Unlocking the potential of unique genes in cyanobacterial alkane synthesis

Alkanes are considered among the most promising candidates for next-generation biofuels. Amongst various pathways discovered for alkane production, the cyanobacterial AAR (acyl ACP reductase) - ADO (aldehyde deformylating oxygenase) pathway has been the most studied pathway. Considering that cyanobacteria have the innate ability to produce alkanes, they can serve as an excellent chassis for sustainable biofuel production. In the first report of the AAR-ADO pathway, it was recorded that there are 17 unique genes present in the genome of only alkane-producing cyanobacteria. However, except for the role of AAR and ADO, none of the other genes have been implicated in alkane production so far. In this study, we performed overexpression and/or deletion of all 17 unique genes in Synechococcus elongatus PCC7942 and evaluated their role in growth, photosynthetic efficiency and alkane production. Based on the essentiality feature of genes in the cell survival of PCC7942, 9 essential genes were overexpressed, and 8 non-essential genes were knocked out in PCC7942. Among the essential genes that made a significant impact on alkane production, the overexpression of Synpcc7942_1772 (encoding small subunit ribosomal protein) and Synpcc7942_2212 (encoding large subunit ribosomal protein) led to ∼3.3-fold and ∼4.1-fold increased alkane production, respectively, suggesting a previously unrecognized link between translational machinery and metabolic pathway, while co-expression of aar and ado together increased alkane production by ∼5-fold. For the non-essential genes, the deletion of Synpcc7942_0452 (encoding hypothetical protein), Synpcc7942_1223 (encoding DevC), and Synpcc7942_1918 (encoding UDP-glucose: tetrahydro biopterin glucosyltransferase) led to the complete abolition of alkane production, indicating their critical roles. On the other hand, the deletion of Synpcc7942_0544 and Synpcc7942_0619, both encoding hypothetical proteins, led to a ∼5.6-fold and ∼4.4-fold increase in intracellular alkane production, respectively. Measurement of photosynthetic efficiency via Dual PAM (Pulse-Amplitude Modulated) fluorometry revealed a correlation between higher alkane production and increased photosynthetic efficiency, which the genome-scale metabolic model of PCC7942 also validated. Upon further detailed investigation of the genes making a large impact on alkane production, we identified Synpcc7942_0619 and Synpcc7942_1223 gene products as potential transporters based on the AlphaFold structure model and TMHMM (Transmembrane Hidden Markov Model) plot. The Synpcc7942_0619 encodes a DedA family transporter whose overexpression led to ∼1.5-fold higher extracellular alkane production, while deletion had a reverse effect. On the other hand, Synpcc7942_1223 encodes the DevC component of the tripartite efflux system, whose overexpression increased ∼13-fold intracellular alkane and deletion led to abrogation of both extra and intracellular alkane, indicating its vital interaction with the core enzymes of alkane synthesis pathway. These findings highlight the complex interplay between translation, photosynthesis, transport, and alkane biosynthesis, and provide a foundation for developing genetically engineered strains with improved alkane production through systems-level metabolic engineering.