). Assimilation of nitrogen from these sources in S. coelicolor remains largely unstudied. Using microbiological, biochemical and in silico approaches, it was recently possible to postulate polyamine and monoamine (ethanolamine) utilization pathways in S. coelicolor. Glutamine synthetase-like enzymes (GS-like) play a central role in these pathways. Extensive studies have revealed that these enzymes are able to detoxify polyamines or monoamines and allow the survival of S. coelicolor in soil containing an excess of these compounds. On the other hand, at low concentrations, polyamines and monoamines can be utilized as nitrogen and carbon sources. It has been demonstrated that the first step in poly-/monoamine assimilation is catalyzed by GlnA3 (a γ-glutamylpolyamine synthetase) and GlnA4 (a γ-glutamylethanolamide synthetase), respectively. First insights into the regulation of polyamine and ethanolamine metabolism have revealed that the expression of the glnA3 and the glnA4 gene are controlled on the transcriptional level.

NADP+-Dependent Dehydrogenase SCO3486 and Cycloisomerase SCO3480: Key Enzymes for 3,6-Anhydro-L-Galactose Catabolism in Streptomyces coelicolor A3(2).

Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-L-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-L-galactonate, and SCO3480 is a 3,6-anhydro-L-galactonate cycloisomerase that converts 3,6-anhydro-L-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50°C, increased activity in the presence of iron ions, and activity against various aldehyde substrates, which is quite distinct from AHG-specific H2IFE7.1 in Vibrio sp. EJY3. Therefore, the catabolic pathway of AHG seems to be similar in most agar-degrading microorganisms, but the enzymes involved appear to be very diverse.

The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy.

The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center (TNC). The TNC comprises a type-3 (T3) and a type-2 (T2) copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor (SLAC) without the substrate shows a mixture of two catalytic states, the resting oxidized (RO) state and the native intermediate (NI) state. An analysis of the resonances of the RO state is reported. In this state, hydrogen resonances only of the T3 copper ligands can be found, in the region of 12-22 ppm. Signals from all six histidine ligands are found and can be attributed to Hδ1, Hß or backbone amide HN nuclei. Two sequence-specific assignments are proposed on the basis of a second-coordination shell variant that also lacks the copper ion at the T1 site, SLAC-T1D/Q291E. This double mutant is found to be exclusively in the RO state, revealing a subtle balance between the RO and the NI states.

Mechanistic divergence between (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol synthases from Dictyostelium purpureum and Streptomyces coelicolor.

The main product of DpTPS9 from the social amoeba Dictyostelium purpureum was identified as (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol that is also known as an intermediate of bacterial geosmin synthase, but the experimentally verified cyclisation mechanisms differ. Together with the low sequence identity this points to convergent evolution. The functionality of selected residues in DpTPS9 was investigated via site-directed mutagenesis experiments.

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine.

Saturation mutagenesis at seven first-sphere residues of the cytochrome P450 monooxygenase 154E1 (CYP154E1) from Thermobifida fusca YX was applied to construct a variant with only three substitutions that enabled the effective two-step synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine. A recombinant E. coli whole-cell system was essential for GC/MS based medium-throughput screening and at the same time facilitated the oxidation of the substrate (R)-ketamine at a higher scale for product isolation and subsequent NMR analysis.

An Aromatic Cluster in the Active Site of epi-Isozizaene Synthase Is an Electrostatic Toggle for Divergent Terpene Cyclization Pathways.

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic precursor of the antibiotic albaflavenone. The hydrophobic active site is largely defined by aromatic residues that direct a multistep reaction sequence through multiple carbocation intermediates. The previous substitution of polar residues for a key aromatic residue, F96, converts EIZS into a high-fidelity sesquisabinene synthase: the F96S, F96M, and F96Q variants generate 78%, 91%, and 97% sesquisabinene A, respectively. Here, we report high-resolution X-ray crystal structures of two of these reprogrammed cyclases. The structures of the F96M EIZS-Mg2+3-risedronate and F96M EIZS-Mg2+3-inorganic pyrophosphate-benzyltriethylammonium cation complexes reveal structural changes in the F96 aromatic cluster that redirect the cyclization pathway leading from the bisabolyl carbocation intermediate in catalysis. The structure of the F96S EIZS-Mg2+3-neridronate complex reveals a partially occupied inhibitor and an enzyme active site caught in transition between open and closed states. Finally, three structures of wild-type EIZS complexed with the bisphosphonate inhibitors neridronate, pamidronate, and risedronate provide a foundation for understanding binding differences between wild-type and variant enzymes. These structures provide new insight regarding active site flexibility, particularly with regard to the potential for subtle expansion and contraction to accommodate ligands of varying sizes as well as bound water molecules. Additionally, these structures highlight the importance of conformational changes in the F96 aromatic cluster that could influence cation-π interactions with carbocation intermediates in catalysis.

Cofactor Complexes of DesD, a Model Enzyme in the Virulence-related NIS Synthetase Family.

The understudied nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase family has been increasingly associated with virulence in bacterial species due to its key role in the synthesis of hydroxamate and carboxylate "stealth" siderophores. We have identified a model family member, DesD, from Streptomyces coelicolor, to structurally characterize using a combination of a wild-type and a Arg306Gln variant in apo, cofactor product AMP-bound, and cofactor reactant ATP-bound complexes. The kinetics in the family has been limited by solubility and reporter assays, so we have developed a label-free kinetics assay utilizing a single-injection isothermal-titration-calorimetry-based method. We report second-order rate constants that are 50 times higher than the previous estimations for DesD. Our Arg306Gln DesD variant was also tested under identical buffer and substrate conditions, and its undetectable activity was confirmed. These are the first reported structures for DesD, and they describe the critical cofactor coordination. This is also the first label-free assay to unambiguously determine the kinetics for an NIS synthetase.

Structural characterization of three noncanonical NTF2-like superfamily proteins: implications for polyketide biosynthesis.

Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp. CM020, a protein in the biosynthetic pathway of alnumycin. The presence of a solvent-accessible cavity and the conservation of the His/Asp dyad that is characteristic of many polyketide cyclases suggest a potential enzymatic role for these enzymes in polyketide biosynthesis.

Constructing a Novel Biosynthetic Pathway for the Production of Glycolate from Glycerol in Escherichia coli.

Glycolate is an important α-hydroxy acid with a wide range of industrial applications. The current industrial production of glycolate mainly depends on chemical synthesis, but biochemical production from renewable resources using engineered microorganisms is increasingly viewed as an attractive alternative. Crude glycerol is an abundant byproduct of biodiesel production and a widely investigated potential sustainable feedstock. Here, we constructed a novel biosynthetic pathway for the production of glycolate from glycerol in Escherichia coli. The pathway starts from the oxidation of glycerol to d-glycerate by alditol oxidase, followed by sequential enzymatic dehydrogenation and decarboxylation as well as reduction reactions. We screened and characterized the catalytic activity of candidate enzymes, and a variant of alditol oxidase from Streptomyces coelicolor A3(2), 2-hydroxyglutarate-pyruvate transhydrogenase from Saccharomyces cerevisiae, α-ketoisovalerate decarboxylase from Lactococcus lactis, and aldehyde dehydrogenase from Escherichia coli were selected and assembled to create an artificial operon for the biosynthetic production of glycolate from glycerol. We also characterized the native strong constitutive promoter Plpp from E. coli and compared it with the PT7 promoter, which was employed to express the artificial operon on the plasmid pSC105-ADKA. To redirect glycerol flux toward glycolate synthesis, we deleted key genes of the native glycerol assimilation pathways and other branches of native E. coli metabolism, and we introduced a second plasmid expressing Dld3 to reduce the accumulation of the intermediate d-glycerate. Finally, the engineered strain TZ-108 harboring pSC105-ADKA and pACYC184-Plpp-Dld3 produced 0.64 g/L glycolate in shake flasks, which was increased to 4.74 g/L in fed-batch fermentation. This study provides an alternative pathway for glycolate synthesis and demonstrates the potential for producing other commodity chemicals by redesigning glycerol metabolism.

Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation.

OBJECTIVES: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. RESULTS: Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3-. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3- reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. CONCLUSIONS: Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.

Analyzing and Engineering the Product Selectivity of a 2-Methylenebornane Synthase.

Terpenes constitute the largest class of natural products with more than 70 000 compounds. Many different terpenes find applications in the flavor and fragrance industry or can be used as fine chemicals or drugs. In some bacteria, noncanonical terpenes with 11 carbon atoms are synthesized via a GPP-C2-methyltransferase and the subsequent conversion of 2-methyl-GPP by certain terpene synthases into mainly 2-methylisoborneol and 2-methylenebornane. Many other C11-terpenes were reported as side products, but they are synthesized only in minor amounts by the bacterial C11-terpene biosynthesis pathway. To enable biotechnological synthesis of these largely unexplored natural products, we changed the product selectivity of the 2-methylenebornane synthase from Pseudomonas fluorescens by a semirational protein engineering approach. Active site amino acids with impact on the product selectivity were identified and variants with completely altered product spectra could be identified and characterized. The gathered data provide new insights into the structure-function relationship for C11-terpene synthases and demonstrate the production of formerly inaccessible noncanonical terpenes.

Characterization of alditol oxidase from Streptomyces coelicolor and its application in the production of rare sugars.

A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor d-glyceraldehyde was directly produced from glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and d-glyceraldehyde was performed by l-rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phosphate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars d-sorbose and d-psicose. To accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldOS.coe) was expressed in E. coli and the purified AldOS.coe was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes including AldOS.coe was constructed. Under the optimized conditions, it produced 7.9 g/L of d-sorbose and d-psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective method for the synthesis of rare sugars.