Engineering yeast for de-novo synthesis of the insect repellent - nepetalactone

ABSTRACTWhile nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe a platform for de novo nepetalactone production in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introduction of eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, which represented the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, as well as copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity, with a higher geraniol-8-hydroxylase ratio further increasing 8-hydroxygeraniol titers. Another roadblock was the accumulation of an unwanted metabolite that implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologs were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in a final de novo accumulation of 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. Our pathway optimization will aid in the development of high yielding monoterpene S. cerevisiae strains.

Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

A review on stability improvement of sugarcane juice by using natural preservatives

Sugarcane juice is liquid extract as a drinking beverage in India, possesses therapeutic value. Stability or shelf life is very less due to spoilage or degradation of sugarcane juice because of presence of simple sugar in sugarcane juice. Microorganisms like bacteria prone to degradation of sugarcane juice. Which convert sucrose into dextran as deteriorating agent. Shelf life or stability can be improved by using natural preservatives also chemical preservatives; having a therapeutic value. In this article improvement of stability of sugarcane juice by using natural preservatives such as lemon extract, ginger extract, also may be moringa extract over the chemical preservatives. Citric acid in lemon extract acts as antimicrobial agent while ascorbic acid in ginger extract both improves stability of sugarcane juice. Stabilization of sugarcane juice improved by using naturally obtained preservatives up to several days with good quality.

Not Just a Simple Sugar: Arabinose Metabolism and Function in Plants

Growth, development, structure as well as dynamic adaptations and remodelling processes in plants are largely controlled by the properties of their cell walls. These intricate wall structures are mostly made up of different sugars connected through specific glycosidic linkages but also contain many glycosylated proteins. A key plant sugar that is present throughout the plantae, even before the divergence of the land plant lineage, but is not found in animals, is L-Arabinose (L-Ara). Here, we summarize and discuss the processes and proteins involved in L-Ara de novo synthesis, L-Ara interconversion, and the assembly and recycling of L-Ara-containing cell wall polymers and proteins. We also discuss the biological function of L-Ara in a context focused manner, mainly addressing cell wall related functions that are conferred by the basic physical properties of arabinose containing polymers/compounds. In this article we explore these processes with the goal of directing future research efforts to the many exciting yet unanswered questions in this research area.

Production Of Bioplastics From Renewable And Sustainable Feedstock Resources

This study illustrates the potential opportunity for the utilization of hemp to produce PHB (poly(3-hydroxybutyrate). The objective of the study was to optimize simple sugar availability from hemp for Ralstonia eutropha. The use of three pre-treatment methods (grinded – 5% NaOH – Autoclave at 121 oC for 60 minutes) was able to provide a better fractional insoluble solids (FIS) of ≃ 61 % that was significantly better compared to other combinations of pre-treatments studied. Optimum enzyme dosage was also determined by comparing different enzyme concentrations and found that three enzymes should contain a dose of 1.5 g /L. The optimum pretreatment and hydrolysis conditions resulted in a better enzyme hydrolysis yield of 10.9 % and PHB yield of ≃ 43 %. Results also demonstrate that sonification did not improve PHB recovery, while pH control increased PHB recovery. Keywords: Hemp, Ralstonia eutropha, PHB, Pre-treatment, Enzyme Hydrolysis

Production Of Bioplastics From Renewable And Sustainable Feedstock Resources

This study illustrates the potential opportunity for the utilization of hemp to produce PHB (poly(3-hydroxybutyrate). The objective of the study was to optimize simple sugar availability from hemp for Ralstonia eutropha. The use of three pre-treatment methods (grinded – 5% NaOH – Autoclave at 121 oC for 60 minutes) was able to provide a better fractional insoluble solids (FIS) of ≃ 61 % that was significantly better compared to other combinations of pre-treatments studied. Optimum enzyme dosage was also determined by comparing different enzyme concentrations and found that three enzymes should contain a dose of 1.5 g /L. The optimum pretreatment and hydrolysis conditions resulted in a better enzyme hydrolysis yield of 10.9 % and PHB yield of ≃ 43 %. Results also demonstrate that sonification did not improve PHB recovery, while pH control increased PHB recovery. Keywords: Hemp, Ralstonia eutropha, PHB, Pre-treatment, Enzyme Hydrolysis

Production, Extraction, and Characterization of Green Biodegradable Bioplastics Polyhydroxybutyrates (PHB) for Biomedical Applications

Biodegradable plastics are that kind of plastics that will decompose naturally, when environmental microorganisms metabolize and break down the chemical bonds present in the structure of biopolymer. Bio-plastics offer an advantage to earth by reducing carbon footprint and use of fossil fuel. Bio-plastics are completely biodegradable and can be recycled. The focus of the study was to produce green bioplastics by utilizing the sugar hydrolysate of Hemp fibers. Producing of Bioplastic was pursued in fermentation experiments, where HEMP was utilized as feedstock to produce PHB. Hemp fibers were initially pretreated, and then enzymatically hydrolyzed to produce PHB. Hemp can produce PHB (poly(3-hydroxybutyrate) when the environmental conditions are controlled. The objective of the study was to optimize simple sugar availability from hemp for Ralstonia eutropha. Powedered hemp with the contration of 5% NaOH, and 1.5g/ L of enzyme addition was found to yield the best restluts in the production process of Bioplastics as the fractional insoluble solids (FIS) of ≃ 61 % was significantly better compared to other combinations of pre-treatments studied. The enzyme hydrolysis yield of 10.9 % and PHB yield of ≃ 43 % were also found in this study.

Production, Extraction, and Characterization of Green Biodegradable Bioplastics Polyhydroxybutyrates (PHB) for Biomedical Applications

Biodegradable plastics are that kind of plastics that will decompose naturally, when environmental microorganisms metabolize and break down the chemical bonds present in the structure of biopolymer. Bio-plastics offer an advantage to earth by reducing carbon footprint and use of fossil fuel. Bio-plastics are completely biodegradable and can be recycled. The focus of the study was to produce green bioplastics by utilizing the sugar hydrolysate of Hemp fibers. Producing of Bioplastic was pursued in fermentation experiments, where HEMP was utilized as feedstock to produce PHB. Hemp fibers were initially pretreated, and then enzymatically hydrolyzed to produce PHB. Hemp can produce PHB (poly(3-hydroxybutyrate) when the environmental conditions are controlled. The objective of the study was to optimize simple sugar availability from hemp for Ralstonia eutropha. Powedered hemp with the contration of 5% NaOH, and 1.5g/ L of enzyme addition was found to yield the best restluts in the production process of Bioplastics as the fractional insoluble solids (FIS) of ≃ 61 % was significantly better compared to other combinations of pre-treatments studied. The enzyme hydrolysis yield of 10.9 % and PHB yield of ≃ 43 % were also found in this study.

The Role of Fructose in Non-Alcoholic Steatohepatitis: Old Relationship and New Insights

Non-alcoholic fatty liver disease (NAFLD) represents the result of hepatic fat overload not due to alcohol consumption and potentially evolving to advanced fibrosis, cirrhosis, and hepatocellular carcinoma. Fructose is a naturally occurring simple sugar widely used in food industry linked to glucose to form sucrose, largely contained in hypercaloric food and beverages. An increasing amount of evidence in scientific literature highlighted a detrimental effect of dietary fructose consumption on metabolic disorders such as insulin resistance, obesity, hepatic steatosis, and NAFLD-related fibrosis as well. An excessive fructose consumption has been associated with NAFLD development and progression to more clinically severe phenotypes by exerting various toxic effects, including increased fatty acid production, oxidative stress, and worsening insulin resistance. Furthermore, some studies in this context demonstrated even a crucial role in liver cancer progression. Despite this compelling evidence, the molecular mechanisms by which fructose elicits those effects on liver metabolism remain unclear. Emerging data suggest that dietary fructose may directly alter the expression of genes involved in lipid metabolism, including those that increase hepatic fat accumulation or reduce hepatic fat removal. This review aimed to summarize the current understanding of fructose metabolism on NAFLD pathogenesis and progression.