Engineering a synthetic pathway for propane biosynthesis

The final precursor for propane biosynthesis is butyraldehyde. Several Clostridium species naturally harbour a CoA-dependent pathway for 1-butanol synthesis that also proceeds via a butyraldehyde intermediate13. The final step in 1-butanol biosynthesis is catalysed by AdhE2, a bi-functional acyl-CoA/aldehyde reductase14, using butyryl-CoA as the immediate precursor. As AdhE2 directly converts butyryl-CoA to 1-butanol, without releasing the butyraldehyde intermediate needed for the ADO reaction, alkane biosynthesis would not be possible with an AdhE2-dependent pathway. In addition, AdhE2 is reported to be sensitive to O 2 (ref. 14) an essential co-substrate for ADO7. Although an O 2 -tolerant butyraldehyde dehydrogenase (PduP) was recently shown to generate free butyraldehyde in cyanobacteria15, we instead exploited an ATP-dependent carboxylic acid reductase (CAR; Fig. 1; step 3) that catalyses the conversion of a broad range of fatty acids to their corresponding aldehydes, including butyrate to butyraldehyde10.

In order to supply butyrate for butyraldehyde synthesis, we sought to redirect native fatty acid biosynthesis (FASII; Fig. 1; step 1) of the host as demonstrated earlier for medium-chain alcohols and alkanes10. To achieve this, a thioesterase with high specificity for C4 butyryl-ACP was required. Heterologous expression of a bacterial acyl-ACP thioesterase from Bacteroides fragilis (Tes4) in E. coli K27 was recently reported to result in the accumulation of low quantities of butyrate, although >85% of the identified products were longer chain-length acids such as 8:0, 12:1 and 14:1 (ref. 16). Surprisingly, in our hands the expression of Tes4 (Fig. 1; step 2) in E.coli BL21(DE3) (Supplementary Table 2; strains Tes4 and Tes4Car) resulted in accumulation of over 5 mM butyrate as the main product, whereas only low amount of octanoate and minor quantities of >C8 chain-length acids were detected (Fig. 2a, Supplementary Fig. 1). There are two main differences between our experiments and those of Jing et al.16, which may explain the contrasting results. First, they used an E. coli K strain, which differs markedly17 from the B strain we used in our study. Second, FadD, an acyl CoA synthetase that catalyses the first step in the fatty acid β-oxidation pathway, and which therefore may compete for the fatty acid substrate of CAR, is missing in K27. Although the deletion of fadD was earlier found to be beneficial for free fatty acid production in a BL21(DE3) derivative strain using the C12–C16-specific E. coli thioesterase TesA18, recent studies using the same thioesterase and strain background suggested the opposite19. Therefore, the marked difference in the product profile between our work and the study of Jing et al.16 remains unknown and in need of further studies.

Figure 2: Propane and metabolite production under various environmental conditions using different strains. (a) Tes4 and Tes4Car strains were compared for fatty acid and alcohol production in shake flask fermentation under aerobic conditions; metabolites were analysed from culture supernatant after 24 h cultivation. (b) GC–MS chromatogram of a typical sample injected from the headspace of the propane-producing strain ProFΔA and a 1% (v/v) propane standard. (c) Supplementing the reaction culture headspace with oxygen to the final concentration of 51, 80 and 100% (v/v) using Pro strain (generating propane from glucose) and CarAdo strain (fed with butyrate). (d) Co-expression of Fpr and PetF with and without O 2 supplementation using strains Pro and ProF. (e) The effect of aldehyde reductase knock-outs with and without O 2 supplementation with strains ProFΔA versus ProF. Error bars, mean±s.d. (n=4). Full size image

Nevertheless, the availability of a C4-specific thioesterase allowed the assembly of a synthetic pathway for propane biosynthesis (Fig. 1) in BL21(DE3) by co-expression of Tes4 with CAR (Mycobacterium marinum)10, the CAR maturation factor phosphopantetheinyl transferase (Sfp; Bacillus subtilis)20, ADO (Prochlorococcus marinus)6 and ferredoxin (PetF; Synechocystis sp. PCC6803; Supplementary Table 2; strain Pro). The 2Fe-2S ferredoxin (PetF) (Fig. 1; Step 6) from cyanobacteria was introduced in order to enhance electron-supply, as the availability of ferredoxin or ferredoxin-like electron acceptor/donors was previously observed to be inadequate for a synthetic H 2 -pathway in E. coli BL21(DE3)21. A gas-tight cultivation method was used in order to allow propane to accumulate in the reaction vial headspace. Careful optimization of the analytical setup enabled reproducible gas chromatograph–mass spectrometer (GC–MS)-based quantification of even subtle changes in product formation. Mass fragment product analysis of cultures fed with 13C-labelled glucose confirmed that the synthesized propane (Fig. 2b) was derived from glucose supplemented to the medium (Supplementary Fig. 2).

Propane synthesis is dependent upon oxygen availability

Although the FASII-dependent propane pathway in the Pro strain was functional, continuous production could not be maintained and a plateau was reached within an hour after the culture vials were sealed (Supplementary Fig. 3). A similar trend was also noted for the pathway intermediate, butyrate (Supplementary Fig. 4A), in addition to the unwanted side-product, 1-butanol (Supplementary Fig. 4B). In order to understand and address the limiting factors affecting propane biosynthesis, a systematic evaluation of the effect of precursor supply, O 2 availability, H 2 O 2 -metabolism, electron-supply and competing native pathways was carried out using a combination of different strains (Supplementary Tables 2 and 3) and conditions (Supplementary Table 4).

Propane synthesis in the closed-vial systems was enhanced by the presence of additional O 2 (Fig. 2c, Supplementary Fig. 5A), increased relative head-space volume (Supplementary Fig. 6), or periodic regeneration of the head-space with air (Supplementary Fig. 7). This response to O 2 was unlikely due to changes in cell density as additional supply of oxygen had a negligible effect on growth during the reaction (Supplementary Fig. 5B), and increasing the cell density in the sealed vials had very little impact on propane production (Supplementary Fig. 8). When butyrate was supplied directly to the culture medium (Supplementary Table 2; strain CarAdo), the plateau in propane accumulation was delayed (Supplementary Fig. 9) and yields were higher (Fig. 2c). Together these findings suggested that (i) the limitation observed with glucose-dependent propane biosynthesis in the closed system was caused by insufficient availability of butyrate and not the downstream biosynthetic steps, and (ii) the amount of FASII-derived butyrate was limited by the availability of oxygen. This hypothesis was further corroborated experimentally by demonstrating that the pool of free butyrate (Supplementary Table 2; strain Tes4) increased in relation to the amount of supplied O 2 (Supplementary Fig. 10). In parallel, lack of O 2 may also directly limit the ADO-catalysed conversion of butyraldehyde to propane as was previously demonstrated in vitro7. This may explain why complete depletion of O 2 from the culture by flushing with N 2 severely compromised propane biosynthesis even in the presence of butyrate (Supplementary Fig. 11).

Even though oxygen is a prerequisite for alkane biosynthesis, an excess supply of pure O 2 was found to have a negative impact on production (Fig. 2c). As ferredoxins have been shown to generate superoxide radicals22 and ADO can be inactivated in the presence of H 2 O 2 (ref. 23), we hypothesized that the increase in O 2 content, and/or the effect of O 2 on electron supply, interferes with ADO activity through an increase in H 2 O 2 . One possible strategy for resolving this issue is to over-express a catalase, which breaks down H 2 O 2 , as was shown previously23. Despite a comprehensive evaluation, however, we did not observe any clear positive effect on alkane accumulation by over-expression of the catalase KatE (Fig. 1; Step 9) from E. coli (Supplementary Fig. 12).

Increased electron supply enhances propane synthesis

For the conversion of aldehydes to alkanes, ADO forms a peroxo-intermediate as part of its reaction mechanism24,25. This requires an input of four electrons for every alkane that is formed; this electron supply chain has been previously reconstituted (non-chemically) in vitro by the use of non-native surrogate ferredoxins and/or ferredoxin reductases6,7,23,26 and recently, by a combination of native enzymes from Synechococcus elongatus PCC7942 (ref. 27). The previously reported in vivo E. coli systems, however, have relied exclusively upon the endogenous capacity of the host for ADO reduction6,9,10,11. In order to provide sufficient supply of electrons to ADO in our system, 2Fe-2S ferredoxin PetF from Synechocystis sp. PCC 6803 was over-expressed as part of the pathway. In parallel, to ensure that insufficient reduction of ferredoxin was not limiting pathway flux as was previously observed with a synthetic H 2 -pathway21, a ferredoxin-oxidoreductase was further introduced into the system. Even though native cyanobacterial redox partners have been shown to confer slightly higher ADO activity in vitro27, the E. coli counterpart was selected for this study for maximal compatibility with the overexpression host. In E. coli, there are two annotated flavodoxin/ferredoxin-oxidoreductases: (i) the pyruvate/ferredoxin-oxidoreductase YdbK and (ii) NADPH/ferredoxin/flavodoxin-oxidoreductase Fpr. The extreme O 2 -sensitivity of YdbK28 ruled out its involvement in the aerobic formation of propane leaving Fpr as the most likely endogenous candidate. The overexpression of Fpr (Fig. 1; step 7) resulted in only a small increase in propane under standard conditions (Fig. 2d; 21% (v/v)). In contrast, when the O 2 concentration was increased to 80% (v/v), there was a five- to eightfold increase in propane accumulation, enhancing the titre to over 3 mg l−1 of propane (Fig. 2d, Supplementary Figs 13–15).

Competition with native host metabolism

E. coli has an efficient system for removing potentially harmful intracellular aldehydes by the action of aldehyde reductases and dehydrogenases. This native detoxification mechanism by the host is likely to compete with the ADO enzyme for the aldehyde substrate29. The accumulation of 1-butanol with the propane-generating strains Pro, and its parent strain Tes4Car (Fig. 2a, Supplementary Fig. 4B), suggested that this indeed was the case. In order to efficiently channel the butyraldehyde precursor towards propane, two aldehyde reductase genes ahr and yqhD (Fig. 1; Step 8) that had previously been shown to be most important for the conversion of isobutyraldehyde to isobutanol29 were deleted. The resulting strains (Supplementary Table 2; strains ProFΔA and ProFKΔA) showed enhanced propane synthesis at both atmospheric (21% (v/v)) and elevated oxygen concentrations (80% (v/v)), resulting in approximately three- and twofold improvement, respectively (Figs 2e and 3a). As expected, the increase in propane afforded by the aldehyde reductase deletions (Supplementary Fig. 16A) was followed by a concomitant decrease in the production of butanol (Supplementary Fig. 16B).

Figure 3: Comparison of the pathways for propane and heptane production. (a) The effect of aldehyde reductase knock-outs in ΔyqhD Δahr strains ProFKΔA and HepFKΔA versus the corresponding strains ProFK and HepFK, respectively, with supplemented oxygen. (b) Octanoate and butyrate production with strains Tes3 and Tes4, respectively, using conditions optimized for propane production (80% v/v oxygen). (c) Supplementing the reaction culture headspace with oxygen to the final concentration of 51, 80 and 100% (v/v) using strains Pro and Hep. (d) Overexpression of KatE in strains ProK and HepK versus Pro and Hep with supplemented oxygen; values are represented as relative % for comparison. Error bars, mean±s.d. (n=4). Full size image

Enzyme constraints for alkane biosynthesis

Although both the CAR and ADO enzymes can accept four-carbon substrates, earlier in vitro characterization suggest that they exhibit superior kinetics for longer chain substrates. For example, the K M values for CAR are ~90 times lower for C8 versus C4 (ref. 10) whereas the K app values for ADO are ~190 times higher for C8 versus C4 (ref. 30). In order to test whether poor enzyme kinetics was compromising the performance of the propane pathway in vivo, we constructed a C7 heptane pathway, by exchanging the thioesterase gene tes4, with tes3 (Anaerococcus tetradius), that has been reported to generate primarily the C8 fatty acid, octanoate16. Induction of this modified pathway resulted in the accumulation of heptane (Supplementary Fig. 17A) from glucose (Supplementary Fig. 17B) within the headspace of the sealed cultivation vessels, although the majority of the product is expected to exist in the liquid state under the conditions employed. Using the optimal conditions established for propane synthesis, heptane titres were found to be almost twofold greater than propane (5 versus 9 mg l−1; Fig. 3a). However, the quantity of the heptane precursor (octanoate) released by the Tes3 strain was only about one-fourth of the amount of the propane precursor (butyrate) released by Tes4 (Fig. 3b), which confirmed that the kinetic properties of the CAR and ADO enzymes used in this study indeed were limiting propane productivity. The effect of supplemented oxygen on heptane production was similar as with propane, although the overall positive impact was less significant (<50% improvement) and inhibitory effects were observed at lower oxygen concentrations (Fig. 3c). Catalase over-expression did not improve heptane productivity and generally only had a slight negative effect (Fig. 3d, Supplementary Fig. 18). Co-expression of Fpr (Supplementary Fig. 19) and deletion of the aldehyde reductases (Supplementary Fig. 20), however, had a stimulating impact.

Potential for large-scale production of propane

Throughout this study, cultivations were performed with small-scale cultures (0.5 ml) using gas-tight crimped GC vials (2 ml). Although this allowed high-throughput evaluation with a large number of replicates, it was obviously not ideal for evaluating the potential productivity. For example, termination of propane accumulation within the first 3 h of cultivation was repeatedly observed, which raised doubts about the longevity of the propane-generating system. To address this issue, while also assessing the system in larger batch cultures, the culture volume was scaled up 40-fold to 20 ml, whereas the volume of the cultivation vessel was increased 80-fold to 160 ml. Overall, this resulted in a greater headspace/liquid ratio (from 3:1 to 7:1) for which the gas-environment was expected to be less variable. Indeed, propane production continued up to 19 h, resulting in a total titre of 32 mg l−1 (Supplementary Table 2; strain ProFΔA and 80% (v/v) O 2 supplementation; Fig. 4a). From the initial development of the propane-producing pathway, the overall titre was improved more than two orders of magnitude (from 0.3 to 32 mg l−1). The step-wise improvement in propane production from genetic modification to physical optimization is summarized in Fig. 4a,b. Figure 4c,d show the detailed production profile of the up-scaled system over a 19-h cultivation illustrating the parallel formation of the end-products (i) propane, (ii) heptane, the corresponding fatty acids (iii) butyrate and (iv) octanoate, the alcohols (v) butanol and (vi) octanol, respectively, in addition to (vii) glucose concentration and (viii) cell density. Interestingly, the accumulation of butanol stalled soon after the 8 h point, whereas propane accumulation continued and eventually became dominant over the other C4 products.