Engineered isoprene production from Chlamydomonas reinhardtii using herbicide selection markers and CO2-fed cultivation optimization through multi-parallel photobioreactor headspace gas analysis

Metabolic engineering requires selection markers for transformant generation. However, antibiotic resistance is of concern for potential horizontal gene transfer. Herbicide resistance markers are an alternative for photosynthetic cell engineering as plant-specific agents with resistance mechanisms that can come from mutations of endogenous genes. Here, we developed norflurazon and oxyfluorfen resistance markers for nuclear genome transformant selection in the alga Chlamydomonas reinhardtii . These were used to facilitate robust isoprene biosynthesis engineered by overexpression of a yeast isopentenyl-diphosphate delta-isomerase ( Sc IDI), the alga's own beta carotene ketolase ( Cr BKT), and the sweet potato isoprene synthase ( Ib IspS). Further UV-C mutagenesis and selection were employed to improve yields to ~334 mg isoprene L −1 culture on organic carbon. It was then possible to optimize CO₂-driven cultivation for isoprene biosynthesis in batch and continuous processes using multi-port, real-time, in-line mass spectrometry coupled to parallel photobioreactors. Highest isoprene yields in batch were under 900 μE illumination and 33 °C. In turbidostat mode, ~51 mg isoprene L culture −1 day −1 was achieved for ~2.5 days concomitant with algal biomass production. Cultivation of the engineered alga directly in effluent from an anaerobic membrane bioreactor was also conducted. Isoprene production was concomitant with removal of ammonium and phosphate from the wastewater, and biomass production was similar to that in replete medium. Isoprene yields exhibited gradual reduction after each successive repetitive refresh, which was mitigated by supplementation of trace elements. The results demonstrate that engineered algae could be used as a secondary wastewater valorization step while generating both biomass and volatile co-products like isoprene. • Herbicide selection markers enabled engineering algal isoprene production • Isoprene production enhanced through UV-C mutagenesis • 20-parallel photobioreactors coupled to real-time off-gas analysis for first time • Determined optimal temperature and light conditions for CO 2 to isoprene from algae • Isoprene production from algae grown on secondary effluent in repetitive batch