Development of the biotechnological production of (+)-zizaene : enzymology, metabolic engineering and in situ product recovery

The sesquiterpene (+)-zizaene is the immediate precursor of khusimol, the main compound of the vetiver essential oil from the vetiver grass, which grants its characteristic woody scent. Among its distinct applications, this oil is relevant for the formulation of cosmetics and used in approximately 20% of all men’s perfumery. The traditional supply of the vetiver essential oil had suffered shortages due to natural disasters. Consequently, the biotechnological production of khusimol is an alternative towards a more reliable supply. In this study, we provide new insights towards the microbial production of khusimol by characterizing the zizaene synthase, engineering the metabolic pathway of (+)-zizaene in Escherichia coli and analyzing the in situ recovery of (+)-zizaene from fermentation. In the first chapter, the zizaene synthase, the critical enzyme for khusimol biosynthesis, was characterized. A SUMO-fused zizaene synthase variant was overexpressed in E. coli, and in vitro reactions yielded 90% (+)-zizaene. Furthermore, enzyme characterization comprised enzyme kinetics, optimal reaction conditions, substrate specificity and reaction mechanisms. The in vitro reactions showed high stability through varying pH and temperature values. By in silico docking model, this was explained due to the hydrophobicity of the surrounding loops, which stabilized the closed conformation of the active site. The second chapter addressed the metabolic engineering of the (+)-zizaene biosynthetic pathway in E. coli. A systematic strategy was applied by modulating the substrate FDP and the zizaene synthase to improve the zizaene titers. The optimal (+)-zizaene production was reached by engineering the mevalonate pathway and two copies of the zizaene synthase into a multi-plasmid strain. Optimization of the fermentation conditions such as IPTG, media, pH and temperature improved the production further, achieving a (+)-zizaene titer of 25 mg L‒1. In the third chapter, the in situ recovery of (+)-zizaene from fermentation was analyzed. Initially, liquid-liquid phase partitioning cultivation improved the (+)-zizaene recovery at shake flask scale. Subsequently, solid-liquid phase partitioning cultivation was evaluated by screening polymeric adsorbers, where Diaion HP20 obtained the highest recovery ratio. The bioprocess was scaled up to 2 L fed-batch bioreactors by integrating in situ recovery and fermentation. External and internal (with and without gas stripping) recovery configurations were tested, where the internal configuration obtained the highest (+)-zizaene recovery of all, achieving a (+)-zizaene titer of 211.13 mg L−1 and a productivity of 3.2 mg L−1 h−1

Use of Essential Oils and Volatile Compounds as Biological Control Agents

Essential oils (EOs) and microbial/plant-based volatile organic compounds (VOCs) are being used in an increasing number of sectors such as health, cosmetics, the food industry and, more recently, agronomy. In agronomy, they are employed as bio-herbicides and bio-pesticides due to their their insecticidal, antifungal, and bactericidal effects. Several EO-based bio-pesticides are already registered. Essential oils and other VOCs are 100% bio-based and present numerous additional advantages. They contain a great number of structurally diverse compounds that frequently act in synergy; they are thus less subject to resistance. As highly volatile compounds are found in EOs and VOCs, they typically cause no residue problems in food products or in soils. Indeed, the supply of EOs can be really challenging because they are frequently produced in restricted areas of the world with prices and chemical composition fluctuations. Besides, while the high volatility of EOs and VOCs is interesting for some specific applications, it can be a problem when developing a bio-pesticide with long lasting effects. Finally, EOs are frequently phytotoxic, which is perfect for herbicide formulations, but not for other applications. In both cases, the development of a proper formulation is essential. Owing to the current attraction for natural products, a better understanding of their modes of biological action is of importance for the development of new and optimal applications

Towards the Use of Natural Compounds for Crop Protection and Food Safety

In consideration of the ever-increasing global population, the demand for food—and on food-production—is massive. It is critical that we are able to meet this demand and mitigate the risks and factors that challenge our ability to do so, including pestilence to food crops and biological threats to food safety, before food reaches the consumer. As such, the advancement of measures to both protect crops and facilitate the surety of safe food products to end-users is a research area of great interest and growing development. This book details exciting new research into the use of natural compounds for the protection of crops and food products. From essential oils and their potential uses as naturally derived antimicrobial agents to the use of carbon dioxide as a pesticide and the use of biofertilisers, the articles herein describe and review cutting edge research in this area to help facilitate a more sustainable future

Production of natural vanillin from cymbopogon citratus using phanerochaete chrysosporium

Malaysia as one of the important agricultural countries in the world producing 70 million tonnes of lignocellulosic biomass. One of the abundant agricultural wastes which has high lignocellulose content is lemongrass leaves (Cymbopogon citratus). A total of 8,000 tonnes of dry leaves are produced annually from 1,150 hectares of C. citratus plantation in Malaysia. Parts of the leave wastes are burned for electric generation while the rest are left in the fields to decompose naturally. The use of lemongrass leaves as ruminant feedstock is, however, not favoured due to animal rejection against its aroma. C. citratus leaves have 58% hemicellulose and lignin content and are also rich in ferulic acid. Ferulic acid is the precursor for vanillin production. The C. citratus leave wastes that contain ferulic acid could be potentially used for vanillin production via microbial approach, which is currently less investigated. The main purpose of this research was to investigate the potential of a one-step natural vanillin production from C. citratus leaves hydrolysate by Phanerochaete chrysosporium, the white-rot fungi of basidiomycetes. The research work focused on the recovery of ferulic acid as well as the optimization of natural vanillin production from C. citratus leaves. Leaves within size of 125-249 µm appeared to be suitable for ferulic acid extraction, with 1.12g/L total recovery of ferulic acid by 55 minutes of boiling. A total of 27 strains of fungi were screened for natural vanillin and vanillic acid production. Among these fungi, Phanerochaete chrysosporium was chosen due to its high natural vanillin productivity. The effect of different nitrogen source in vanillin production was investigated using the General Factorial Design. A combination of ammonium chloride with yeast extract (ratio: 75:4) increased the vanillin production to 15-fold. To further optimize the process using 2-Level Factorial Design, the aforementioned nitrogen sources and 5 others independent variables (incubation temperature, pH, incubation time, agitation and inoculum size) were studied. The ammonium chloride concentration, temperature and pH were found to be the key factors for natural vanillin production and these factors were further examined using the Central Composite Design (CCD). The maximum vanillin production was observed with media composition of 1.0 g/L ferulic acid from hydrolysate of C. citratus leaves, 4.43 g/L ammonium chloride (inorganic nitrogen), 0.25 g/L yeast extract (organic nitrogen), 0.2 g/L KH2PO4, 0.013 g/L CaCl22.H2O, 0.5 g/L MgSO4.7H2O and 0.0025 g/L thiamin hydrochloride, at 36.3 °C and pH 6.84 under shaking condition at 150 rpm with inoculum size of 7 % (w/v). The generated model fitted well to the data set with R2 of 0.9059. The actual optimum conditions yielded 0.20 g/L vanillin (17-fold higher than non-optimised condition). The actual experimental data showed 2-fold higher yield as compared to previous reported studies. Artificial Neural Network model were also found to provide accurate validation and prediction result based on feed-forward back propagation as similar to Response Surface Methodology (RSM). A network with one hidden layer and 25 neurons were found to be the most optimum model with the lowest root mean squared error (RMSE; 0.0002415), absolute average deviation (AAD; 0.0004358) and the highest R2 (0.9024). In this study, it was confirmed that C. citratus leaves could serve as a promising alternative source for vanillin production with potential to scale-up for commercial production in Malaysia

Discovery and characterization of a novel class of metabolic regulators in the malaria parasite Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, infects hundreds of millions of people per year and causes hundreds of thousands of deaths. Within the host red blood cell, the parasite relies on glycolysis for energy and synthesis of essential biomolecules. One such anabolic fate of glucose is the synthesis of isoprenoids, a broad and essential class of compounds that participate in a variety of cellular functions. In the face of ever-evolving drug resistance, new inhibitors and better understanding of parasite metabolism are required. The antibiotic fosmidomycin (FSM) targets the methylerythritol phosphate pathway for isoprenoid synthesis and is a well-validated inhibitor of P. falciparum growth. A forward selection for FSM resistance generated a number of parasite strains with increased drug tolerance. We identify mutations in two members of the haloacid dehalogenase-like hydrolase (HAD) superfamily, PfHAD1 and PfHAD2, as causal for resistance. Enzymatic characterization and metabolic profiling reveal that these mutations are deleterious and confirm the role of PfHAD1 and PfHAD2 as novel negative regulators of glucose and isoprenoid metabolism. Despite their homology and shared role in FSM resistance, PfHAD1, a sugar phosphatase, and PfHAD2, a purine nucleotidase, appear to mediate FSM resistance via distinct enzymatic mechanisms. To further understand the role of PfHADs as metabolic regulators, we harness a growth defect in FSM-resistant PfHAD2 mutants to select for suppressors of FSM resistance. We identify suppressor mutations in the key glycolytic enzyme phosphofructokinase (PfPFK9) and describe the effect of these mutations on enzyme function and parasite metabolism

Use of Essential Oils and Volatile Compounds as Biological Control Agents

peer reviewedMDP

Plant Essential Oil with Biological Activity

Plant essential oils (PEOs) are hydrophobic liquids that contain volatile chemical components that are derived from various plant parts. They are among the most important plant natural products because of their diverse biological features as well as their therapeutic and nutritional applications. In addition, several aromatic PEOs are used to flavor food and add aromas to incense in the culinary sector. Recently, many PEOs have demonstrated promising antimicrobial activity against different post-harvest diseases and have been considered as possible natural alternatives for chemical treatments. This Special Issue titled “Plant Essential Oil with Biological Activity” provided an overview of several elements of PEOs, including their biological applications, antimicrobial activities, bio-pharmaceutical properties, principal single constituents, and mechanisms of action. This Special Issues fills in knowledge gaps and aids in the advancement of EO applications around the world. This issue contains thirteen research articles and two review papers that address a wide range of topics and applications relevant to the bioactivity of PEOs

Biological and Pharmacological Activity of Plant Natural Compounds II

The Special issue "Biological and Pharmacological Activity of Plant Natural Compounds II" is continuing the intriguing research on the use of natural plant products. The second edition follows the aim of the first one