Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering

Milling and bread baking techniques significantly affect the mycotoxin (deoxynivalenol and nivalenol) level in bread

The influence of three milling techniques (MT1: industrial roller-grinder, MT2: grain hammer crasher, and MT3: traditional millstone) and two baking methods (BM1: industrial oven, BM2: traditional ceramic stove heated by wood (log fire oven)) on mycotoxin deoxynivalenol (DON) and nivalenol (NIV) levels in bread were investigated. The DON and NIV concentrations in wheat grain, flour, and bread were analysed using high performance liquid chromatography with UV-detection methods. The 2 500 kg lot of wheat grain containing 1 400–1 900 μg kg−1 deoxynivalenol and 130–200 μg kg−1 nivalenol was divided into sub-lots which were processed to get three types of flour (F1: industrial bread flour, F2: industrial wholegrain flour and F3: traditional wholegrain flour). The concentrations of DON and NIV measured after milling the grain according to MT1 (yielding F1) amounted to 310–370 \g kg−1 and <50–70 μg kgt1, respectively. After applying MT2 to the grain (yielding F2), the DON and NIV levels were measured to be 1 060–1 400 μg kg−1 and 60–87 μg kg−1, respectively. Applying MT3 (yielding F3) produced a DON level of 1 100–1 770 μg kg−1 and a NIV level of 80–95 μg kg−1. Six types of bread were baked from the three types of flour according to BM1 or BM2, and the mycotoxin levels were analysed. The average reduction in DON concentration after baking (70 min at 195–235 °C) was 47.2% for bread baked in the industrial oven and 48.7% for bread baked in the log fire oven. Concentrations of DON in bread prepared by the industrial MT1 were under the permitted limit of 500 μg kg−1 stated in EC (2006) regulation, despite the fact that the bread was baked from grains highly contaminated with mycotoxins. In the bread baked from traditional wholegrain flour, mycotoxin concentrations were higher (850–950 μg kg−1)

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea : a platform for producing novel tetracycline antibiotics

Tetracyclines (TCs) are medically important antibiotics from the polyketide family of natural products. Chelocardin (CHD), produced by Amycolatopsis sulphurea, is a broad-spectrum tetracyclic antibiotic with potent bacteriolytic activity against a number of Gram-positive and Gram-negative multi-resistant pathogens. CHD has an unknown mode of action that is different from TCs. It has some structural features that define it as 'atypical' and, notably, is active against tetracycline-resistant pathogens. Identification and characterization of the chelocardin biosynthetic gene cluster from A. sulphurea revealed 18 putative open reading frames including a type II polyketide synthase. Compared to typical TCs, the chd cluster contains a number of features that relate to its classification as 'atypical': an additional gene for a putative two-component cyclase/aromatase that may be responsible for the different aromatization pattern, a gene for a putative aminotransferase for C-4 with the opposite stereochemistry to TCs and a gene for a putative C-9 methylase that is a unique feature of this biosynthetic cluster within the TCs. Collectively, these enzymes deliver a molecule with different aromatization of ring C that results in an unusual planar structure of the TC backbone. This is a likely contributor to its different mode of action. In addition CHD biosynthesis is primed with acetate, unlike the TCs, which are primed with malonamate, and offers a biosynthetic engineering platform that represents a unique opportunity for efficient generation of novel tetracyclic backbones using combinatorial biosynthesis