Functional analysis of Hypoxylon pulicicidum genes required for heterologous biosynthesis of nodulisporic acids

<p>Nature holds some of the greatest secrets in drug design and development and the ability to access these trade secrets has been revolutionised by modern bioengineering technologies. In order to exploit these technologies it is essential to understand what genes are involved in compound production and the enzymatic steps that limit flux to the desired product. This thesis describes the discovery of four secondary-metabolic enzymatic steps involved in the biosynthesis of a group of valuable natural products known as nodulisporic acids. Nodulisporic acids are known for their potent insecticidal activities; however, biosynthesis of these compounds by the natural fungal producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult and has prevented the commercial development of novel nodulisporic acid-containing veterinary medicines and crop protects. To discover how nodulisporic acids are biosynthesized: 1. the H. pulicicidum genome was sequenced 2. a gene cluster responsible for nodulisporic acid production was predicted 3. genes in the cluster were functionally characterised by pathway reconstitution in a common, fast growing mould, Penicillium paxilli In turn, four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F, have been functionally characterised. The four genes encode a geranylgeranyl transferase (NodC), a flavin adenine dinucleotide-dependent oxygenase (NodM), an indole diterpene cyclase (NodB) and a cytochrome P450 oxygenase (NodW). Two of the gene products (NodM and NodW) catalyse two previously unreported reactions that provide the enzymatic basis of the biosynthetic branch point unique to nodulisporic acid biosynthesis. From here, future efforts will explore how these genes can be engineered to overcome flux bottlenecks and enable production of significantly increased, and even industrially relevant, product titres.</p>

Functional analysis of Hypoxylon pulicicidum genes required for heterologous biosynthesis of nodulisporic acids

<p>Nature holds some of the greatest secrets in drug design and development and the ability to access these trade secrets has been revolutionised by modern bioengineering technologies. In order to exploit these technologies it is essential to understand what genes are involved in compound production and the enzymatic steps that limit flux to the desired product. This thesis describes the discovery of four secondary-metabolic enzymatic steps involved in the biosynthesis of a group of valuable natural products known as nodulisporic acids. Nodulisporic acids are known for their potent insecticidal activities; however, biosynthesis of these compounds by the natural fungal producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult and has prevented the commercial development of novel nodulisporic acid-containing veterinary medicines and crop protects. To discover how nodulisporic acids are biosynthesized: 1. the H. pulicicidum genome was sequenced 2. a gene cluster responsible for nodulisporic acid production was predicted 3. genes in the cluster were functionally characterised by pathway reconstitution in a common, fast growing mould, Penicillium paxilli In turn, four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F, have been functionally characterised. The four genes encode a geranylgeranyl transferase (NodC), a flavin adenine dinucleotide-dependent oxygenase (NodM), an indole diterpene cyclase (NodB) and a cytochrome P450 oxygenase (NodW). Two of the gene products (NodM and NodW) catalyse two previously unreported reactions that provide the enzymatic basis of the biosynthetic branch point unique to nodulisporic acid biosynthesis. From here, future efforts will explore how these genes can be engineered to overcome flux bottlenecks and enable production of significantly increased, and even industrially relevant, product titres.</p>

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism

Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension. The mechanism of activation by excitation and reduction of the chromophore flavin adenine dinucleotide has been controversially discussed for many years. Various spectroscopic techniques have contributed to our understanding of plant cryptochromes by providing high time resolution, ambient conditions and even in-cell approaches. As a result, unifying and differing aspects of photoreaction and signal propagation have been revealed in comparison to members from other cryptochrome subfamilies. Here, we review the insight from spectroscopy on the flavin photoreaction in plant cryptochromes and present the current models on the signal propagation from flavin reduction to dissociation of the C-terminal extension.

Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: A FADH2-Dependent Enzyme with Remarkable Industrial Potential

Recently, we described the preparation of the recombinant oleate hydratase from Lactobacillus rhamnosus ATCC 53103. We observed that the purified C-terminal His-tagged enzyme was completely inactive and the catalytic activity was partially restored only in presence of a large amount of flavin adenine dinucleotide (FAD). In the present work, we assess that this hydratase in the presence of the reduced form of flavin adenine dinucleotide (FADH2) is at least one hundred times as active as in the presence of the same concentration of FAD. By means of two different biochemical processes, we demonstrated unambiguously that oleate hydratase from Lactobacillus rhamnosus ATCC 53103 is a FADH2-dependent enzyme. As a first relevant application of this discovery, we devised a preparative procedure for the stereoselective synthesis of (R)-10-hydroxystearic acid. Accordingly, the hydration of oleic acid (up to 50 g/L) is performed on a multigram scale using the recombinant hydratase and FADH2 generated in situ as cofactor. The produced (R)-10-hydroxystearic acid (ee > 97%) precipitates from the reaction solvent (water/glycerol/ethanol) and is conveniently recovered by simple filtration (>90% yield).

An Update of Lysine Specific Demethylase 1 Inhibitor: A Patent Review (2016-2020)

Background: As a FAD (Flavin Adenine Dinucleotide) - dependent histone demethylase discovered in 2004, LSD1 (lysine specific demethylase 1) was reported to be overexpressed in diverse tumors, regulating target genes transcription associated with cancer development. Hence, LSD1 targeted inhibitors may represent a new insight in anticancer drug discovery. For these reasons, researchers in both the pharmaceutical industry and academia have been actively pursuing LSD1 inhibitors in the quest for new anti-cancer drugs. Objectives: This review summaries patents about LSD1 inhibitors in recent 5 years in hope of providing a reference for LSD1 researchers to develop new modulators of LSD1 with higher potency and fewer adverse effects. Methods: This review collects LSD1 inhibitors disclosed in patents since 2016. The primary ways of patent searching are Espacenet®, Google Patents, and CNKI. Results: This review covers dozens of patents related to LSD1 inhibitors in recent five years. The compound structures are mainly divided into TCP (Tranylcypromine) derivatives, imidazole derivatives, pyrimidine derivatives, and other natural products and peptides. Meanwhile, the compounds that have entered the clinical phase are also described. Conclusion: Most of the compounds in these patents have been subjected to activity analysis with LSD1 and multi-cell lines, showing good antitumor activity in vitro and in vivo. These patents exhibited the structural diversity of LSD1 inhibitors and the potential of natural products as novel LSD1 inhibitors.

An rfuABCD -like operon and its relationship to riboflavin utilization and mammalian

Riboflavin is an essential micronutrient, but its transport and utilization has remained largely understudied among pathogenic spirochetes. Here we show that Borrelia burgdorferi , the zoonotic spirochete that causes Lyme disease, is able to import riboflavin via products of its rfuABCD -like operon as well as synthesize flavin mononucleotide and flavin adenine dinucleotide despite lacking canonical genes for their synthesis. Additionally, a mutant deficient in the rfuABCD -like operon is resistant to the antimicrobial effect of roseoflavin, a natural riboflavin analog, and is attenuated in a murine model of Lyme borreliosis. Our combined results indicate that not only are riboflavin and the maintenance of flavin pools essential for B. burgdorferi growth, but that flavin utilization and its downstream products (e.g., flavoproteins) may play a more prominent role in B. burgdorferi pathogenesis than previously appreciated.