ATP drives direct photosynthetic production
of 1-butanol in cyanobacteria
Ethan I. Lana,band James C. Liaoa,b,c,1
aDepartment of Chemical and Biomolecular Engineering,
Proteomics, University of California, Los Angeles, CA 90095
bBiomedical Engineering Interdepartmental Program, and
cInstitute for Genomics and
Edited by Lonnie O. Ingram, University of Florida, Gainesville, FL, and approved February 21, 2012 (received for review January 3, 2012)
While conservation of ATP is often a desirable trait for microbial
production of chemicals, we demonstrate that additional consump-
tion of ATP may be beneficial to drive product formation in a
nonnatural pathway. Although production of 1-butanol by the fer-
organisms, the first step of the pathway, condensation of two
molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically
unfavorable. Here, we show that artificially engineered ATP con-
sumption through a pathway modification can drive this reaction
forward and enables for the first time the direct photosynthetic pro-
duction of 1-butanol from cyanobacteria Synechococcus elongatus
aldehyde/alcohol dehydrogenase (AdhE2) with separate butyralde-
hyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehy-
drogenase (YqhD) increased 1-butanol production by 4-fold. These
results demonstrated the importance of ATP and cofactor driving
forces as a design principle to alter metabolic flux.
biofuel ∣ malonyl-CoA ∣ metabolic engineering ∣ synthetic biology
In particular, 1-butanol has received increasing attention be-
cause it is a potential fuel substitute and an important chemical
feedstock. 1-Butanol can be produced by two distinct routes: the
coenzyme A (CoA)-dependent pathway (1, 2) and the keto acid
pathway (3–5). The CoA-dependent pathway follows the chem-
istry of β-oxidation in the reverse direction, in which acetyl-CoA
is condensed to form acetoacetyl-CoA and then reduced to 1-bu-
tanol. The keto acid pathway utilizes 2-ketobutyrate as an inter-
mediate, which goes through keto acid chain elongation to
2-ketovalarate using the leucine biosynthesis enzymes. 2-Ketova-
larate is then decarboxylated and reduced into 1-butanol. In each
case, the pathway can be extended to produce 1-hexanol and
other higher alcohols (6–8).
The CoA-dependent reverse β-oxidation is a natural fermen-
tation pathway used by Clostridium species (9–11) and has been
transferred to various recombinant heterotrophs, resulting in
1-butanol titers ranging from 2.5 mg∕L to 1.2 g∕L with glucose
as the substrate (12–16). One of the challenges in transferring this
pathway to other organisms lies in the hydrogenation of crotonyl-
CoA to butyryl-CoA catalyzed by the butyryl-CoA dehydrogen-
ase/electron transferring flavoprotein (Bcd/EtfAB) complex.
Bcd/EtfAB complex is difficult to express in recombinant systems,
is presumably oxygen sensitive (12, 17), and possibly requires re-
duced ferredoxin as the electron donor (18). This difficulty was
overcome by expressing trans-2-enoyl-CoA reductase (Ter) (19,
20), which is readily expressed in Escherichia coli and directly re-
duces crotonyl-CoA using NADH. This modified 1-butanol path-
way (Fig. 1; outlined in blue) is catalyzed by five enzymes: thiolase
(AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase
(Crt), Ter, and bifunctional aldehyde/alcohol dehydrogenase
(AdhE2). Simultaneously expressing these enzymes and engi-
neering NADH and acetyl-CoA accumulation as driving forces,
1-butanol production with a high titer of 15 g∕L and 88% of
iological production of chemicals and fuel from renewable
resources is an attractive approach to a sustainable future.
theoretical yield has been achieved using E. coli in flasks without
product removal (19). This result demonstrates the feasibility of
transferring the CoA-dependent pathway to nonnative organisms
for high-titer 1-butanol fermentation from glucose.
However, the success of the CoA-dependent pathway in
E. coli is not directly transferrable to photoautotrophs. By
expressing the same enzymes in cyanobacteria Synechococcus
elongatus PCC 7942, photosynthetic 1-butanol production from
CO2 was barely detectable (21). 1-Butanol production was
achieved by this strain only when internal carbon storage made
by CO2fixation in light conditions was fermented under anoxic
conditions (21). We hypothesized that both the acetyl-CoA and
NADH pools in this organism under photosynthetic conditions
may be insufficient to drive 1-butanol formation. Acetyl-CoA is
the precursor for fermentation pathway and the TCA cycle, both
of which are not active in light conditions. Furthermore, photo-
synthesis generates NADPH, but not NADH, and the intercon-
version between the two may not be efficient enough. Without a
significant driving force against the unfavorable thermodynamic
gradient, 1-butanol production cannot be achieved. The diffi-
culty of direct photosynthetic production of 1-butanol is in sharp
contrast to the production of isobutanol (450 mg∕L) and isobu-
tyraldehyde (1;100 mg∕L) by S. elongatus PCC 7942 (22), which
has an irreversible decarboxylation step as the first committed
reaction to drive the flux toward the products. This difference
suggests the importance of driving forces in altering the direc-
tion of metabolic flux.
We reason that instead of the acetyl-CoA pool, ATP may be
used to drive the thermodynamically unfavorable condensation
of two acetyl-coA molecules under photosynthetic conditions.
Thus, we engineered the ATP-driven malonyl-CoA synthesis and
decarboxylative carbon chain elongation used in fatty acid synth-
esis to drive the carbon flux into the formation of acetoacetyl-
CoA, which then undergoes the reverse β-oxidation to synthesize
1-butanol. We further replaced the subsequent NADH-depen-
dent enzymes with NADPH-dependent ones and successfully
achieved 1-butanol synthesis under photosynthetic conditions.
In theory, excess ATP consumption in the cell might cause a de-
crease in biomass. Thus, with notable exceptions (23–26), most
metabolic engineering design do not choose to increase ATP con-
sumption. Although many natural examples of microbes using
ATP to drive reactions, most of them are highly regulated. There-
fore, it is unpredictable whether it is feasible to use ATP con-
sumption to push flux in a nonnative pathway, for which no
Author contributions: E.I.L. and J.C.L. designed research; E.I.L. performed research;
E.I.L. and J.C.L. analyzed data; and E.I.L. and J.C.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed: E-mail: firstname.lastname@example.org.
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6018–6023 ∣ PNAS ∣ April 17, 2012 ∣ vol. 109 ∣ no. 16www.pnas.org/cgi/doi/10.1073/pnas.1200074109
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Lan and Liao PNAS
April 17, 2012