Biosynthesis of Carotenoids and Apocarotenoids by Microorganisms and Their Industrial Potential

Carotenoids are a large group of natural pigments, ranging from red, to orange, to yellow colors. Synthesized by plants and some microorganisms (e.g., microalgae, fungi and bacteria), carotenoids have important physiological functions (e.g., light harvesting). Apocarotenoids are carotenoid-derived compounds and play important roles in various biological activities (e.g., plant hormones). Many carotenoids and apocarotenoids have high economic value in feed, food, supplements, cosmetics and pharmaceutical industries. Despite high commercial values, they are severely undersupplied because of low abundance in natural hosts (usually a few milligrams per kilogram of raw materials). Furthermore, plants or microbes usually produce mixtures of these molecules with very similar physical and chemical properties (such as α- and β-carotenes). All these features render the extraction from natural hosts rather difficult and also very costly both from process economics and sustainable land-use viewpoints. Chemical synthesis is also expensive due to structural complexity (e.g., astaxanthin has many unsaturated bonds and two chiral regions). Biotechnology via the rapidly advancing metabolic engineering and synthetic biology approaches has led to alternative ways to attain several carotenoids and apocarotenoids at relatively high titers and yields using fast-growing microorganisms. This chapter briefly reviews the biosynthesis of carotenoids and apocarotenoids by microorganisms and their industrial potential

METABOLIC ENGINEERING OF THE DEOXYXYLULOSE PHOSPHATE PATHWAY FOR ISOPRENOID PRODUCTION IN E.COLI

Ph.DDOCTOR OF PHILOSOPH

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.Singapore-MIT Alliance for Research and Technology (SMART

Engineering an artificial pathway for Cis-alpha-irone biosynthesis

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Spatial organisation of enzymes in the biosynthetic limonene production pathway in Escherichia coli

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Structural understanding of fungal terpene synthases for terpene product cyclization

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Advances and applications of CRISPR/Cas-mediated interference in Escherichia coli

The bacterium Escherichia coli (E. coli) is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds. In the past decade, the metabolic engineering of E. coli has undergone significant advances, although further productivity improvements will require extensive genome modification, multi-dimensional regulation, and multiple metabolic-pathway coordination. In this context, clustered regularly interspaced short palindromic repeats (CRISPR), along with CRISPR-associated protein (Cas) and its inactive variant (dCas), have emerged as notable recombination and transcriptional regulation tools that are particularly useful for multiplex metabolic engineering in E. coli. In this review, we briefly describe the CRISPR/Cas9 technology in E. coli, and then summarize the recent advances in CRISPR/dCas9 interference (CRISPRi) systems in E. coli, particularly the strategies designed to effectively regulate gene repression and overcome retroactivity during multiplexing. Moreover, we discuss recent applications of the CRISPRi system for enhancing metabolite production in E. coli, and finally highlight the major challenges and future perspectives of this technology

Metabolic Engineering Strategies for Sustainable Terpenoid Flavor and Fragrance Synthesis

Terpenoids derived from plant material are widely applied in the flavor and fragrance industry. Traditional extraction methods are unsustainable, but microbial synthesis offers a promising solution to attain efficient production of natural-identical terpenoids. Overproduction of terpenoids in microbes requires careful balancing of the synthesis pathway constituents within the constraints of host cell metabolism. Advances in metabolic engineering have greatly facilitated overcoming the challenges of achieving high titers, rates, and yields (TRYs). The review summarizes recent development in the molecular biology toolbox to achieve high TRYs for terpenoid biosynthesis, mainly in the two industrial platform microorganisms: Escherichia coli and Saccharomyces cerevisiae. The biosynthetic pathways, including alternative pathway designs, are briefly introduced, followed by recently developed methodologies used for pathway, genome, and strain optimization. Integrated applications of these tools are important to achieve high "TRYs" of terpenoid production and pave the way for translating laboratory research into successful commercial manufacturing