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Author

Peter Mrak

Other affiliations: Novartis
Bio: Peter Mrak is an academic researcher from University of Ljubljana. The author has contributed to research in topics: Streptomyces tsukubaensis & Nonribosomal peptide. The author has an hindex of 6, co-authored 18 publications receiving 191 citations. Previous affiliations of Peter Mrak include Novartis.

Papers
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Journal ArticleDOI
TL;DR: A biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex is proposed.

74 citations

Journal ArticleDOI
TL;DR: Using a combination of metabolic engineering and chemobiosynthetic approach, the exclusive production of FK506 is achieved, representing a significant step towards development of an advanced industrial bioprocess.

39 citations

Journal ArticleDOI
07 May 2013-PLOS ONE
TL;DR: To the authors' knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme.
Abstract: Employing DERA (2-deoxyribose-5-phosphate aldolase), we developed the first whole-cell biotransformation process for production of chiral lactol intermediates useful for synthesis of optically pure super-statins such as rosuvastatin and pitavastatin. Herein, we report the development of a fed-batch, high-density fermentation with Escherichia coli BL21 (DE3) overexpressing the native E. coli deoC gene. High activity of this biomass allows direct utilization of the fermentation broth as a whole-cell DERA biocatalyst. We further show a highly productive bioconversion processes with this biocatalyst for conversion of 2-substituted acetaldehydes to the corresponding lactols. The process is evaluated in detail for conversion of acetyloxy-acetaldehyde with the first insight into the dynamics of reaction intermediates, side products and enzyme activity, allowing optimization of the feeding strategy of the aldehyde substrates for improved productivities, yields and purities. The resulting process for production of ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate (acetyloxymethylene-lactol) has a volumetric productivity exceeding 40 g L−1 h−1 (up to 50 g L−1 h−1) with >80% yield and >80% chromatographic purity with titers reaching 100 g L−1. Stereochemical selectivity of DERA allows excellent enantiomeric purities (ee >99.9%), which were demonstrated on downstream advanced intermediates. The presented process is highly cost effective and environmentally friendly. To our knowledge, this is the first asymmetric aldol condensation process achieved with whole-cell DERA catalysis and it simplifies and extends previously developed DERA-catalyzed approaches based on the isolated enzyme. Finally, applicability of the presented process is demonstrated by efficient preparation of a key lactol precursor, which fits directly into the lactone pathway to optically pure super-statins.

39 citations

Journal ArticleDOI
TL;DR: The combined overexpression of the endogenous DERA and the membrane-bound, PQQ-dependent glucose dehydrogenase, the latter being coupled to the respiratory chain, allows direct biosynthesis of 6-substituted lactones in a highly productive, high-yield, cost-effective and industrially scalable process.

19 citations

Journal ArticleDOI
TL;DR: A stepwise biosynthesis of APL is proposed, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into an NP, and a strong synergism of these compounds in antifungal assays is confirmed.

11 citations


Cited by
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Journal ArticleDOI
TL;DR: This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK505 analogues.
Abstract: The allyl moiety of the immunosuppressive agent FK506 is structurally unique among polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical, and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multistep enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogues, 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the c...

152 citations

Journal ArticleDOI
TL;DR: This review covers the emerging biosynthetic role of crotonyl-CoA carboxylase/reductase (CCR) homologs in extending the structural and functional diversity of polyketide natural products.

125 citations

Journal ArticleDOI
TL;DR: This new metabolite, produced by a marine sediment-derived bacterium of the genus Streptomyces, possesses a novel spiro γ-lactam moiety and a distinctive isobutyryl polyketide fragment observed for the first time in this class of natural products.
Abstract: Reported is the structure and biosynthesis of ansalactam A, an ansamycin class polyketide produced by an unusual modification of the polyketide pathway This new metabolite, produced by a marine sediment-derived bacterium of the genus Streptomyces, possesses a novel spiro γ-lactam moiety and a distinctive isobutyryl polyketide fragment observed for the first time in this class of natural products The structure of ansalactam A was defined by spectroscopic methods including X-ray crystallographic analysis Biosynthetic studies with stable isotopes further led to the discovery of a new, branched chain polyketide synthase extender unit derived from (E)-4-methyl-2-pentenoic acid for polyketide assembly observed for the first time in this class of natural products

88 citations

Journal ArticleDOI
TL;DR: An overview of the origins of antibiotic resistance is provided to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms.

84 citations

Journal ArticleDOI
11 Jul 2013-PLOS ONE
TL;DR: The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.
Abstract: We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L-1 to 5.5 mg L-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

76 citations