Engineering cyanobacteria to improve photosynthetic production of alka(e)nes

Abstract

Background: Cyanobacteria can utilize solar energy and convert carbon dioxide into biofuel molecules in one single biological system. Synechocystis sp. PCC 6803 is a model cyanobacterium for basic and applied research. Alkanes are the major constituents of gasoline, diesel and jet fuels. A two-step alkane biosynthetic pathway was identified in cyanobacteria recently. It opens a door to achieve photosynthetic production of alka(e)nes with high efficiency by genetically engineering cyanobacteria.

Results: A series of Synechocystis sp. PCC6803 mutant strains have been constructed and confirmed. Overexpression of both acyl-acyl carrier protein reductase and aldehyde-deformylating oxygenase from several cyanobacteria strains led to a doubled alka(e)ne production. Redirecting the carbon flux to acyl- ACP can provide larger precursor pool for further conversion to alka(e)nes. In combination with the overexpression of alkane biosynthetic genes, alka(e)ne production was significantly improved in these engineered strains. Alka(e)ne content in a Synechocystis mutant harboring alkane biosynthetic genes over-expressed in both slr0168 and slr1556 gene loci (LX56) was 1.3% of cell dry weight, which was enhanced by 8.3 times compared with wildtype strain (0.14% of cell dry weight) cultivated in shake flasks. Both LX56 mutant and the wildtype strain were cultivated in column photo-bioreactors, and the alka(e)ne production in LX56 mutant was 26 mg/L (1.1% of cell dry weight), which was enhanced by 8 times compared with wildtype strain (0.13% of cell dry weight).

Conclusions: The extent of alka(e)ne production could correlate positively with the expression level of alkane biosynthetic genes. Redirecting the carbon flux to acyl-ACP and overexpressing alkane biosynthetic genes simultaneously can enhance alka(e)ne production in cyanobacteria effectively.

Figure 1

Schematic overview of fatty acid, alkane (alkene) and main competing metabolic pathways in Synechocystis sp. PCC6803. Key enzyme genes in those pathways are indicated. 3-PGA, glyceraldehyde 3-phosphate; PYR, pyruvate; PHB, poly-β-hydroxybutyrate; acyl-ACP, acyl- acyl carrier protein; ddh, 2-hydroxyacid dehydrogenase gene; phaA, polyhydroxyalkanoates-specific beta-ketothiolase gene; accBCDA, multi-subunit acetyl-CoA carboxylase gene;lipA, lipolytic enzyme gene; aas, acyl-ACP synthetase gene; aar, acyl-ACP reductase gene; ado, aldehyde-deformylating oxygenase.

Figure 2

Alka(e)ne production in Synechocystis mutants overexpressing cyanobacteria alkane biosynthetic genes. (A) Alka(e)ne production in Synechocystis mutants overexpressing sll0208 (LX31), sll0209 (LX33) and both genes (LX32) compared with Synechocystis sp. PCC6803 (6803yu). Error bars represent the standard deviation of three replicates. (B) Alka(e)ne production in Synechocystis mutants overexpressing orf1594 and Npun_R1711 (LX39), alkane biosynthetic genes from Synechococcus elongatus PCC7942 (LX34) and Nostoc punctiforme PCC73102 (LX35) compared with wildtype strain (6803yu) and LX32 mutant. Error bars represent the standard deviation of three replicates.

Figure 3

Alka(e)ne production can be enhanced effectively by redirecting the carbon flux to acyl-ACP. (A) Alka(e)ne production in Synechocystis mutants overexpressing sll0208 and sll0209 in slr1609 over-producing strain (LX38) and phaA gene deletion mutant (LX40) compared with wildtype strain (6803yu) and LX32 mutant. Error bars represent the standard deviation of three replicates. (B) Alka(e)ne production in Synechocystis mutants overexpressing sll0208 and sll0209 in acetyl-CoA carboxylase genes (LX57) and lipolytic enzyme gene (LX58) over-producing strain compared with wildtype strain (6803yu) and LX32 mutant. Error bars represent the standard deviation of three replicates.

Figure 4

Alka(e)ne production in Synechocystis mutants overexpressing two copies of alkane biosynthetic genes. (A) Alka(e)ne production in the Synechocystis mutant overexpressing sll0208 and sll0209 in both slr0168 and ddh gene sites (LX56) compared with wildtype strain (6803yu) and the Synechocystis mutant overexpressing two copies of sll0208 and sll0209 in slr0168 site (LX70). Error bars represent the standard deviation of three replicates. (B) Growth curves of wildtype strain and the LX56 strain in the bubble column photo-bioreactors. Error bars represent the standard deviation of three replicates. (C) Alka(e)ne production calculated as a percentage of DW of LX56 strain was enhanced by 8 times compared with wildtype strain when cultivated in the bubble column photo-bioreactors. Error bars represent the standard deviation of three replicates.

Figure 5

Semi-quantitative reverse transcription PCR analysis of the transcriptional levels of sll0208 and sll0209 in wildtype, LX32, LX70 and LX56 mutant. The rnpB gene was used as the external standards. Lane 1, LX56; Lane 2, LX70; Lane 3, LX32; Lane 4: wildtype.