Analysis of the genome sequence of Streptomyces sp. KCB13F003 showed the presence of a cryptic gene cluster encoding flavin-dependent halogenase and nonribosomal peptide synthetase. Pleiotropic approaches using multiple culture media followed by LC-MS-guided isolation and spectroscopic analysis enabled the identification of two new chlorinated cyclic hexapeptides, ulleungmycins A and B (1 and 2). Their structures, including absolute configurations, were determined by 1D and 2D NMR techniques, advanced Marfey's analysis, and GITC derivatization. The new peptides, featuring unusual amino acids 5-chloro-l-tryptophan and d-homoleucine, exhibited moderate antibacterial activities against Gram-positive pathogenic bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus.
BackgroundPterostilbene, a structural analog of resveratrol, has higher oral bioavailability and bioactivity than that of the parent compound; but is far less abundant in natural sources. Thus, to efficiently obtain this bioactive resveratrol analog, it is necessary to develop new bioproduction systems.ResultsWe identified a resveratrol O-methyltransferase (ROMT) function from a multifunctional caffeic acid O-methyltransferase (COMT) originating from Arabidopsis, which catalyzes the transfer of a methyl group to resveratrol resulting in pterostilbene production. In addition, we constructed a biological platform to produce pterostilbene with this ROMT gene. Pterostilbene can be synthesized from intracellular l-tyrosine, which requires the activities of four enzymes: tyrosine ammonia lyase (TAL), p-coumarate:CoA ligase (CCL), stilbene synthase (STS) and resveratrol O-methyltransferase (ROMT). For the efficient production of pterostilbene in E. coli, we used an engineered E. coli strain to increase the intracellular pool of l-tyrosine, which is the initial precursor of pterostilbene. Next, we tried to produce pterostilbene in the engineered E. coli strain using l-methionine containing media, which is used to increase the intracellular pool of S-adenosyl-l-methionine (SAM). According to this result, pterostilbene production as high as 33.6 ± 4.1 mg/L was achieved, which was about 3.6-fold higher compared with that in the parental E. coli strain harboring a plasmid for pterostilbene biosynthesis.ConclusionAs a potential phytonutrient, pterostilbene was successfully produced in E. coli from a glucose medium using a single vector system, and its production titer was also significantly increased using a l-methionine containing medium in combination with a strain that had an engineered metabolic pathway for l-tyrosine. Additionally, we provide insights into the dual functions of COMT from A. thaliana which was characterized as a ROMT enzyme.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0644-6) contains supplementary material, which is available to authorized users.
One of the optimization strategies of an artificial biosynthetic metabolic flux with a multienzyme pathway is when the enzyme concentrations are present at the appropriate ratios rather than at their maximum expression. Thus, many recent research efforts have focused on the development of tools that fine-tune the enzyme expression, and these research efforts have facilitated the search for the optimum balance between pathway expression and cell viability. However, the rational approach has some limitations in finding the most optimized expression ratio in in vivo systems. In our study, we focused on fine-tuning the expression level of a six-enzyme reaction for the artificial biosynthesis of curcumin by screening a library of 5'-untranslational region (UTR) sequence mutants made by a multiplex automatic genome engineering (MAGE) tool. From the screening results, a variant (6M08rv) showed about a 38.2-fold improvement in the production of curcumin compared to the parent strain, in which the calculated expression levels of 4-coumarate:CoA ligase (4CL) and phenyldiketide-CoA synthase (DCS), two of the six enzymes, were much lower than those of the parent strain.
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