Introduction
α-Farnesene, one of the simplest sesquiterpenes, has an enormous application in nature and industry. For example, α-farnesene works as chemical signaling molecule to signal danger and to implicate the orientation of aphids and termites in nature.1 In addition, α-farnesene acts as the intermediate to produce biofuel, vitamin E, vitamin K1, squalane and other high value-added products in industry.2-4Therefore, α-farnesene has important economic value in agriculture, chemical, bioenergy, medicine, and cosmetics.1, 3Since α-farnesene is abundant in plants (e.g., apple and Artemisia annua )1, 5, plant extraction is the major method for producing α-farnesene.4 However, the weaknesses of plant extraction limit the application in industry, such as the low yield, the high production cost, the limited feedstock and the serious environmental pollution.3, 5-6 Therefore, researchers turn their attention to use microbial fermentation to produce α-farnesene,4 and many effective strategies have been used in modifying microorganisms to enhance the biosynthesis of α-farnesene, including enhancing α-farnesene biosynthesis pathway, blocking the downstream α-farnesene biosynthesis pathway, rewriting the central carbon metabolism, compartmentalizing the supply ways of precursors, relieving the cell growth inhibition and optimizing the medium components and culture conditions.3-4, 7-9 In the previous study, we constructed a α-farnesene high-producing strainPichia pastoris X33-30 by dual regulation of cytoplasm and peroxisomes.3 PCAT1 promoters were replaced with PGAP promoters in X33-30 to obtain strain X33-30*, which produced 2.18 ± 0.04 g/L of α-farnesene in shake flasks. The strain P. pastoris X33-30* was enhanced the supply of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Although there are two pathways for producing IPP and DMAPP, i.e., the mevalonate (MVA) pathway and the methylerythritol-4-phosphate (MEP) pathway,5 however, no fewer than 6 molecules of ATP and NADPH are need to produce 1 molecule of α-farnesene (Fig. 1). The overall stoichiometry of α-farnesene biosynthesis via the MVA pathway is: 9 acetyl-CoA + 9 ATP + 3 H2O + 6 NADPH + 6 H+ → 1 α-farnesene + 9 CoA + 6 NADP++ 9 ADP + 3 Pi + 3 PPi + 3 CO2.10 The above equation indicates that the cofactors ATP and NADPH are also important for increasing α-farnesene production except for the precursor acetyl-CoA. And yet, very little research has addressed ATP and NADPH in α-farnesene production.
NADPH acts as cofactor for catalyzing the formation of mevalonate from 3-hydroxy-3-methyl glutaryl coenzyme A (i.e., HMG-CoA) (Fig. 1). Besides, the extra demand of NADPH has been discussed to be responsible for the heterologous protein production. Previous research indicated that NADPH availability is closely related to the yield of biomass and heterologous proteins.11-12 In addition, NADPH is also used to protect cells against endoplasmic reticulum (ER) stress and oxidative stress.11, 13 It should be noted that MVA pathway is the major pathway for producing IPP and DMAPP in P. pastoris ,3 and thus 6 molecules of NADPH and 9 molecules of ATP are need to produce 1 molecule of α-farnesene. However, NADH is the predominant reduced cofactor of catabolism rather than NADPH in yeast and bacteria.4 Thus, increasing the intracellular NADPH level or eliminating NADPH consumption is a common strategy to facilitate NADPH-dependent products, including terpenoid.14-15 For example, Liu et al. compared the effect of six native enzymes involved in NAPDH regeneration inYarrowia lipolytica and found that mannitol dehydrogenase benefits to increase squalene production.16 In addition, introduction of a synthetic version of the Entner-Doudoroff pathway from Zymomonas mobilis in E. coli MG1655 has been shown to be able to increase the NADPH regeneration rate by 25-fold and thus increasing terpenoid production.17 In P. pastoris , there are two inherent routes for NADPH generation, i.e., the oxidative branch of pentose phosphate pathway (oxiPPP) and the acetate biosynthetic pathway.9, 11, 18 However, the key enzymes in oxiPPP were negatively controlled by NADPH and ATP at the transcriptional and/or the translational level.11, 19Although heterogeneous expression of NADH kinase (i.e., POS5, catalyzed NADH to form NADPH) would increase the NADPH regeneration in P. pastoris ,11 NADH plays pivotal roles in ATP regeneration.18, 20 ATP acts as a key factor for α-farnesene biosynthesis (Fig. 1), except for as the energy currency in cells.21 Therefore, the ATP availability is extremely important for cell growth and α-farnesene biosynthesis so that adequate supplies of NADH are need for cell because ATP is mainly produced by NADH oxidation via electron transport phosphorylation (ETP) under aerobic conditions.22 For these, how to efficiently supply NADPH and ATP already becomes an important research direction in developing a α-farnesene high-producing strain.
In this work, the biosynthetic pathways of NADPH and ATP were rationally reconstructed in an α-farnesene high-producing strain P. pastoris X33-30*, which was reconstructed the carbon’s metabolic pathways in P. pastoris X33, to further increase the α-farnesene production. To do this, the native oxiPPP was firstly reconstructed in strain X33-30* by over-expressing the key enzymes in oxiPPP or/and inactivating the glucose-6-phosphate isomerase in glycolysis. Subsequently, the heterologous POS5 fromS. cerevisiae was introduced into P. pastoris and controlled by different intensity of promoters to further optimize the NADPH supply. Finally, the ATP availability was tried to increase by enhancing the supply of adenosine monophosphate (AMP) for the synthesis of ATP and decreasing the consumption of NADH in shunt pathway. As a result, the resultant strain P. pastoris X33-38 produced 3.09±0.37 g/L of α-farnesene after 72 h in shake-flask fermentation. These results demonstrate the effectiveness of increasing the availability of NADPH and ATP in P. pastoris for increasing the α-farnesene production and provide a new perspective to construct industrial-strength α-farnesene producer by rational modification of NADPH and ATP regeneration pathway in P. pastoris .