Plants produce a large array of natural products of biotechnological interest. In many cases, these compounds are naturally produced at low titers and involve complex biosynthetic pathways, which often include cytochrome P450 enzymes. P450s are known to be difficult to express in traditional heterotrophic chassis. However, cyanobacteria have shown promise as a sustainable alternative for the heterologous expression of P450s and light-driven product biosynthesis. In this study, we explore strategies for improving plant P450 stability and membrane insertion in cyanobacteria. The widely used model cyanobacterium Synechocystis sp. PCC 6803 was chosen as the host, and the well-studied P450 CYP79A1 from the dhurrin pathway of Sorghum bicolor was chosen as the model enzyme. Combinations of the P450fused with individual elements (e.g., signal peptide, transmembrane domain) or the full length cyanobacterial, thylakoid-localized,protein PetC1 were designed. All generated CYP79A1 variants led to oxime production. Our data show that strains producingCYP79A1 variants with elements of PetC1 improved thylakoid targeting. In addition, chlorophyll-normalized oxime levels increased, on average, up to 18 times compared to the unmodified CYP79A1. These findings offer promising strategies to improve heterologous P450 expression in cyanobacteria and can ultimately contribute to advancing light-driven biocatalysis in cyanobacterial chassis.

Cyanobacteria are ancient prokaryotes known for their ability to perform oxygenic photosynthesis. The photoautotrophic lifestyle of these organisms requires the efficient synthesis, translocation and assembly of photosynthetic proteins into supramolecular photosynthetic complexes which are typically localised in the thylakoid membranes. Co- and posttranslational translocation of proteins across and into cyanobacterial membranes is carried out by multiple protein translocation systems. These systems, which include the general secretory pathway, the twin-arginine translocation pathway and the signal recognition pathway, are highly conserved across prokaryotes and organelles of endosymbiotic origin. However, cyanobacteria are unique in the requirement to target proteins to and across multiple membranes (cytoplasmic and thylakoid) with a single set of machinery for each translocation pathway. Therefore, one of the current challenges is to understand how photosynthetic proteins and cofactors are synthesized, targeted, and assembled into supramolecular photosynthetic complexes. Recent studies have provided an unprecedented view into thylakoid biogenesis and organisation of photosynthetic complexes in cyanobacteria; however, open questions regarding targeting mechanisms and the existence of a physical connection between membrane systems remain. This chapter will summarize the existing knowledge on protein trafficking in cyanobacteria with a focus on photosynthetic proteins. This information will then be contextualised to discuss similarities and differences between protein targeting in cyanobacteria, other prokaryotes and chloroplast thylakoid membranes.

Cyanobacteria are photosynthetic prokaryotes of high ecological and biotechnological relevance that have been cultivated in laboratories around the world for more than 70 years. Prolonged laboratory culturing has led to multiple microevolutionary events and the appearance of a large number of 'domesticated' substrains among model cyanobacteria. Despite its widespread occurrence, strain domestication is still largely ignored. In this work we describe Synechococcus elongatus PCC 7942-KU, a novel domesticated substrain of the model cyanobacterium S. elongatus PCC 7942, which presents a fast-sedimenting phenotype. Under higher ionic strengths the sedimentation rate increased leading to complete sedimentation in just 12 h. Through whole genome sequencing and gene deletion, we demonstrated that the Group 3 alternative sigma factor F plays a key role in cell sedimentation. Further analysis showed that significant changes in cell surface structures and a three-fold increase in released polysaccharides lead to the appearance of a fast-sedimenting phenotype. This work sheds light on the determinants of the planktonic to benthic transitions and provides genetic targets to generate fast-sedimenting strains that could unlock cost-effective cyanobacterial harvesting at scale.

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts but, so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that overexpression of a modified bacterial microcompartment shell protein, PduA*, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly only had a minor impact on cellular metabolism. Finally, we targeted the fluorescent reporter mCitrine to the nanofilaments using an encapsulation peptide that natively interacts with PduA. To our knowledge, this is the first study to apply bacterial microcompartment based nanotechnology in cyanobacteria. The establishment of nanofilaments in cyanobacterial cells is an important step towards cellular organization of heterologous pathways and the establishment of cyanobacteria as next generation hosts.