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 .