An EntD-like phosphopantetheinyl transferase gene from Photobacterium profundum SS9 complements pfa genes of Moritella marina strain MP-1 involved in biosynthesis of docosahexaenoic acid

The EntD-like phosphopantetheinyl transferase (PPTase) gene, cloned from the eicosapentaenoic acid-producing bacterium Photobacterium profundum strain SS9, has an ORF of 690 bp encoding a 230-amino acid protein. When this PPTase gene was expressed in Escherichia coli with pfaA, pfaB, pfaC and pfaD derived from Moritella marina MP-1, which were four of five essential genes for biosynthesis of docosahexaenoic acid (DHA), the DHA production of the recombinant was 2% (w/w) of total fatty acids. This is the first report showing that the EntD-like PPTase is involved in producing n-3 polyunsaturated fatty acids. Shinji Sugihara and Yoshitake Orikasa contributed equally to this work.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Cloning and sequencing of clustered genes involved in fatty acid biosynthesis from the docosahexaenoic acid-producing bacterium, Vibrio marinus strain MP-1

A cluster of genes involved in fatty acid biosynthesis (fab) was isolated from docosahexaenoic acid (DHA)-producing Vibrio marinus strain MP-1. This fab gene cluster included five genes highly homologous to the Escherichia coli counterparts, and their order in the cluster was the same with that of the E. coli fab gene cluster except that the latter included the additional fabH gene. These fab genes should be involved in early steps of DHA biosynthesis in V. marinus strain MP-1.     

Recombinant production of omega‐3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1

Aim: To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost‐effective, sustainable and convenient alternative sources for fish oils. Methods and Results: A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography‐mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes. Conclusions: The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega‐3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli. Significance and Impact of the Study: These findings have implications for any future use of the EPA and DHA gene cluster in other micro‐organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro‐organism has great potential to add economic value to a variety of industrial and agricultural products.     

A cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2)

The chromosome of Streptomyces coelicolor A3(2), a model organism for the genus Streptomyces, contains a cryptic type I polyketide synthase (PKS) gene cluster which was revealed when the genome was sequenced. The ca. 54-kb cluster contains three large genes, cpkA, cpkB and cpkC, encoding the PKS subunits. In silico analysis showed that the synthase consists of a loading module, five extension modules and a unique reductase as a terminal domain instead of a typical thioesterase. All acyltransferase domains are specific for a malonyl extender, and have a B-type ketoreductase. Tailoring and regulatory genes were also identified within the gene cluster. Surprisingly, some genes show high similarity to primary metabolite genes not commonly identified in any antibiotic biosynthesis cluster. Using western blot analysis with a PKS subunit (CpkC) antibody, CpkC was shown to be expressed in S. coelicolor at transition phase. Disruption of cpkC gave no obvious phenotype.     

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.     

Heterologous expression of the naphthocyclinone hydroxylase gene from Streptomyces arenae for production of novel hybrid polyketides

Streptomyces arenae produces at least four different isochromanequinone antibiotics, the naphthocyclinones, of which the - and -form are active against Gram-positive bacteria. The naphthocyclinone biosynthesis gene cluster was isolated from Streptomyces arenae DSM 40737 and by sequence analysis the minimal polyketide synthase genes and several genes encoding tailoring enzymes were identified. Southern blot analysis of the naphthocyclinone gene cluster indicated that a 3.5 kb BamHI fragment located approximately 9 kb downstream of the minimal PKS genes hybridizes to the schC hydroxylase DNA probe isolated from S. halstedii. Two complete and one incomplete open reading frames were identified on this fragment. Sequence analysis revealed strong homology to the genes of the actVA region of S. coelicolor, to several (suggested) hydroxylases and a putative FMN-dependent monooxygenase. The proposed hydroxylase, encoded by ncnH, could hydroxylate aloesaponarin II, a molecule that is produced by the actinorhodin minimal polyketide synthase in combination with the actinorhodin ketoreductase, aromatase and cyclase. Furthermore, this enzyme is capable of accepting additional polyketide core structures that contain a 5-hydroxy-1,4-naphthoquinone moiety as substrates which makes it an interesting tailoring enzyme for the modification of polyketide structures.     

Identification of a gene cluster encoding meilingmycin biosynthesis among multiple polyketide synthase contigs isolated from<Emphasis Type="Italic"> Streptomyces nanchangensis</Emphasis> NS3226

A cluster encoding genes for the biosynthesis of meilingmycin, a macrolide antibiotic structurally similar to avermectin and milbemycin 11, was identified among seven uncharacterized polyketide synthase gene clusters isolated from Streptomyces nanchangensis NS3226 by hybridization with PCR products using primers derived from the sequences of aveE, aveF and a thioesterase domain of the avermectin biosynthetic gene cluster. Introduction of a 24.1-kb deletion by targeted gene replacement resulted in a loss of meilingmycin production, confirming that the gene cluster encodes biosynthesis of this important anthelminthic antibiotic compound. A sequenced 8.6-kb fragment had aveC and aveE homologues (meiC and meiE) linked together, as in the avermectin gene cluster, but the arrangement of aveF (meiF) and the thioesterase homologues differed. The results should pave the way to producing novel insecticidal compounds by generating hybrids between the two pathways.     

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter P_kasO* or P_neo. The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

Characterization of a regulatory gene, <Emphasis Type="Italic">aveR</Emphasis>, for the biosynthesis of avermectin in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Avermectin is an important macrocyclic polyketide produced by Streptomyces avermitilis and widely used as an anthelmintic agent in the medical, veterinary, and agricultural fields. The avermectin biosynthetic gene cluster contains aveR, which belongs to the LAL-family of regulatory genes. In this study, aveR was inactivated by gene replacement in the chromosome of S. avermitilis, resulting in the complete loss of avermectin production. The aveR mutant was unable to convert an avermectin intermediate to any avermectin derivatives, and complementation by intact aveR and its proper upstream region restored avermectin production in the mutant, suggesting that AveR is a positive regulator controlling the expression of both polyketide biosynthetic genes and postpolyketide modification genes in avermectin biosynthesis. Despite the general concept that an increased amount of a positive pathway-specific regulator leads to higher production, a higher amount of aveR resulted in complete loss of avermectin, indicating that there is a maximum threshold concentration of aveR for the production of avermectin.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Growth condition optimization for docosahexaenoic acid (DHA) production by Moritella marina MP-1

The marine organism Moritella marina MP-1 produces the polyunsaturated fatty acid docosahexaenoic acid (DHA). While the basic metabolic pathway for DHA production in this organism has been identified, the impact of growth conditions on DHA production is largely unknown. This study examines the effect of supplemental carbon, nitrogen and salts, growth temperature and media composition and pH on DHA and biomass production and the fatty acid profile. The addition of supplemental nitrogen significantly increased the overall DHA titer via an increase in biomass production. Supplemental glucose or glycerol increased biomass production, but decreased the amount of DHA per biomass, resulting in no net change in the DHA titer. Acidification of the baseline media pH to 6.0 increased DHA per biomass. Changes in growth temperature or provision of supplemental sodium or magnesium chloride did not increase DHA titer. This organism was also shown to grow on defined minimal media. For both media types, glycerol enabled more DHA production per biomass than glucose. Combination of these growth findings into marine broth supplemented with glycerol, yeast extract, and tryptone at pH?6.0 resulted in a final titer of 82?±?5 mg/L, a nearly eightfold increase relative to the titer of 11?±?1 mg/L seen in the unsupplemented marine broth. The relative distribution of other fatty acids was relatively robust to growth condition, but the presence of glycerol resulted in a significant increase in myristic acid (C14:0) and decrease in palmitic acid (C16:0). In summary, DHA production by M. marina MP-1 can be increased more than fivefold by changing the growth media. Metabolic engineering of this organism to increase the amount of DHA produced per biomass could result in additional increases in titer.     

Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis

Arachidonic acid (ARA) is a polyunsaturated fatty acid (PUFA) and an essential component of membrane lipids. However, the PUFA synthase required for ARA biosynthesis has not been identified in any organism. To identify the PUFA synthase producing ARA, we determined the draft genome sequence of the marine bacterium Aureispira marina, which produces a high level of ARA, and found a gene cluster encoding a putative PUFA synthase for ARA production. Expression of the gene cluster in Escherichia coli induced production of ARA, demonstrating that the gene cluster encodes a PUFA synthase required for ARA biosynthesis.     

Algicide production by the filamentous cyanobacteriumFischerellasp. CENA 19

The biosynthesis of algicides produced by a novelFischerellastrain was investigated. Two allelochemicals were identified, the aminoacylpolyketide fischerellin A (FsA) and the alkaloid 12-epi-hapalindole F (HapF). Based on the structure of FsA, genes that could be involved in its biosynthesis, including those encoding nonribosomal peptide synthetases (NRPSs) and a polyketide synthase (PKS), were identified by the polymerase chain reaction (PCR). By showing that the expression of NRPSs and PKSs is concomitant with algicide production we suggest that the identified genes may be involved in algicide biosynthesis. Analysis of an algicide preparation of the Brazilian-Amazonian strainFischerellasp. CENA 19 revealed the production of FsA,m/z409 (MH⁺), HapF,m/z370 (MH⁺), and other potential isoforms of the latter compounds, which were identified by high-performance liquid chromatography (HPLC) and matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass-spectrometry. The production of HapF was confirmed after purification by HPLC, analysis by NMR, and high-resolution mass-spectrometry (HRMS). Two-NRPS and a PKS gene were identified after specific amplification using a degenerate PCR. The expression of these synthetases was confirmed by Western blot analysis employing enzyme family-specific antibodies. These analyses revealed the presence of three NRPSs and a single PKS inFischerellasp. CENA 19. The structure of FsA indicates both aminoacyl- and polyketide moeities, suggesting that its biosynthesis may require an integrated NRPS/PKS enzyme system, possibly involving the genes and the synthetases identified.     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046

Saprolmycins A–E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography–mass spectrometry analysis of the extract.     

Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes

Summary There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning theactI, actIII, actIV, andactVII genes (pANT12) of actinorhodin biosynthesis pathway fromStreptomyces coelicolor in anthracycline producing streptomycetes.Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kbXhoI fragment containing only theactI andactVII loci was necessary for aloesaponarin II biosynthesis byS. galilaeus 31 133. WhenS. galilaeus 31 671 was transformed with theactI, actVII, andactIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-0-methyldesoxyerythrolaccin. WhenS. galilaeus 31671 was transformed with only the intactactIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for theactIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci,dauG, dnrR1, andasaA, on the production of natural and hybrid polyketides were also shown.