UC Berkeley

Heterologous cyanobacterial production of isoprene (C₅H₈) presents an opportunity to develop renewable resources for fuel and industrial chemicals. Isoprene can be generated photosynthetically in these microorganisms from dimethylallyl-diphosphate (DMAPP) by the recombinant enzyme isoprene synthase (ISPS), as a transgenic product of the isoprenoid biosynthetic pathway. The present work sought to combine recent enhancements in the cellular level of reactant (DMAPP) and enzyme (ISPS), as a means in the further development of this technology. This objective was approached upon the heterologous overexpression of fni, an isopentenyl isomerase from Streptococcus pneumoniae, which increased the amount of the DMAPP reactant at the expense of its isomer, isopentenyl-diphosphate (IPP), in the cells. In addition, the cellular concentration of ISPS was substantially enhanced upon expression of the ISPS gene, as a fusion construct with the highly expressed in cyanobacteria cpcB gene, encoding the abundant β-subunit of phycocyanin. Synergy between these two modifications, i.e., enhancement in DMAPP substrate availability and enhancement in the concentration of the ISPS enzyme, improved the isoprene-to-biomass production ratio in cyanobacteria from 0.2:1 mg g^-1 (w:w), attained with the ISPS transgene alone, up to 12.3:1 mg g^-1 (w:w), measured when the combined two modifications were applied to the same cell. This is the highest verifiable yield of heterologous photosynthetic isoprene production reported so far. Findings in this work constitute a step forward in the development of the cyanobacterial biotechnology for isoprene production.