Geranyl Diphosphate Synthase (CrtE) Inhibition Using Alendronate Enhances Isoprene Production in Recombinant <i>Synechococcus elongatus</i> UTEX 2973: A Step towards Isoprene Biorefinery

A hemiterpene, isoprene, is commercially produced from crude oil refining processes. As a result of fossil fuel depletion, isoprene production process development is gaining attention from recombinant cyanobacteria and other microbial systems for its industrial and biofuel applications. In the present study, a fast-growing and CO₂-tolerant cyanobacteria, <i>Synechococcus elongatus</i> UTEX 2973, is engineered with <i>Pueraria montana</i> isoprene synthase (<i>IspS</i>) at neutral site I (NSI) in the genome of <i>S. elongatus</i> UTEX 2973. Furthermore, to enhance isoprene production a key enzyme (isopentenyl diphosphate isomerase, <i>IDI</i>) of the methyl-D-erythritol 4-phosphate (MEP) pathway is also overexpressed at neutral site III (NSIII). Wild-type and recombinant strains of <i>S. elongatus</i> UTEX 2973 (UTEX <i>IspS</i> and UTEX <i>IspS.IDI</i>) are studied for growth and isoprene production in the presence of an inducer (IPTG) and/or inhibitor (alendronate). Alendronate is used for the inhibition of geranyl diphosphate synthase (CrtE), downstream of the MEP pathway that catalyzes dimethylallyl diphosphate/isopentenyl pyrophosphate (DMAPP/IPP) condensation in the recombinant UTEX 2973 strains. The docking studies on SeCrtE (CrtE of <i>Synechcoccus elongatus</i> PCC 7942) and alendronate as an inhibitor have revealed that alendronate binds more tightly than IPP in the cavity of SeCrtE, with a higher number of intermolecular interactions and energy. The UTEX <i>IspS</i> strain has shown isoprene production below the limit of detection in the presence of an inducer and/or inhibitor; however, production studies using UTEX <i>IspS</i>.<i>IDI</i> showed a maximum production of 79.97 and 411.51 µg/g dry cell weight (DCW) in a single day in the presence of an inducer only and an inducer along with an inhibitor, respectively. The UTEX <i>IspS</i>.<i>IDI</i> strain produced 0.41 mg/g DCW of cumulative isoprene in the presence of an inducer and 1.92 mg/g DCW in the presence of an inducer as well as an inhibitor during six days of production. The yield improvement of isoprene is observed as being 4.7-fold by using the inhibition strategy, which is used for the first time in the recombinant cyanobacterial system. The average productivities of isoprene obtained from UTEX <i>IspS</i>.<i>IDI</i> are observed to be 2.8 μg/g DCW/h in the presence of an inducer and 13.35 μg/g DCW/h in the presence of an inducer as well as an inhibitor. This study provides a basis for the process development and yield improvement in isoprene production using a novel inhibition strategy in fast-growing recombinant cyanobacteria. Recombinant strains and metabolic pathway inhibition studies can be used in future attempts to photosynthetically produce hemiterpenes.