Engineering the fast growing and highly productive cyanobacterium Synechococcus sp. PCC 11901

Abstract

Synechococcus sp. PCC 11901 (PCC 11901) is a new cyanobacteria strain exhibiting fast and sustained growth and biomass accumulation, making it an interesting and potentially revolutionary host strain for biotechnology. At the time of starting the PhD project, very limited information was available apart from a first report published in literature (Wlodarczyk et al. 2020). The overarching goal of my thesis is to develop PCC 11901 as a chassis strain for cyanobacterial biotechnology.

In this thesis I first reviewed the current state of the art in cyanobacteria biotechnology, with emphasis on where new fast-growing strains like PCC 11901 can excel and what molecular and computational tools are needed to maximise their use towards carbon negative emissions technologies (NETs). I highlight the potential of cyanobacterial biorefineries based on these new strains in making NETs more cost-effective, as this has been the main bottleneck in the uptake of cyanobacteria-based solutions by industry.

I next developed a CyanoGate-based synthetic biology toolkit which significantly expanded our ability to engineer PCC 11901 by characterising new and existing standard parts (neutral sites, constitutive and inducible promoters, transcriptional terminators). I performed a proof-of-concept study of conditional knockdown of essential genes using CRISPRi and a novel markerless genome editing strategy using CRISPR/Cas12a. This extensive toolkit is a major milestone in engineering PCC 11901 and has been made available through Addgene, an open vector repository for the research community. This toolkit chapter is complemented by collaborative published papers (Mills et al., 2022; Mager et al., 2023).

I then performed an RNA-seq study of PCC 11901 to understand the transcriptomic landscape of this fast-growing strain and find out differentially expressed genes across different growth phases/densities in comparison to Synechococcus sp. PCC 7002, a strain with 96% genome similarity but does not exhibit the maximum growth densities reached by PCC 11901. In this chapter I generated the first RNA-seq dataset of PCC 11901 at different growth densities, which will ultimately be a valuable resource to inform future engineering work this strain.

Finally, to demonstrate the potential of PCC 11901 for biotechnology, I explored its capability as a platform for the bioproduction of high-value plant-derived products. First is the small, taste-modifying protein monellin, and second is the terpenoid α-bisabolene. I adapted and developed a phycobiliprotein (cpcB) fusion strategy to improve protein expression, and optimised growth conditions which led to increased protein and enzyme production. The results in this chapter set the stage for PCC 11901 as a viable photoautotrophic host for sustainable bioproduction.