|Other Abstract||The monoterpene geraniol, which is emitted from flowers, takes an important role in flavor and fragrance industries due to its pleasant rose-like odor. Geraniol also exhibits huge potential in pharmacy and agrochemistry. Growing world demand for aroma chemicals and fuels has led to an increased demand for geraniol. Fractional distillation of plant essential oils is the major method for geraniol manufacture, but high cost and other limitations, such as weather dependence and plant diseases, limited the supplies of geraniol. For the chemical-synthesis method, synthesis from petrochemicals is not sustainable and often lacks of substrate selectivity, which may cause the formation of undesirable racemic mixtures. The disadvantages of both methods and the rising interest in natural products have sparked people to seek sustainable technologies for geraniol production.
Converting renewable resources into monoterpene products by engineered microorganisms was interesting technology and developed quickly, which have the advantages of fast growth, no need for land during their growth and sustainable development. In this study, we tried to assemble a new pathway for geraniol products in E. coli, and several strategies to increase the production efficiency and selectivity were tested.
(1) Utilization of phosphatase in the bioproduction of geraniol. Geraniol is likely to be synthesized from geranyl diphosphate (GPP). It has been hypothesized that phosphatases can catalyze geranyl diphosphate (GPP) into geraniol. But, whether and which phosphatases can transform GPP to geraniol has remained unanswered up to now. In this paper，the catalysis ability of four different types of phosphatases were studied with GPP as substrate in vitro, and just alkaline phosphatase (PhoA) from Escherichia coli can catalyze GPP into geraniol. Moreover, in order to confirm the ability of PhoA in vivo, the heterologous mevalonate pathway and geranyl diphosphate synthase gene from Abies grandis were co-overexpressed in E. coli with PhoA gene and 5.3±0.2 mg/l geraniol was produced from glucose in flask-culture.
(2) Biotransformation between geranyl acetate and geraniol by E.coli. For the first time, the biotransformation between geranyl acetate and geraniol by E.coli was proved. More than 40% of fed geraniol was converted into geranyl acetate by E. coli BL21 (DE3). Moreover, we revealed the role of acetylesterase (Aes, EC 184.108.40.206) from E. coli in hydrolyzing of geranyl acetate to geraniol and about 75% of geranyl acetate was converted into geraniol after 2 h of incubation in vitro.
(3) Engineering E. coli for high-yield geraniol production. Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6±3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4% of fed geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, fermentation condition were optimized and geraniol production reached up to 2.0 g/L with biotransformation of 88.8% geranyl acetate to geraniol by our strategy.
Mevalonate is an intermediate metabolites in MVA pathway, which is the precursor of geraniol, isoprenen, carotenoids, artemisinin, paclitaxel and other high value-added products. MVA can be biosynthesized from acetyl coenzyme A, which is produced from glucose through EMP pathway. However, there is carbon dioxide producted in this process and caused carbon loss. In addition, the pervious studies on terpenoid biosynthesis were most carried out under aerobic conditions, which will increase the difficulty and costs of terpenoid collection for its volatility. Overexpression of heterologous genes maybe lead to the unblance of cell under anaerobic conditions, which will result in the lower production and productivity. According to the above problems, this study explores the ways for carbon utilization under the anaerobic condition. The main results as follows:
(1) Use NOG pathway for MVA biosynthesis under anaerobic fermentation. NOG pathway was discovered in 2013. Compared with the traditional EMP pathway, 1 mol glucose can be convert to 3 mol acetyl phosphate and without carbon dioxide produced under anaerobic conditions by NOG pathway. In this study, we tried to improve the yield of the MVA and MVA biosynthesis with NOG pathway was constructed in E. coli BL21 (DE3). However, when introducing NOG way in E. coli, the yield of the MVA decreased to 1/3 of control. Overexpression of acetyl transferase gene pta or ptaF3, which removal of P - loop NTPase part of pta, MVA yeild down to 0.053 times and 0.09 times of control.
(2) Co-production of succinic acid and MVA to increase the comprehensive utilization of carbon. pyc gene and MVA upstream were co-expression in E.coli to co-production of succinic acid and MVA. Under anaerobic conditions, the co-production strains LWPYC1 synthetic 1.8 g/L MVA and 5.1 g/L succinic acid. The total production carbon conversion rate increase to 2 times of control. Moreover, adding exogenous carbon dioxide to fermentation process and MVA production increased to 5.9 g/L and succinic acid production reached 6 g/L while total carbon conversion rate increased to three times of control. Finally, lactic acid dehydrogenase gene ldhA was knockout, which lead to the total carbon conversion rate increase to 10 times of control while MVA prodction up to 12.2 g/L and succinic acid reach 7.8 g/L.|