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>

The Rab Geranylgeranyl Transferase Beta Subunit Is Essential for Embryo and Seed Development in Arabidopsis thaliana

Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.

Analysis of Genetic Potential of Banyuwangi Local Rice (Oryza sativa L.) Based on Relative Expression of Homogentisate Geranylgeranyl Transferase (HGGT) and Granule-Bound Starch Synthase I (GBSSI) Gene

Rice nutrition including vitamin and amylose contents become important aspect for many people around the world. Rice with high amylose content (low glycemic index) is good for those with Diabetes mellitus. Tocotrienol, one precursor of Vitamin-E biosynthesis is catalyzed by enzymes encoded HGGT, while amylose biosynthesis is catalyzed by enzymes encoded GBSSI. The objective of this study was to find rice varieties with high tocotrienol and/or amylose content based on the expression of HGGT and GBSSI among eight Banyuwangi local rice varieties. Relative expression of HGGT and GBSSI was measured by qRT-PCR and analyzed using 2ΔCt method. Statistical analysis resulted in the significantly different of HGGT and GBSSI relative expression among samples. Relative expression of HGGT from the highest to the lowest were demonstrated by Hitam Melik, Hitam Pekat, Blambangan A3, Merah Bali, Blambangan A2, Berlian, Janur Kuning, and SOJ A3, respectively; while relative expression of GBSSI from the highest to the lowest were demonstrated by Hitam Melik, Hitam Pekat, SOJ A3, Janur Kuning, Berlian, Merah Bali, Blambangan A3, and Blambangan A2, respectively. Based on this research we conclude that Hitam Melik potentially produces higher tocotrienol and lower glycemic index than other studied varieties.

Synthesis of the 6-Substituted Imidazo[1,2-a]Pyridine-3-yl-2- Phosphonopropionic Acids as Potential Inhibitors of Rab Geranylgeranyl Transferase

Twelve phosphonopropionates derived from 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC) were synthesized and evaluated for their activity as inhibitors of protein geranylgeranylation. The nature of the substituent in the C6 position of imidazo[1,2-a]pyridine ring was responsible for the compound's activity against Rab geranylgeranyl transferase (RGGT). The most active inhibitors disrupted Rab11A prenylation in the human cervical carcinoma HeLa cell line. The esterification of carboxylic acid in the phosphonopropionate moiety turned the inhibitor into an inactive analog.

Rab-dependent vesicular traffic affects female gametophyte development in Arabidopsis

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.

Geranylgeranyl Transferase-I Knockout Inhibits Oxidative Injury of Vascular Smooth Muscle Cells and Attenuates Diabetes-Accelerated Atherosclerosis

The proliferation of vascular smooth muscle cells (VSMCs) induced by oxidative injury is one of the main features in diabetes-accelerated atherosclerosis. Geranylgeranyl transferase-I (GGTase-I) is an essential enzyme mediating posttranslational modification, especially the geranylgeranylation of small GTPase, Rac1. Our previous studies found that GGTase-I played an important role in diabetes-accelerated atherosclerosis. However, its exact role is largely unclear. In this study, mouse conditional knockout of VSMC GGTase-I (Pggt1bΔ/Δ mice) was generated using the CRISPR/Cas9 system. The mouse model of diabetes-accelerated atherosclerosis was induced by streptozotocin injections and an atherogenic diet. We found that GGTase-I knockout attenuated diabetes-accelerated atherosclerosis in vivo and suppressed high-glucose-induced VSMC proliferation in vitro. Moreover, after a 16-week duration of diabetes, Pggt1bΔ/Δ mice exhibited lower α-smooth muscle actin (α-SMA) and nitrotyrosine level, Rac1 activity, p47phox and NOXO1 expression, and phospho-ERK1/2 and phosphor-JNK content than wild-type mice. Meanwhile, the same changes were found in Pggt1bΔ/Δ VSMCs cultured with high glucose (22.2 mM) in vitro. In conclusion, GGTase-I knockout efficiently blocked diabetes-accelerated atherosclerosis, and this protective effect must be related to the inhibition of VSMC proliferation. The potential mechanisms probably involved interfering Rac1 geranylgeranylation, inhibiting the assembly of NADPH oxidase cytosolic regulatory subunits, reducing oxidative injury, and decreasing ERK1/2 and JNK phosphorylation.