A Membrane‐Bound Prenyltransferase Catalyzes the O‐Prenylation of 1,6‐Dihydroxyphenazine in the Marine Bacterium Streptomyces sp. CNQ‐509

Streptomyces sp. CNQ‐509 produces the rare O‐prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O‐prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ‐509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine‐1‐carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6‐dihydroxyphenazine geranyltransferase, which catalyzes the C?O bond formation between C‐1 of the geranyl moiety and O‐6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O‐prenyl transfer to a phenazine.     

Highly Efficient Chemoenzymatic Synthesis of Methyl (R)‐o‐Chloromandelate,a Key Intermediate for Clopidogrel,via Asymmetric Reduction with Recombinant Escherichia coli

Methyl (R)‐o‐chloromandelate [(R)‐ 1 ], which is an intermediate for a platelet aggregation inhibitor named clopidogrel, was obtained in >99% ee by the asymmetric reduction of methyl o‐chlorobenzoylformate ( 2 ) with recombinant Escherichia coli overproducing a versatile carbonyl reductase. A remarkable temperature effect on productivity was observed in the whole‐cell reduction of 2 , and the optimum productivity as high as 178 g/L was attained at 20 °C on a 2‐g scale (1.0 M). The optimized reaction could be scaled up easily to transform 20 g of 2 in 100 mL of buffer. Three synthetic methods for 2 are compared.     

Genome Mining of Streptomyces sp. Tü 6176: Characterization of the Nataxazole Biosynthesis Pathway

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary‐metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O‐methyltransferase gene required to convert AJI9561 into nataxazole. This O‐methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3‐hydroxyanthranilate and an iterative type I PKS to generate 6‐methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5‐hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5‐Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross‐talk between the nataxazole biosynthesis pathway and an unknown pathway.     

一株梅花鹿粪便链霉菌化学成分的分离与鉴定

对一株梅花鹿粪便链霉菌(Streptomyces sp.)的化学成分进行了分析鉴定。从链霉菌发酵液的95%乙醇提取物中分离得到6个已知化合物,分别鉴定为6-N,N-二甲基腺苷(Ⅰ)、5′-甲硫基腺苷(Ⅱ)、胸腺嘧啶脱氧核苷(Ⅲ)、环-(苯丙氨酸-羟脯氨酸)-二肽(Ⅳ)、吲哚-3-甲酸(Ⅴ)、对羟基苯乙醇(Ⅵ);6个化合物均为首次从该菌株中分离得到。     

Recombinant Δ4,5‐Steroid 5 β‐Reductases as Biocatalysts for the Reduction of Activated CC‐Double Bonds in Monocyclic and Acyclic Molecules

It was found that Δ^4,5‐steroid 5β‐reductases are capable of reducing also small molecules bearing an activated CC double bond such as monocyclic enones and acyclic enoate esters. As preferred Δ^4,5‐steroid 5β‐reductase (5β‐StR) for this purpose, 5β‐StR from Arabidopsis thaliana was used. In part, enzyme activities are even higher than that for progesterone. Successful preliminary biotransformations with enzymatic in situ cofactor recycling were also carried out. When using the prochiral compound isophorone as a substrate, a high enantioselective reaction course (>99% ee) was observed.     

Epoxide Hydrolase‐Catalysed Kinetic Resolution of a Spiroepoxide,a Key Building Block of Various 11‐Heterosteroids

Two microbial epoxide hydrolases – i.e., Aspergillus niger (AnEH) and Rhodococcus erythropolis (the so‐called “Limonene EH”: LEH) were used to achieve, for the first time, the biocatalysed hydrolytic kinetic resolution (BHKR) of spiroepoxide rac‐ 1 . This compound is a strategic key building block allowing the synthesis of 11‐heterosteroids. Interestingly enough, the two enzymes exhibited opposite and therefore complementary enantioselectivity allowing us to isolate the residual (R,R)‐ 1 (from AnEH) and the residual (S,S)‐ 1 (from LEH) in nearly enantiopure forms (>98 %). Their absolute configurations were determined by X‐ray crystallography. An opposite regioselectivity of the oxirane ring opening for both enantiomers of substrate 1 , determined using H₂¹⁸O labelling and chiral GC‐MS analysis, was also observed, corresponding to an attack at the less substituted carbon atom using AnEH, and at the most substituted carbon atom using LEH. A chemical process‐improving methodology was also developed. This allowed us to obtain both enantiomers of the substrate in high enantiomeric purity (99 %) and optimised quantity. In the case of the AnEH, the use of a biphasic (water/isooctane) reaction medium allowed us to increase the global substrate concentration up to 200 g/ L. The preparation of both enantiomers of 1 clearly paves the way to the preparative scale synthesis and biochemical evaluation of the corresponding 11‐heterosteroid enantiomers.     

Desymmetrisations of 1‐Alkylbicyclo[3.3.0]octane‐2,8‐diones by Enzymatic Retro‐Claisen Reaction Yield Optically Enriched 2,3‐Substituted Cyclopentanones

A series of 1‐alkylbicyclo[3.3.0]octane‐2,8‐diones was transformed by enzymatic retro‐Claisen reaction using recombinant 6‐oxocamphor hydrolase (OCH) overexpressed in Escherichia coli, to yield optically active 2,3‐substituted cyclopentanones with enantiomeric excesses of up to >95 %. Whilst the parent substrate, bicyclo[3.3.0]octane‐2,8‐dione 12 , was transformed only very slowly, derivatives 13, 14, 15, 16 and 30 with alkyl chains of varying length in the 1‐position were converted rapidly to optically active products with typically 82 % de and up to >95 % enantiomeric excess. The results confirm the apparent requirement of OCH for non‐enolisable diketone substrates, and offer a potential route to decorated cyclopentanone derivatives of multiple chiral centres. Computer modelling of 1‐methylbicyclo[3.3.0]octane‐2,8‐dione into the active site of OCH suggested that the bicyclic [3.3.0] series substrates were accommodated in the active site in similar orientation with the natural enzyme substrate, 6‐oxocamphor, and would thus yield the (2S,3S)‐product series.     

Efficient Biosynthesis of (R)‐ or (S)‐2‐Hydroxybutyrate from l‐Threonine through a Synthetic Biology Approach

An efficient multi‐enzyme cascade reaction for the synthesis of (R)‐ or (S)‐2‐hydroxybutyric acid [(R)‐ or (S)‐2‐HB] from l ‐threonine was developed by using recombinant Escherichia coli cells expressing separately or co‐expressing l ‐threonine deaminase from Escherichia coli K‐12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l ‐lactate dehydrogenase (l ‐LDH) from Oryctolagus cuniculus or d ‐lactate dehydrogenase (d ‐LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l ‐threonine were completely transformed to (R)‐ or (S)‐2‐HB in optically pure form (>99% ee) with high isolated yields. This one‐pot multi‐enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds.

     

Genome Mining of the Hitachimycin Biosynthetic Gene Cluster: Involvement of a Phenylalanine‐2,3‐aminomutase in Biosynthesis

Hitachimycin is a macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β‐Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine‐2,3‐aminomutase (PAM), five polyketide synthases, four β‐amino‐acid‐carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)‐β‐Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Kinetic Resolution of 1‐Biaryl‐ and 1‐(Pyridylphenyl)alkan‐1‐ols Catalysed by the Lipase B from Candida antarctica

Lipase B from Candida antarctica (CAL‐B) catalyses the highly enantioselective (E>200) transesterification of some 1‐biaryl‐2‐yl‐, ‐3‐yl‐, and ‐4‐ylethanols and ‐propan‐1‐ols, as well as 1‐(o‐, m‐, and p‐pyridylphenyl)ethanols, 6 , with vinyl acetate, Kazlauskas' rule being obeyed in all cases. meta and para‐Substituted substrates were transformed within several hours (conversion degree ranging from 23–50%), reaction rates for propan‐1‐ol derivatives being slower than those for ethanol derivatives. Transesterifications of ortho‐substituted alcohols took several days and were accompanied by a chemoenzymatic side reaction: the formation of another acetate derived from the hemiacetal between 6 and acetaldehyde coming from vinyl acetate. This side reaction was suppressed in the presence of isopropenyl acetate as acyl donor, conversion degrees for transesterification ranging from 20–40% after ten days (E>200). The usefulness of (R)‐ 6p as ligand in the asymmetric addition of diethylzinc to benzaldehyde was also demonstrated.     

Antifungal Activity and Biosynthetic Potential of New Streptomyces sp. MW-W600-10 Strain Isolated from Coal Mine Water

Crop infections by fungi lead to severe losses in food production and pose risks for human health. The increasing resistance of pathogens to fungicides has led to the higher usage of these chemicals, which burdens the environment and highlights the need to find novel natural biocontrol agents. Members of the genus Streptomyces are known to produce a plethora of bioactive compounds. Recently, researchers have turned to extreme and previously unexplored niches in the search for new strains with antimicrobial activities. One such niche are underground coal mine environments. We isolated the new Streptomyces sp. MW-W600-10 strain from coal mine water samples collected at 665 m below ground level. We examined the antifungal activity of the strain against plant pathogens Fusarium culmorum DSM62188 and Nigrospora oryzae roseF7. Furthermore, we analyzed the strain’s biosynthetic potential with the antiSMASH tool. The strain showed inhibitory activity against both fungi strains. Genome mining revealed that it has 39 BGCs, among which 13 did not show similarity to those in databases. Additionally, we examined the activity of the Streptomyces sp. S-2 strain isolated from black soot against F. culmorum DSM62188. These results show that coal-related strains could be a source of novel bioactive compounds. Future studies will elucidate their full biotechnological potential.     

Identification of Specific Variations in a Non-Motile Strain of Cyanobacterium Synechocystis sp. PCC 6803 Originated from ATCC 27184 by Whole Genome Resequencing

Cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism in basic research and biofuel biotechnology application. Here, we report the genomic sequence of chromosome and seven plasmids of a glucose-tolerant, non-motile strain originated from ATCC 27184, GT-G, in use at Guangzhou. Through high-throughput genome re-sequencing and verification by Sanger sequencing, eight novel variants were identified in its chromosome and plasmids. The eight novel variants, especially the five non-silent mutations might have interesting effects on the phenotype of GT-G strains, for example the truncated Sll1895 and Slr0322 protein. These resequencing data provide background information for further research and application based on the GT-G strain and also provide evidence to study the evolution and divergence of Synechocystis 6803 globally.     

Production of fructooligosaccharides by β‐fructofuranosidase from Aspergillus sp 27H

β‐fructofuranosidase (EC 3.2.1.26) from Aspergillus sp 27H isolated from soil was investigated for production of fructooligosaccharides (FOS) using whole cells. It possesses hydrolytic and transfructosylating activities that can be altered by modifying the reaction conditions. The optimal conditions for the transfructosylating activity occur in the pH range 5.5–6.0 and at 60 °C, while hydrolytic activity was highest at pH 4.0 and 55 °C. At low sucrose concentration (10 g dm^?3) there was rapid conversion of sucrose to glucose and fructose and very low concentrations of FOS were obtained. However, at sucrose concentrations higher than 216 g dm^?3 the concentrations of hydrolysis products were reduced. Under the following conditions: pH 5.5, temperature 40 °C, sucrose concentration 615 g dm^?3 and enzyme concentration 20β‐fructofuranosidase units g^?1 of sucrose, the FOS concentration reached a maximum value of 376 g dm^?3 (234 g dm^?3 1‐kestose and 142 g dm^?3 nystose) and the proportion of FOS in the solids in the reaction mixture was 600–620 g kg^?1 at 6 h. These results suggest that β‐fructofuranosidase from Aspergillus sp 27H could be an appropriate enzyme for the commercial production of FOS. Copyright © 2004 Society of Chemical Industry     

α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

We report the first heterologous production of a fungal rutinosidase (6‐O‐α‐L ‐rhamnopyranosyl‐β‐D ‐glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the α‐L ‐rhamnosidase‐catalyzed synthesis of a chromogenic substrate for a rutinosidase assay – para‐nitrophenyl β‐rutinoside – is described.