Establishment of an Efficient Expression and Regulation System in Streptomyces for Economical and High-Level Production of the Natural Blue Pigment Indigoidine.

Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceuticals and is mainly synthesized from l-glutamine via a condensation reaction by indigoidine synthetases, most of which originates from Streptomyces species. However, due to the complex metabolic switches of Streptomyces, most of the researchers choose to overexpress indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine. Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, here, an updated regulation system derived from the Streptomyces self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in Streptomyces lividans TK24. The updated system was constructed via promoter mining and σhrdB expression optimization, and this system was applied to precisely and continuously regulate the expression of indigoidine synthetase IndC derived from Streptomyces albus J1704. Finally, the engineered strain was cultured with cheap industrial glycerol as a supplementary carbon source, and 14.3 and 46.27 g/L indigoidine could be achieved in a flask and a 4 L fermentor, respectively, reaching the highest level of microbial synthesis of indigoidine. This study will lay a foundation for the industrial application of Streptomyces cell factories to produce indigoidine.

Microbial Degradation, Spectral analysis and Toxicological Assessment of Malachite Green Dye by Streptomyces exfoliatus.

Malachite green (MG) dye is a common environmental pollutant that threatens human health and the integrity of the Earth's ecosystem. The aim of this study was to investigate the potential biodegradation of MG dye by actinomycetes species isolated from planted soil near an industrial water effluent in Cairo, Egypt. The Streptomyces isolate St 45 was selected according to its high efficiency for laccase production. It was identified as S. exfoliatus based on phenotype and 16S rRNA molecular analysis and was deposited in the NCBI GenBank with the gene accession number OL720220. Its growth kinetics were studied during an incubation time of 144 h, during which the growth rate was 0.4232 (µ/h), the duplication time (td) was 1.64 d, and multiplication rate (MR) was 0.61 h, with an MG decolorization value of 96% after 120 h of incubation at 25 °C. Eleven physical and nutritional factors (mannitol, frying oil waste, MgSO4, NH4NO3, NH4Cl, dye concentration, pH, agitation, temperature, inoculum size, and incubation time) were screened for significance in the biodegradation of MG by S. exfoliatus using PBD. Out of the eleven factors screened in PBD, five (dye concentration, frying oil waste, MgSO4, inoculum size, and pH) were shown to be significant in the decolorization process. Central composite design (CCD) was applied to optimize the biodegradation of MG. Maximum decolorization was attained using the following optimal conditions: food oil waste, 7.5 mL/L; MgSO4, 0.35 g/L; dye concentration, 0.04 g/L; pH, 4.0; and inoculum size, 12.5%. The products from the degradation of MG by S. exfoliatus were characterized using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The results revealed the presence of several compounds, including leuco-malachite green, di(tert-butyl)(2-phenylethoxy) silane, 1,3-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 1,4-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 1,2-benzenedicarboxylic acid, di-n-octyl phthalate, and 1,2-benzenedicarboxylic acid, dioctyl ester. Moreover, the phytotoxicity, microbial toxicity, and cytotoxicity tests confirmed that the byproducts of MG degradation were not toxic to plants, microbes, or human cells. The results of this work implicate S. exfoliatus as a novel strain for MG biodegradation in different environments.

Multi-omic characterisation of Streptomyces hygroscopicus NRRL 30439: detailed assessment of its secondary metabolic potential.

The emergence of multidrug-resistant pathogenic bacteria creates a demand for novel antibiotics with distinct mechanisms of action. Advances in next-generation genome sequencing promised a paradigm shift in the quest to find new bioactive secondary metabolites. Genome mining has proven successful for predicting putative biosynthetic elements in secondary metabolite superproducers such as Streptomycetes. However, genome mining approaches do not inform whether biosynthetic gene clusters are dormant or active under given culture conditions. Here we show that using a multi-omics approach in combination with antiSMASH, it is possible to assess the secondary metabolic potential of a Streptomyces strain capable of producing mannopeptimycin, an important cyclic peptide effective against Gram-positive infections. The genome of Streptomyces hygroscopicus NRRL 30439 was first sequenced using PacBio RSII to obtain a closed genome. A chemically defined medium was then used to elicit a nutrient stress response in S. hygroscopicus NRRL 30439. Detailed extracellular metabolomics and intracellular proteomics were used to profile and segregate primary and secondary metabolism. Our results demonstrate that the combination of genomics, proteomics and metabolomics enables rapid evaluation of a strain's performance in bioreactors for industrial production of secondary metabolites.

Assessment of the phylogenetic analysis and antimicrobial, antiviral, and anticancer activities of marine endophytic Streptomyces species of the soft coral Sarcophyton convolutum.

In the present work, the extensive biological activities of marine endophytic Streptomyces strains isolated from marine soft coral Sarcophyton convolutum have been demonstrated. Within fifty-one Streptomyces isolates evaluated for their hydrolytic enzymes and enzyme inhibitors productivities, six isolates showed the hyperactivities. Pharmaceutical metabolites productivities evaluated include enzymes (alkaline protease, L-asparaginase, L-glutaminase, tyrosinase, and L-methioninase) and enzyme inhibitors (inhibitors of α-amylase, hyaluronidase, ß-lactamase, α-glucosidase, and ß-glucosidase). These isolates were identified based on their morphological, biochemical, and genetic characteristics as Streptomyces sp. MORSY 17, Streptomyces sp. MORSY 22, Streptomyces sp. MORSY 25, Streptomyces sp. MORSY 36, Streptomyces sp. MORSY 45, and Streptomyces sp. MORSY 50. Moreover, in further evaluation, these strains exhibited wide spectrum of antimicrobial (against bacteria and fungi), antiviral (against hepatitis C virus), antibiofilm against biofilm-forming bacteria (methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas species), and anti-proliferative activities (against liver and colon carcinoma cell lines). The GC-MS analysis of the hyperactive strains MORSY 17 and MORSY 22 revealed the presence of different bioactive agents in the ethyl acetate extract of both strains.

Enhancement of undecylprodigiosin production from marine endophytic recombinant strain Streptomyces sp. ALAA-R20 through low-cost induction strategy.

Genetic manipulation of the undecylprodigiosin-producing strains and engineered culture medium approaches were applied as the most economical induction strategy for improving production. The hyper-producing recombinant strain ALAA-R20 was obtained after applying protoplast fusion strategy between the potent producer marine endophytic strains Streptomyces sp. ESRAA-10 (P1) and Streptomyces sp. ESRAA-31 (P2) of Dendronephthya hemprichi. Recombinant strain ALAA-R20 produced undecylprodigiosin yield higher than its parental strains ESRAA-10 and ESRAA-31 by 82.45% and 105.52% under submerged fermentation using modified R2YE medium. In order to reduce the costs of producing undecylprodigiosin, a solid-state fermentation (SSF) was applied. Scaled-up of optimized SSF parameters consisting of groundnut oil cake (GOC) sized to 3 mm, initial moisture content 80% with a mixture of dairy mill and fruit processing wastewaters (1:1), pH 7.0, inoculum size equal to 3 × 105 spores/g dry substrate (gds), incubation temperature 30 °C, and 7-day incubation period yielded the highest yield of 181.78 mg/gds of undecylprodigiosin by the recombinant strain Streptomyces sp. ALAA-R20. Extraction and purification of the pigment using the chromatographic techniques as well as mass spectral analysis exhibited maximum absorbance at 539 nm which is physiological property of the undecylprodigiosin. Undecylprodigiosin was stable over a wide temperature ranged from - 20 to 35 °C even after storage for 6 months. The maximum yield and stability of pigment was obtained at the acidic pH (acidified methanol, pH 4.0). Undecylprodigiosin obtained from the recombinant strain Streptomyces sp. ALAA-R20 demonstrated strong antimicrobial activity against all multidrug-resistant bacterial and fungal strains tested with minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations ranged between 0.5 and 4.0, 0.5 to 4.0, and 1.0 to 8.0 µg/mL, respectively. It also showed complete inhibition of cancer cells; HCT-116, HepG-2, MCF-7 and A-549 at 5, 8, 4, and 7 µM with IC50 equal to 2.0, 4.7, 1.2, and 2.8 µM, respectively.

Aqueous two-phase partitioning and characterization of xylanase produced by Streptomyces geysiriensis from low cost lignocellulosic substrates.

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100 rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0 at 40 °C. The scale-up was done based on the results of optimized parameters using 3 L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K2HPO4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5-8.5, with thermal stability from 20 °C to 60 °C up to 180 min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4 °C and room temperature (35 °C) for 30 and 9 days respectively. The kinetic parameters Km (0.48 mg/ml) and Vmax (8.33 U/mg) were determined using birchwood xylan as substrate.

Bioactivity Assessment of Indian Origin-Mangrove Actinobacteria against Candida albicans.

Actinobacteria is found to have a potent metabolic activity against pathogens. The present study reveals the assessment of potent antifungal secondary metabolites from actinobacteria isolated from Indian marine mangrove sediments. The samples were collected from the coastal regions of Muthupet, Andaman and the Nicobar Islands. Identification was carried out using 16S rRNA analysis and biosynthetic genes (Polyketide synthase type I/II and Non-ribosomal peptide synthase) were screened. Actinobacteria were assayed for their antifungal activity against 16 clinical Candida albicans and the compound analysis was performed using gas chromatography-mass spectrometry GC-MS. The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this ecosystem is rich on actinobacteria, with Streptomyces as the predominant genus. The PCR based screening of biosynthetic genes revealed the presence of PKS-I in six strains, PKS-II in four strains and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates) were found to have a potential antifungal activity against all the tested C. albicans. GC-MS results revealed that the actinobacterial compounds were belonging to heterocyclic, polyketides and peptides. Overall, the strains possess a wide spectrum of antifungal properties which affords the production of significant bioactive metabolites as potential antibiotics.

Biodiversity of Actinobacteria from the South Pacific and the Assessment of Streptomyces Chemical Diversity with Metabolic Profiling.

Recently, bioprospecting in underexplored habitats has gained enhanced focus, since new taxa of marine actinobacteria can be found, and thus possible new metabolites. Actinobacteria are in the foreground due to their versatile production of secondary metabolites that present various biological activities, such as antibacterials, antitumorals and antifungals. Chilean marine ecosystems remain largely unexplored and may represent an important source for the discovery of bioactive compounds. Various culture conditions to enrich the growth of this phylum were used and 232 bacterial strains were isolated. Comparative analysis of the 16S rRNA gene sequences led to identifying genetic affiliations of 32 genera, belonging to 20 families. This study shows a remarkable culturable diversity of actinobacteria, associated to marine environments along Chile. Furthermore, 30 streptomycete strains were studied to establish their antibacterial activities against five model strains, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa, demonstrating abilities to inhibit bacterial growth of Gram-positive bacteria. To gain insight into their metabolic profiles, crude extracts were submitted to liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis to assess the selection of streptomycete strains with potentials of producing novel bioactive metabolites. The combined approach allowed for the identification of three streptomycete strains to pursue further investigations. Our Chilean marine actinobacterial culture collection represents an important resource for the bioprospection of novel marine actinomycetes and its metabolites, evidencing their potential as producers of natural bioproducts.

Environmental monitoring and assessment of antibacterial metabolite producing actinobacteria screened from marine sediments in south coastal regions of Karnataka, India.

Assessment of the therapeutic potential of secondary metabolite producing microorganisms from the marine coastal areas imparts scope and application in the field of environmental monitoring. The present study aims to screen metabolites with antibacterial potential from actionbacteria associated with marine sediments collected from south coastal regions of Karnataka, India. The actinobacteria were isolated and characterized from marine sediments by standard protocol. The metabolites were extracted, and antibacterial potential was analyzed against eight hospital associated bacteria. The selected metabolites were partially characterized by proximate analysis, SDS-PAGE, and FTIR-spectroscopy. The antibiogram of the test clinical isolates revealed that they were emerged as multidrug-resistant strains (P ≤ 0.05). Among six actinobacteria (IS1-1S6) screened, 100 µl-1 metabolite from IS1 showed significant antibacterial activities against all the clinical isolates except Pseudomonas aeruginosa. IS2 demonstrated antimicrobial potential towards Proteus mirabilis, Streptococcus pyogenes, and Escherichia coli. The metabolite from IS3 showed activity against Strep. pyogenes and E. coli. The metabolites from IS4, IS5, and IS6 exhibited antimicrobial activities against Ps. aeruginosa (P ≤ 0.05). The two metabolites that depicted highest antibacterial activities against the test strains were suggested to be antimicrobial peptides with low molecular weight. These isolates were characterized and designated as Streptomyces sp. strain mangaluru01 and Streptomyces sp. mangaloreK01 by 16S ribosomal DNA (rDNA) sequencing. This study suggests that south coastal regions of Karnataka, India, are one of the richest sources of antibacterial metabolites producing actinobacteria and monitoring of these regions for therapeutic intervention plays profound role in healthcare management.

Automated identification of depsipeptide natural products by an informatic search algorithm.

Nonribosomal depsipeptides are a class of potent microbial natural products, which include several clinically approved pharmaceutical agents. Genome sequencing has revealed a large number of uninvestigated natural-product biosynthetic gene clusters. However, while novel informatic search methods to access these gene clusters have been developed to identify peptide natural products, depsipeptide detection has proven challenging. Herein, we present an improved version of our informatic search algorithm for natural products (iSNAP), which facilitates the detection of known and genetically predicted depsipeptides in complex microbial culture extracts. We validated this technology by identifying several depsipeptides from novel producers, and located a large number of novel depsipeptide gene clusters for future study. This approach highlights the value of chemoinformatic search methods for the discovery of genetically encoded metabolites by targeting specific areas of chemical space.

Assessment of resistomycin, as an anticancer compound isolated and characterized from Streptomyces aurantiacus AAA5.

A new actinomycete strain, isolated from humus soils in the Western Ghats, was found to be an efficient pigment producer. The strain, designated AAA5, was identified as a putative Streptomyces aurantiacus strain based on cultural properties, morphology, carbon source utilization, and analysis of the 16S rRNA gene. The strain produced a reddish-brown pigmented compound during the secondary metabolites phase. A yellow compound was derived from the extracted pigment and was identified as the quinone-related antibiotic resistomycin based on ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, liquid chromatography and mass spectroscopy, and nuclear magnetic resonance analyses. The AAA5 strain was found to produce large quantities of resistomycin (52.5 mg/L). It showed potent cytotoxic activity against cell lines viz. HepG2 (hepatic carcinoma) and HeLa (cervical carcinoma) in vitro, with growth inhibition (GI(50)) of 0.006 and 0.005 µg/ml, respectively. The strain also exhibited broad antimicrobial activities against both Gram-positive and Gram-negative bacteria. Therefore, AAA5 may have great potential as an industrial resistomycin-producing strain.

On-line identification of 4''-isovalerylspiramycin I in the genetic engineered strain of S. spiramyceticus F21 by liquid chromatography with electrospray ionization tandem mass spectrometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry.

LC-hyphenated techniques were applied to the on-line identification of isovalerylspiramycin I (isp I), a spiramycin-like macrolide in the crude extract of fermentation broth from a genetically engineered strain of S. spiramyceticus F21. In the structural characterization of the large molecular secondary metabolite of isp I, LC-DAD-UV-ESI-MS(n) analysis played a crucial role, and stop-flow LC-(1)H NMR measurement, with bitespiramycin used as reference, was a valuable complement approach. This rational approach proved to be an efficient means for the rapid and accurate structural determination of known microbial secondary metabolites, by which targeted isolation of component(s) of interest can be subsequently performed for further biological and pharmacological studies in drug development.

Disruption of cagA, the apoprotein gene of chromoprotein antibiotic C-1027, eliminates holo-antibiotic production, but not the cytotoxic chromophore.

C-1027 is a chromoprotein of the nine-membered enediyne antitumour antibiotic family, comprising apoprotein to stabilize and transport the enediyne chromophore. The disruption of apoprotein gene cagA within the C-1027 biosynthetic gene cluster abolished C-1027 holo-antibiotic production detected by an antibacterial assay, as well as the expression of the apoprotein and C-1027 chromophore extracted following protein precipitation of the culture supernatant. Complementation of the cagA-disrupted mutant AKO with the intact cagA gene restored C-1027 production, suggesting that cagA is indispensable for holo-antibiotic production. Overexpression of cagA in the wild-type strain resulted in a significant increase in C-1027 production as expected. Surprisingly, electrospray ionization (ESI)-MS and ESI-MS/MS analyses suggested that the AKO mutant still produced the C-1027 enediyne chromophore [m/z=844 (M+H)(+)] and its aromatized product [m/z=846 (M+H)(+)]. Consistent with this, the results from gene expression analysis using real-time reverse transcriptase-PCR showed that transcripts of the positive regulator sgcR3 and the structural genes sgcA1, sgcC4, sgcD6 and sgcE were readily detected in the AKO mutant as well as in the wild-type and the complementation strain. These results provided, for the first time, evidence suggesting that the apoprotein of C-1027 is not essential in the self-resistance mechanism for the enediyne chromophore.

Nutritional regulation of actinomycin-D production by a new isolate of Streptomyces sindenensis using statistical methods.

Production of actinomycin-D, by an isolate, S. sindenensis, was optimized by statistical methods. Fructose peptone and NaNO3 were found to be critical for antibiotic production. In the second step, their concentrations were optimized with Central Composite Design and Response Surface Methodology. Fructose, peptone and NaNO3 at 2.55, 0.309 and 0.114% respectively gave approximately 261% higher yield (289 mg/l). Cultivation in fermentor at 600 rpm agitation and 1.5 vvm aeration with optimized medium gave 3.56 folds higher yield (365 mg/l) as compared to the yields in shake flasks using normal production medium (80 mg/l).

Differential analysis of secondary metabolites by LC-MS following strain improvement of Streptomyces lydicus AS 4.2501.

Metabolite variations in a high-yielding mutant and its parent strain were studied by comparative LC-MS analysis after strain improvement. Streptomyces lydicus AS 4.2501-P28, a propionate-resistant mutant isolated by the high-frequency screening method using the principle of eliminating precursor inhibition effects, showed an increase of 267% in streptolydigin titre over the starting strain. Culture extracts of this mutant and its parent strain were analysed in parallel by an LC-MS technique, including full scan and extracted-ion scan, ESI-MS (electrospray-ionization MS) detection, DAD (diode-array detection) and MS2 (tandem MS) measurement. The main metabolic variations were obviously found in intermediates, metabolites and biosynthetic pathways: two unknown metabolites with the molecular [M-H]- ions at m/z 423.3 and 687.2, corresponding to two branch pathways, were blocked in the mutant, and the accumulation of a significant intermediate at m/z 363.1 [M-H]- decreased dramatically in the mutant cultures, resulting in the overproduction of streptolydigin (an antibiotic that inhibits prokaryotic RNA polymerase) in the mutant. Ion fragmentations of the tandem-MS spectra provided experimental evidence for the structural characterization of the three compounds obtained. In comparison with the traditional methods, comparative LC-MS analysis was rapid, sensitive and suitable for characterizing intermediates, metabolites and pathways for elucidation of the metabolic alterations after the isolation of improved strains.

Plasticity of the streptomyces genome-evolution and engineering of new antibiotics.

Streptomyces is a genus of soil dwelling bacteria with the ability to produce natural products that have found widespread use in medicine. Annotation of Streptomyces genome sequences has revealed far more biosynthetic gene clusters than previously imagined, offering exciting possibilities for future combinatorial biosynthesis. Experiments to manipulate modular biosynthetic clusters to create novel chemistries often result in no detectable product or product yield is extremely low. Understanding the coupling between components in these hybrid enzymes will be crucial for efficient synthesis of new compounds. We are using new algebraic approaches to predict protein properties, and homologous recombination to exploit natural evolutionary constraints to generate novel functional enzymes. The methods and techniques developed could easily be adapted to study modular, multi-interacting complex systems where appreciable biochemical and comparative sequence data are available, for example, clinically significant non-ribosomally synthesised peptides and polyketides.

Reverse engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays.

Transcript levels in production cultures of wildtype and classically improved strains of the actinomycete bacteria Saccharopolyspora erythraea and Streptomyces fradiae were monitored using microarrays of the sequenced actinomycete S. coelicolor. Sac. erythraea and S. fradiae synthesize the polyketide antibiotics erythromycin and tylosin, respectively, and the classically improved strains contain unknown overproduction mutations. The Sac. erythraea overproducer was found to express the entire 56-kb erythromycin gene cluster several days longer than the wildtype strain. In contrast, the S. fradiae wildtype and overproducer strains expressed the 85-kb tylosin biosynthetic gene cluster similarly, while they expressed several tens of other S. fradiae genes and S. coelicolor homologs differently, including the acyl-CoA dehydrogenase gene aco and the S. coelicolor isobutyryl-CoA mutase homolog icmA. These observations indicated that overproduction mechanisms in classically improved strains can affect both the timing and rate of antibiotic synthesis, and alter the regulation of antibiotic biosynthetic enzymes and enzymes involved in precursor metabolism.

Rational affinity purification of native Streptomyces family 10 xylanase.

Xylanase SoXyn10A from Streptomyces olivaceoviridis E-86 comprises a family 10 catalytic module linked to a family 13 carbohydrate-binding module (SoCBM13). The SoCBM13 has a beta-trefoil structure, with binding sites in each subdomain (alpha, beta and gamma). Subdomain alpha, but not subdomains beta and gamma, binds tightly to lactose. It was, therefore, thought that immobilized lactose could be used for the affinity purification of SoXyn10A. Lactosyl-Sepharose was prepared and tested as an affinity matrix. SoXyn10A produced from the cloned xyn10A gene by Escherichia coli, and native SoXyn10A in culture supernatants from S. olivaceoviridis, were purified to homogeneity in a single step by affinity chromatography using this matrix. This simple purification of SoXyn10A makes the enzyme an attractive candidate for applications requiring xylanase. The CBM also has the potential for use as an affinity tag for the purification of other proteins.

Streptomyces serine protease (DHP-A) as a new biocatalyst capable of forming chiral intermediates of 1,4-dihydropyridine calcium antagonists.

Streptomyces viridosporus A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4R) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized. Cloning and sequence analysis of the gene for DHP-A (dhpA) revealed that the enzyme was a serine protease that is highly similar in both structural and enzymatic feature to SAM-P45, which is known as a target enzyme of Streptomyces subtilisin inhibitor (SSI), from Streptomyces albogriseolus. In a batch reaction test, DHP-A produced a higher yield of a chiral intermediate of 1,4-dihydropyridine than the commercially available protease P6. Homologous or heterologous expression of dhpA resulted in overproduction of the enzyme in culture supernatants, with 2.4- to 4.2-fold higher specific activities than in the parent S. viridosporus A-914. This indicates that DHP-A is suitable for use in reactions forming chiral intermediates of calcium antagonists and suggests the feasibility of developing DHP-A as a new commercial enzyme for use in the chiral drug industry.

Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering.

Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.