Characterization of the Ohmyungsamycin Biosynthetic Pathway and Generation of Derivatives with Improved Antituberculosis Activity.

The cyclic depsipeptides ohmyungsamycin (OMS) A (1) and B (2), isolated from the marine-derived Streptomyces sp. SNJ042, contain two non-proteinogenic amino acid residues, ß-hydroxy-l-phenylalanine (ß-hydroxy-l-Phe) and 4-methoxy-l-tryptophan (4-methoxy-l-Trp). Draft genome sequencing of Streptomyces sp. SNJ042 revealed the OMS biosynthetic gene cluster consisting of a nonribosomal peptide synthetase (NRPS) gene and three genes for amino acid modification. By gene inactivation and analysis of the accumulated products, we found that OhmL, encoding a P450 gene, is an l-Phe ß-hydroxylase. Furthermore, OhmK, encoding a Trp 2,3-dioxygenase homolog, and OhmJ, encoding an O-methyltransferase, are suggested to be involved in hydroxylation and O-methylation reactions, respectively, in the biosynthesis of 4-methoxy-l-Trp. In addition, the antiproliferative and antituberculosis activities of the OMS derivatives dehydroxy-OMS A (4) and demethoxy-OMS A (6) obtained from the mutant strains were evaluated in vitro. Interestingly, dehydroxy-OMS A (4) displayed significantly improved antituberculosis activity and decreased cytotoxicity compared to wild-type OMS A.

High-yield production of multiple O-methylated phenylpropanoids by the engineered Escherichia coli-Streptomyces cocultivation system.

BACKGROUND: O-Methylated phenylpropanoids, which are generally present in small amounts in plants, have improved or distinct biological activities and pharmacological properties as opposed to their unmethylated counterparts. Although microbial production could be a useful tool for the efficient and environment-friendly production of methylated phenylpropanoids, a high-yield microbial production of neither tri-methylated stilbenes nor di-/tri-methylated flavonoids has been achieved to date. RESULTS: A methyltransferase from Streptomyces avermitilis (SaOMT2), which has been known to possess 7-O-methylation activity toward several flavonoids, exhibited more diverse regiospecificity and catalyzed mono-, di-, and tri-methylation of stilbene, flavanone, and flavone when it was expressed in Streptomyces venezuelae. For the efficient production of multi-methylated phenylpropanoids, a cocultivation system was developed by employing engineered Escherichia coli strains producing pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant. Consequently, high-yield microbial production of tri-methylated stilbenes and di-/tri-methylated flavonoids (including 3,5,4'-trimethoxystilbene, 5-hydroxy-7,4'-dimethoxyflavanone, 4'-hydroxy-5,7-dimethoxyflavanone, 5,7,4'-trimethoxyflavanone, 5-hydroxy-7,4'-dimethoxyflavone, and 5,7,4'-trimethoxyflavone) has been demonstrated for the first time. CONCLUSIONS: This cocultivation system based on the phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae provides an efficient tool for producing scarce and potentially valuable multi-methylated phenylpropanoids and will enable further development of these compounds as pharmaceuticals and nutraceuticals.

The N3 subdomain in a domain of fibronectin-binding protein B isotype I is an independent risk determinant predictive for biofilm formation of Staphylococcus aureus clinical isolates.

Fibronectin-binding proteins (FnBP), FnBPA and FnBPB, are purported to be involved in biofilm formation of Staphylococcus aureus. This study was performed to find which of three consecutive N subdomains of the A domain in the FnBP is the key domain in FnBP. A total of 465 clinical isolates of S. aureus were examined for the biofilm forming capacity and the presence of N subdomains of FnBP. In the biofilm-positive strains, N2 and N3 subdomains of FnBPA, and N1 and N3 subdomains of FnBPB were significantly more prevalent. Multivariate logistic regression analysis of 246 biofilm-positive and 123 biofilm-negative strains identified only the FnBPB-N3 subdomain as an independent risk determinant predictive for biofilm-positive strains of S. aureus (Odds ratio [OR], 13.174; P<0.001). We also attempted to delete each of the fnbA-N2 and -N3 and fnbB-N1 and -N3 from S. aureus strain 8325-4 and examined the biofilm forming capacity in the derivative mutants. In agreement with the results of the multivariate regression analysis, deletion of either the fnbA-N2 or -N3, or fnbB-N1 did not significantly diminish the capacity of strain 8325-4 to develop a biofilm, while deletion of the fnbB-N3 did. Therefore, it is suggested that the FnBPB-N3 subdomain of isotype I may be a key domain in FnBP which is responsible for the causing biofilm formation in S. aureus clinical isolates.

Effect of gene amplifications in porphyrin pathway on heme biosynthesis in a recombinant Escherichia coli.

A recombinant E. coli co-expressing ALA synthase (hemA), NADP-dependent malic enzyme (maeB), and dicarboxylic acid transporter (dctA) was reported to synthesize porphyrin derivatives including iron-containing heme. To enhance the synthesis of bacterial heme, five genes of the porphyrin biosynthetic pathway [pantothenate kinase (coaA), ALA dehydratase (hemB), 1-hydroxymethylbilane synthase (hemC), uroporphyrinogen III synthase (hemD), and uroporphyrinogen III decarboxylase (hemE)] were amplified in the recombinant E. coli co-expressing hemA-maeB-dctA. Pantothenate kinase expression enabled the recombinant E. coli to accumulate intracellular CoA. Intracellular ALA was the most enhanced by uroporphyrinogen III synthase expression, porphobilinogen by ALA dehydratase expression, and uroporphyrin and coproporphyrin by 1- hydroxymethylbilane synthase expression. The strain coexpressing coaA, hemA, maeB, and dctA produced heme of 0.49 micromol/g-DCW, which was twice as much from the strain without coaA expression. Further strain improvement for the porphyrin derivatives is discussed based on the results.

Porphyrin derivatives from a recombinant Escherichia coli grown on chemically defined medium.

We have reported previously that a recombinant Escherichia coli co-expresses aminolevulinic acid (ALA) synthase, an NADP-dependent malic enzyme, and a dicarboxylate transporter-produced heme, an iron-chelated porphyrin, in a succinate-containing complex medium. To develop an industrially plausible process, a chemically defined medium was formulated based on M9 minimal medium. Heme synthesis was enhanced by adding sodium bicarbonate, which strengthened the C4 metabolism required for the precursor metabolite, although a pH change discouraged cell growth. Increasing the medium pH buffering capacity (100mM phosphate buffer) and adding sodium bicarbonate enabled the recombinant E. coli to produce heme at rates 60% greater than those in M9 minimal medium. Adding growth factors (1 mg/l thiamin, 0.01 mg/l biotin, 5 mg/l nicotinic acid, 1 mg/l pantothenic acid, and 1.4 mg/l cobalamin) also induced positive heme production effects at levels twice of heme production in M9-based medium. Porphyrin derivatives and heme were found in the chemically defined medium, and their presence was confirmed by liquid chromatography/mass spectroscopy (LC/MS). The formulated medium allowed for the production of 0.6 microM heme, 29 microM ALA, 0.07 microM coproporphyrin I, 0.21 microM coproporphyrin III, and 0.23 microM uroporphyrin in a 3 L pH-controlled culture.

Higher biofilm formation in multidrug-resistant clinical isolates of Staphylococcus aureus.

The biofilm-forming capacity of Staphylococcus aureus contributes to antibiotic resistance, but whether antibiotic-resistant strains have the capacity to form biofilms has not yet been determined. Therefore, we recovered 101 clinical isolates of S. aureus and performed antibiotic susceptibility testing for 30 antibiotics using a VITEK II automatic system. We then carried out a biofilm assay on 96-well polystyrene plates. In addition, the presence of IS256 involved in the variation of biofilm phases of S. aureus was determined by polymerase chain reaction. The prevalence of IS256 was significantly related to multidrug resistance as well as biofilm expression, with biofilm positivity in 27 (39.7%) of the 68 IS256-positive strains and 3 (9.1%) of the 33 IS256-negative strains. In our analysis of the relationship between meticillin resistance and biofilm formation, we found that the rate of biofilm positivity was 37.9% (25/66) for meticillin-resistant strains and 14.3% (5/35) for meticillin-susceptible strains (P<0.05). Staphylococcal cassette chromosome mec (SCCmec) typing found that SCCmec type IV was most prevalent, comprising 14 (56.0%) of the 25 biofilm-positive, meticillin-resistant strains. A statistical analysis testing the relationship between multidrug resistance and biofilm formation revealed a significantly higher rate of biofilm development in strains with greater multiresistance compared with strains with less multiresistance. Our results suggest that the multidrug-resistant clinical isolates of S. aureus have a greater likelihood of developing biofilms on medical devices.