Metabolic engineering of Synechocystis sp. strain PCC 6803 for isobutanol production

Abstract

Global warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacterium Synechocystis sp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl β-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employed in situ removal of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions.

Fig 1

(a) Schematic representation showing the integration of the genes kivd and adhA into the genome of Synechocystis 6803. Colony PCR was performed to verify the integration of the insert into the genomic DNA of the mutant (AV03). The vector pTKA3 was used as a template for the positive control, and wild-type cells were used as a negative control. Colony PCR of AV03 showed the presence of a band (8.3 kb) the same size as that of the positive (+ve) control and the absence of the negative-control (WT) band. (b) IB synthesized by engineered Synechocystis 6803 under different IPTG concentrations (n = 3). (Inset) Results of an RT-PCR performed to detect the expression of the heterologous genes kivd (top; 500 bp from kivd) and adhA (bottom; 200 bp from adhA). Lane 1, wild-type 6803; lane 2, AV03 with 0 mM IPTG; lane 3, AV03 with 0.5 mM IPTG; lane 4, AV03 with 1 mM IPTG. The error bars indicate standard deviations.

Fig 2

(A) Growth curves of Synechocystis 6803 WT and AV03 (n = 3; shake flask cultures). ♢, WT under autotrophic conditions; ◆, WT under mixotrophic conditions; ○, AV03 under autotrophic conditions; and ●, AV03 under mixotrophic conditions (note that the growth curve of AV03 under mixotrophic conditions overlaps the autotrophic growth curves of AV03 and the WT). (B) IB synthesized in AV03 under autotrophic conditions (only HCO₃), heterotrophic conditions (only glucose), and mixotrophic conditions (both HCO₃ and glucose) (n = 3; shake flask cultures with closed caps). (C) Percent carbon contribution of glucose for synthesizing amino acids and isobutanol in the WT and the mutant strain (AV03) as measured on day 9 (shake flask cultures with closed caps). Isotopomer analysis (TBDMS-based method) of proteinogenic amino acids confirmed the low [¹³C]glucose utilization by the mutant. The error bars in panels A and B indicate standard deviations, while those in panel C represent the 2% technical error of the instrument.

Fig 3

Toxic effects of IB on the growth of Synechocystis 6803. IB was added to final concentrations (g/liter; n = 2) of 0 (♢), 0.2 (□), 0.5 (△), 1 (○), 2 (■), and 5 (●) to a Synechocystis 6803 culture with an initial OD₇₃₀ of ∼0.8. The error bars indicate standard deviations.

Fig 4

Net concentration of IB synthesized (bars) and biomass growth (curves) by the AV03 culture under different conditions (n = 3). a, IB with 0.5 ml oleyl alcohol (autotrophic); b, IB with 1 ml oleyl alcohol (autotrophic); c, IB with 0.5 ml oleyl alcohol and glucose (mixotrophic); d, IB with no oleyl alcohol (autotrophic, negative control); a1, OD₇₃₀ with 0.5 ml oleyl alcohol (autotrophic); a2, OD₇₃₀ with 1 ml oleyl alcohol (autotrophic); a3, OD₇₃₀ with 0.5 ml oleyl alcohol and glucose (mixotrophic); a4, OD₇₃₀ with no oleyl alcohol (autotrophic, negative control). (Inset) Schematic representation of the in situ IB removal system used to increase the production of IB. The error bars indicate standard deviations.