Direct Photosynthetic Recycling of Carbon Dioxide to Isobutyraldehyde
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LETTERS Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde Shota Atsumi1,3, Wendy Higashide1 & James C Liao1,2 Global climate change has stimulated efforts to reduce Ethanol has also been produced from CO2 and water using an −1 −1 CO2 emissions. One approach to addressing this problem engineered S. elongatus strain at a rate of 0.18 µg l hr (ref. 1). is to recycle CO2 directly into fuels or chemicals using Although these efforts demonstrated the biochemical feasibility of photosynthesis. Here we genetically engineered Synechococcus direct ethanol production from CO2, the cost of separating ethanol elongatus PCC7942 to produce isobutyraldehyde and at low concentration remains a formidable problem. isobutanol directly from CO2 and increased productivity by Productrecovery efficiency is an important determinant of the total overexpression of ribulose 1,5-bisphosphate carboxylase/ production cost and a major limitation of biochemical production from oxygenase (Rubisco). Isobutyraldehyde is a precursor for photosynthetic organisms. In this work, we chose isobutyraldehyde as a the synthesis of other chemicals, and isobutanol can be target because it has a low boiling point (63 °C) and a high vapor pressure used as a gasoline substitute. The high vapor pressure of (66 mm Hg at 4.4 °C), suggesting that it can be readily stripped from isobutyraldehyde allows in situ product recovery and reduces microbial cultures during production. Subsequent purification is also product toxicity. The engineered strain remained active for relatively easy, and the isobutyraldehyde concentration in the production 8 d and produced isobutyraldehyde at a higher rate than medium can remain low. Product removal in situ can reduce the those reported for ethanol1, hydrogen2 or lipid3 production by cytotoxic effects of isobutyraldehyde and enable longterm production. cyanobacteria or algae. These results underscore the promise In addition, isobutyraldehyde can be readily converted to various of direct bioconversion of CO2 into fuels and chemicals, which hydrocarbons currently derived from petroleum (such as isobutanol, bypasses the need for deconstruction of biomass. isobutyric acid, acetal, oxime and imine) using existing chemical catalysis (Fig. 1). In particular, it can be converted either biologically or chemi According to the US Energy Information Administration, world cally to isobutanol, which is suitable for use as a gasoline substitute, thus energy–related CO2 emissions in 2006 were 29 billion metric tons, making isobutyraldehyde a useful chemical feedstock. an increase of 35% from 1990 (ref. 4). As a result of human activ Productivity is a key determinant for the feasibility of direct CO2 ity, atmospheric levels of CO2 have increased by ~25% over the past conversion to chemicals or fuels. As biodiesel production from algae © All rights reserved. 2009 Inc. Nature America, 150 years5. Thus, it has become increasingly important to develop is considered one of the most efficient ways of generating biofuels7, we new technologies to reduce CO2 emissions. estimated the required productivity of isobutyraldehyde by compari Photosynthetic organisms use solar energy to generate reducing son with biodiesel production. The average productivity of microalgal 5 equivalents and incorporate atmospheric CO2 into organic molecules. biodiesel in a welldesigned production system can be ~1 × 10 liter Currently, cellulosic biofuels6 and algal biodiesels7 are prominent bio ha−1 per year (ref. 7). Assuming that onethird of the day (8 h) and logical approaches to sequester and convert CO2. Producing biodiesel 80% of the calendar year (292 d) are dedicated to production and a from algae has been proposed as one of the most efficient ways of generat characteristic dimension of 1 m for converting areal to volumetric ing biofuels7 because the lipid productivity of many algae greatly exceeds productivity, the productivity of isobutyraldehyde would need to be that of the best cellulosic ethanol production. However, these approaches 3,420 µg l−1 h−1 or higher to be competitive with algal biodiesel pro involve several intermediate stages for recycling CO2 into usable fuels duction. This level of productivity serves as our initial benchmark or chemicals, which increases production costs. Many cyanobacteria productivity for direct conversion of CO2 to isobutyraldehyde. and algae have the ability to produce hydrogen, which can be regarded To achieve this goal, we engineered a cyanobacterium, S. elongatus. 8 13 as a way to recycle CO2 indirectly . However, the volumetric hydrogen The ketoacid decarboxylase gene kivd from Lactococcus lactis was productivity reported is still relatively low2. Anaerobic hydrogen produc expressed using an expression cassette under the control of the isopropyl tion using anaerobic photosynthetic bacteria requires a hydrogen source βDthiogalactoside (IPTG) inducible promoter Ptrc (Fig. 1). This other than water9,10, and the productivity remains to be improved8. DNA fragment was integrated into neutral site I (NSI)14 by homologous 11 15 Another approach is direct conversion of CO2 to fuels or chemi recombination , resulting in SA578 (Fig. 2a). To increase the flux to the cals12. Previously, the photosynthetic bacteria Rhodobacrer sphaeroides keto acid precursor, 2ketoisovalerate (KIV)16, we integrated the alsS and Rhodobacrer capsulatus were engineered to produce ethanol from gene from Bacillus subtilis and the ilvC and ilvD genes from Escherichia 11 17 CO2 and a nonwater hydrogen source, with a final titer of ~800 mg/l . coli into neutral site II (NSII) of the SA578 genome, resulting in SA590 1Department of Chemical and Biomolecular Engineering, 2Institute for Genomics and Proteomics, University of California, Los Angeles, USA. 3Present address: Department of Chemistry, University of California, Davis, California, USA. Correspondence should be addressed to J.C.L. ([email protected]). Received 27 May; accepted 7 October; published online 15 November 2009; doi:10.1038/nbt.1586 NATURE BIOTECHNOLOGY VOLUME 27 NUMBER 12 DECEMBER 2009 1177 LETTERS The strain was cultured in a Roux culture bottle at 30 °C. 2PGA PEP Pyruvate O CO2 Isobutyraldehyde concentrations in the culture medium and the trap OH RuBP 3PGA O were measured as described in Online Methods (Fig. 2). The trap was AIsS refreshed daily. The strain produced 723 mg/l isobutyraldehyde in 12 d Calvin cycle 2-acetolactate −1 −1 O O with an average production rate of 2,500 µg l h (Fig. 2d,e). This OH number is very encouraging, as it is already close to our benchmark. The OH Valine biosynthesis isobutyraldehyde production rate remained constant for the first 9 d, but NADP+ IIvC 2,3- dihydroxy-isovalerate the production rate decreased dramatically after the tenth day (Fig. 2e) for O unknown reasons. When the culture was resuspended in fresh medium OH −1 −1 OH OH after 10 d, the bacteria regained their productivity (~60 mg l d ), IIvD suggesting that some inhibitory metabolites18 accumulated during L-valine 2- ketoisovalerate O IIvE O the cultivation. As expected, during the production process the iso OH OH O butyraldehyde concentration in the culture medium remained low, NH2 Kdc around 20 mg/l (Fig. 2f). This low concentration would reduce toxicity Isobutyraldehyde to cells and prolong the production phase. This strain did not produce Biological catalysis isobutanol, indicating that endogenous alcohol dehydrogenase (ADH) O activity toward isobutyraldehyde was not detectable. The enzyme that catalyzes the CO2 fixation reaction in the Calvin 19 Isobutanol Isobutyric acid Acetal Oxime BensonBassham (CBB) cycle is Rubisco , which is implicated as the O OR limiting step in CO fixation because of its poor turnover rate and the OH OH N 2 OR OH 20 competition between O2 and CO2 at the active site . To compensate Imine Diisobutyl phthalate 2-hydroxyisobutyric acid for the inherent limitations of Rubisco, the rbcLS genes from the related Isobutyl acetate Isobutyric acid ester N R S. elongatus strain PCC6301 (ref. 21) were integrated downstream of Isobutylene Acetone Chemical catalysis the endogenous rbcLS genes of SA590, resulting in SA665 (Fig. 3a). The Rubisco activity of SA665 was found to be ~1.4fold higher than that of Figure 1 The pathway for isobutyraldehyde production. The SA590 (Fig. 3b). The strain was cultured in BG11 medium with 50 mM Kdc-dependent synthetic pathway for isobutyraldehyde production. NaHCO3 at 30 °C. With the expression of the additional rbcLS genes, AlsS, acetolactate synthase; IlvC, acetohydroxy acid isomeroreductase; SA665 produced 1.1 g/l of isobutyraldehyde over 8 d with a steady IlvD, dihydroxy-acid dehydratase; Kdc, 2-ketoacid decarboxylase. state (first 7 d) production rate of 6,230 µg l−1 h−1(Fig. 3c–f), which is roughly twofold higher than SA590, which lacks the additional rbcLS (Fig. 2b). All three enzyme assays of SA590 lysates demonstrated higher genes. The increase in Rubisco activity of SA665 and the concomitant activity than those from SA578 (Fig. 2c), indicating that alsS (B. subtilis), increase in isobutyraldehyde production (Fig. 3b,c) suggest that CO2 ilvC (E. coli) and ilvD (E. coli) are expressed and functional in S. elongatus. fixation is one of the bottlenecks of the isobutyraldehyde production. Because the vapor pressure of isobutyraldehyde is relatively high, it can Thus, it is possible that the optimization of CO2 fixation could fur be removed readily from the culture medium during production by the ther improve isobutyraldehyde production. To probe the impact of bubbling of air. Evaporated isobutyraldehyde was then condensed with Rubisco overexpression, photosynthetic O2 production of SA590 and a Graham condenser. SA665 were measured under the same conditions as isobutyraldehyde © All rights reserved. 2009 Inc. Nature America, a b c The neutral site I (NSI) targeting vector The neutral site II (NSII) targeting vector Activity (nmol min–1 mg–1) q P r r PLlacO1 5 -NSI lacl trc kivd TrrnB spec 3 -NSI 5′-NSIIT0kan alsS ilvDilvC T1 ′ ′ 3′-NSII Strain AlsS IIvC IIvD Recombination Recombination SA578 11.1 ± 2.3 2.6 ± 1.3 5.4 ± 0.7 SA590 82.0 ± 7.7 8.1 ± 2.8 21.3 ± 3.9 NSI NSII S.