Diverse hydrocarbon biosynthetic enzymes can substitute for olefin synthase in the cyanobacterium Synechococcus sp. PCC 7002

Abstract

Cyanobacteria are among only a few organisms that naturally synthesize long-chain alkane and alkene hydrocarbons. Cyanobacteria use one of two pathways to synthesize alka/enes, either acyl-ACP reductase (Aar) and aldehyde deformylating oxygenase (Ado) or olefin synthase (Ols). The genomes of cyanobacteria encode one of these pathways but never both, suggesting a mutual exclusivity. We studied hydrocarbon pathway compatibility using the model cyanobacterium Synechococcus sp. PCC 7002 (S7002) by co-expressing Ado/Aar and Ols and by entirely replacing Ols with three other types of hydrocarbon biosynthetic pathways. We find that Ado/Aar and Ols can co-exist and that slower growth occurs only when Ado/Aar are overexpressed at 38 °C. Furthermore, Ado/Aar and the non-cyanobacterial enzymes UndA and fatty acid photodecarboxylase are able to substitute for Ols in a knockout strain and conditionally rescue slow growth. Production of hydrocarbons by UndA in S7002 required a rational mutation to increase substrate range. Expression of the non-native enzymes in S7002 afforded unique hydrocarbon profiles and alka/enes not naturally produced by cyanobacteria. This suggests that the biosynthetic enzyme and the resulting types of hydrocarbons are not critical to supporting growth. Exchanging or mixing hydrocarbon pathways could enable production of novel types of CO₂-derived hydrocarbons in cyanobacteria.

Figure 1

The two major alka/ene hydrocarbon biosynthetic pathways in cyanobacteria are the Ols (red) and Ado/Aar (blue) pathways. Fatty acid (FA) biosynthesis or activation from free fatty acids (FFA) provides acyl-ACP substrates for both pathways.

Figure 2

Representative growth curves of S7002 strains co-expressing Ols and the Ado/Aar pathways. In all panels, 7002::control is shown in red, 7002::6803alk in blue, and 7002::trc7942alk in green. (A) Strains grown at 38 °C. (B) Strains grown at 27 °C. (C) Strains exposed to a temperature and light change during growth. Strains were grown in a bioreactor under 300 μmol LED photons s⁻¹m⁻² and bubbled with air supplement with 1% CO₂ (panels A and B) or only air (panel C). In panel C, the gray box indicates the two-day 15 °C dark period. Cells were grown in a bioreactor under 300 μmol LED photons s⁻¹m⁻² and bubbled with air supplement with 5% CO₂.

Figure 3

Alka/ene hydrocarbon content of the S7002 strains expressing Ols and Ado/Aar pathways and WT references. Hydrocarbon content was normalized as percent of dry cell weight (% DCW). (A) S7942 at 38 °C. (B) S6803 at 30 °C. (C) 7002::control at 38 °C. (D) 7002::6803alk at 38 °C. (E) 7002::trc7942alk at 38 °C. (F) 7002::control at 27 °C. (G) 7002::6803alk at 27 °C. (H) 7002::trc7942alk at 27 °C. (I) 7002::control at 25 °C. (J) 7002::6803alk at 25 °C. (K) 7002::trc7942alk at 25 °C. A to H were cultured under 300 μmol photons s⁻¹ m⁻² and bubbled with 5% CO₂-supplemented air and I to K at 50 μmol photons s⁻¹ m⁻² in air. S6803 was cultured at 30 °C since the strains is not tolerant of 38 °C. Error bars are 1 SD based on at least three biological replicates.

Figure 4

Representative growth curves of WT S7002, 7002ΔOls and Ols-replacement strains at 38 °C and 27 °C. (A) WT S7002 and 7002ΔOls at 38 °C (red and blue traces, respectively) and at 27 °C (yellow and green traces). (B) ΔOls strains grown at 38 °C. ΔOls::control (red trace), ΔOls::6803alk (blue), ΔOls::7942alk (yellow), ΔOls::UndA (green), and ΔOls::FAP (grey). (C) ΔOls strains grown at 27 °C, indicated using the same colors as in panel B. Strains were cultured as in Fig. 3.

Figure 5

Alka/ene content of 7002ΔOls substituted with non-native hydrocarbon biosynthetic genes presented as % DCW. (A) ΔOls::6803alk at 38 °C. (B) ΔOls::7942alk at 38 °C. (C) ΔOls::UndA at 38 °C. (D) ΔOls::FAP at 38 °C. (E) ΔOls::6803alk at 27 °C. (F) ΔOls::7942alk at 27 °C. (G) ΔOls::UndA at 27 °C. (H) ΔOls::FAP at 27 °C. Strains were cultured as in Fig. 3. Error bars are 1 SD based on at least three biological replicates.

Figure 6

Mutation of Pseudomonas aeruginosa UndA to increase substrate range. (A) GC traces of S7002 cell extracts expressing native UndA (red trace) and the UndA-F239A mutant (blue). The green trace shows hydrocarbon standards for C_13:1, C₁₃, C_15:1, and C₁₅ in order of elution. (B) Substrate pocket in WT UndA. (C) Predicted expanded substrate pocket in UndA F239A. In B and C, the substrate pocket is shown as a grey surface, 2,3-dodecenoic acid substrate in orange and the iron ion as a brown sphere. UndA structure images were made using Pymol version 2.0.7, Schrödinger, LLC. Sample inputs for GC in panel A were normalized to dry cell biomass.

Figure 7

Summary of substrates and hydrocarbon products generated by Ols and non-native pathways in S7002. The substrates for Ado/Aar and Ols are acyl-ACPs and those for UndA and FAP are the equivalent fatty acids likely derived from turnover of membrane lipids.