Alkane gene expression and its increased production in recombinant cyanobacteria

Abstract

Background: In cyanobacteria, the mechanism of the biosynthetic pathway of alkanes, which are useful materials for transportation biofuels in automobiles and airplanes, is being elucidated by genetic analytical methods. However, to date, no study has consistently analysed the transcription of alkane genes, molecular structure of mRNA, production of alkanes depending on the culture medium, catalytic activity of enzymes as indicated by the increased production of alkanes by the recombinant strain, or alkane accumulation in cells. Results: Transcription start point (TSP) of alkane genes ado and aar in the representative cyanobacteria Synechocystis sp. PCC 6803 and Limnothrix sp. SK1-2-1 strains producing C₁₇H₃₆ and C₁₅H₃₂, respectively, were determined at -88/-72 (6803_ado/aar) and -70/-20 (SK1-2-1_ado/aar). From the TSPs, consensus promoter sequences were identified, and the transcripts possessed unique stem and loop (S&L) structure at 5’- and 3’-UTR regions, which can regulate mRNA stability, translation initiation, and transcription termination. Alkane gene transcript levels were higher in nitrogen- and phosphorus-depleted BG11 media; however, higher amount of alkanes was produced in BG11 medium because of the reduced biomass. Transconjugants in which 6803_ado/aar or SK1-2-1_ado/aar were cloned into an algal expression vector and inserted into PCC 6803 strain produced approximately 1.6- to 2.2-times more C17 alkanes (C₁₇H₃₆) than that of wild-type strain in both cases. When alkane hyperaccumulator strains were cultured in BG11 or BG11-N/-P/-S medium, TEM observation showed clearly different cell shapes and accumulated products under each condition, but no significant accumulation, such as oil drops, was observed. Conclusion: New transcription start sites and predicted promoter sequences were identified, and unique S&L structures were found in 5'-UTR and 3'-UTR regions of each transcript, indicating mRNA stability and efficiency of translation initiation or termination. Although nutrient-depleted media are effective for alkane gene mRNA accumulation, it is clear that alkane accumulation per unit medium volume is higher when BG11 medium is used, given the reduced biomass. Experiments using recombinant strains for alkane production have shown that not only the enzymatic activity of ADO/AAR but also the intracellular production level of fatty-acyl ACP substrate may be important for mass production of alkanes with the desired carbon chain lengths.