INACTIVATION OF COMPETING PATHWAYS AND INCREASED GENE COPIES FOR ENHANCED ISOPRENE PRODUCTION IN SYNECHOCOCCUS SP. PCC 7002 CYANOBACTERIA

Abstract

Renewable energy resources are necessary for a sustainable environment, energy independence, and national security. Cyanobacteria (blue-green microalgae) hold great potential as sources of sustainable bioproducts because of their ability to convert sunlight and atmospheric carbon-dioxide into energy-rich compounds such as isoprene, a high-value precursor for synthetic rubber and biofuels. Our group has introduced optimized transgenes for an isoprene synthase (IspS) enzyme and a rate-limiting, methyl erythritol phosphate (MEP) pathway isomerase (IDI) from poplar into the fast-growing marine cyanobacterium, Synechococcus sp. PCC 7002, and achieved isoprene synthesis at 4 mg L culture-112 h day-1 (~ 40 to 80-fold higher than previously reported for cyanobacteria). To further improve isoprene yields: 1) a competing glycogen biosynthesis pathway was inactivated, 2) the IspS and IDI genes were targeted to a chromosomal petJ2 site to improve genetic stability, and 3) a second set of IspS-IDI genes was targeted to a high copy plasmid (pAQ1) of Synechococcus. I hypothesized that 1) inactivation of glycogen synthesis, a major carbon-storage pathway, will increase carbon flux to isoprene, 2) targeting the IspS-IDI genes to the chromosome will increase their stability, and 3) increasing the copy number of the IpsS and IDI genes, by targeting both the chromosome and plasmid, will further improve isoprene yield. The glgA1 and glgA2 glycogen synthase genes were inactivated. However, isoprene production in these Synechococcus strains was only ~ 20% higher than in strains with functional glycogen synthesis. This suggested that carbon previously used for glycogen synthesis was directed to pathways other than the MEP pathway. Integration of the IspS-IDI transgenes into the chromosomal petJ2 site (encoding a c6-like cytochrome protein) resulted in continuous, stable isoprene production in these strains (petJ2::IspS-IDI) compared to strains carrying these genes only on the plasmid (pAQ1::IspS-IDI). Inactivation of the petJ2 gene had no observable impact on growth, thus identifying petJ2 as a suitable site for transgene integration. Increased copies of the IspS-IDI transgenes, achieved by targeting these genes to the plasmid as well as the chromosomal petJ2 site, resulted in strains (petJ2::IspS-IDI, pAQ1::IspS-IDI) that produced ~ 1.5 times more isoprene than strains carrying these genes on plasmid pAQ1 only. Reverse transcriptase quantitative polymerase chain reaction gene-expression experiments showed that IspS transcript levels in the petJ2::IspS-IDI strains were several hundred fold higher, especially in old, dense cultures, than in strains with the IspS-IDI genes on plasmid pAQ1 only. These data demonstrate that improved isoprene yields in the petJ2::IspS-IDI strains resulted both from greater stability of the transgenes and increased mRNA transcript levels. This research demonstrates the utility of the petJ2 site and chromosomal integration for transgene stability and expression of isoprene synthase (IspS) and MEP pathway genes for enhanced isoprene production. The study further shows that Synechococccus sp. PCC 7002 is an excellent platform for genetic engineering to achieve improved yields of isoprene and other industrially relevant terpenes or bioproduct-biofuel chemicals.