跳转至内容
Merck
CN
  • Combinatorialization of fungal polyketide synthase-peptide synthetase hybrid proteins.

Combinatorialization of fungal polyketide synthase-peptide synthetase hybrid proteins.

Journal of the American Chemical Society (2014-12-02)
Thomas B Kakule, Zhenjian Lin, Eric W Schmidt
摘要

The programming of the fungal polyketide synthase (PKS) is quite complex, with a simple domain architecture leading to elaborate products. An additional level of complexity has been found within PKS-based pathways where the PKS is fused to a single module nonribosomal peptide synthetase (NRPS) to synthesize polyketides conjugated to amino acids. Here, we sought to understand the communication between these modules that enable correct formation of polyketide-peptide hybrid products. To do so, we fused together the genes that are responsible for forming five highly chemically diverse fungal natural products in a total of 57 different combinations, comprising 34 distinct module swaps. Gene fusions were formed with the idea of testing the connection and compatibility of the PKS and NRPS modules mediated by the acyl carrier protein (ACP), condensation (C) and ketoreductase (KR) domains. The resulting recombinant gene fusions were analyzed in a high-yielding expression platform to avail six new compounds, including the first successful fusion between a PKS and NRPS that make highly divergent products, and four previously reported molecules. Our results show that C domains are highly selective for a subset of substrates. We discovered that within the highly reducing (hr) PKS class, noncognate ACPs of closely related members complement PKS function. We intercepted a pre-Diels-Alder intermediate in lovastatin synthesis for the first time, shedding light on this canonical fungal biochemical reaction. The results of these experiments provide a set of ground rules for the successful engineering of hr-PKS and PKS-NRPS products in fungi.

材料
货号
品牌
产品描述

Sigma-Aldrich
吡啶, ACS reagent, ≥99.0%
Sigma-Aldrich
吡啶, suitable for HPLC, ≥99.9%
Sigma-Aldrich
吡啶, ReagentPlus®, ≥99%
Sigma-Aldrich
乙酸酐, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99% (GC)
Sigma-Aldrich
吡啶, anhydrous, 99.8%
Sigma-Aldrich
乙酸酐, ReagentPlus®, ≥99%
Sigma-Aldrich
乙酸酐, ACS reagent, ≥98.0%
Sigma-Aldrich
吡啶, ACS reagent, ≥99.0%
Sigma-Aldrich
吡啶, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99.5% (GC)
Sigma-Aldrich
吡啶, ≥99%
Supelco
N -甲基-双(三氟乙酰胺), for GC derivatization, LiChropur, ≥97.0% (GC)
Sigma-Aldrich
吡啶, biotech. grade, ≥99.9%
Sigma-Aldrich
吡啶, ReagentPlus®, ≥99%
Supelco
吡啶, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
吡啶, analytical standard
Supelco
乙酸酐, for GC derivatization, LiChropur, ≥99.0%
Supelco
N -甲基-双(三氟乙酰胺), for GC derivatization, LiChropur, ≥99.0% (GC)