A synthetic biology approach to engineer novel commercial variants of cyanobacterial pigment proteins

Phycocyanin (PC) is a high-value blue pigment protein obtained from cyanobacteria and red algae. Inside the cell it is mainly responsible for light absorption at 620 nm as part of the light harvesting phycobilisome (PBS) complex. In addition to its brilliant blue colour, PC has potent anti-oxidant and fluorescence properties, and is therefore in high demand in biopharmaceutical, nutraceutical and food industries. However, the narrow stability range of commercially available PC extracted from Arthrospira platensis limits potential applications. In this thesis I explore a synthetic biology approach to engineer cyanobacterial strains to produce non-native variants of PC with improved temperature and pH stability properties. Firstly, I compiled a detailed review describing PC found in organisms that inhabit extreme environments, such as high temperature and low pH conditions. The review also discusses potential applications of PC that could benefit from improved stability. In parallel, my colleagues and I produced a JoVE transconjugation protocol describing genetic modification of cyanobacteria, which lied in the foundation of the following chapters. In my third and fourth chapters, I used a Synechocystis PCC 6803 (hereafter Synechocystis) mutant ‘Olive’ strain lacking the PC operon (ΔcpcBAC2C1D) as a background to express the PC operon (cpcBACD) from the thermophilic cyanobacterium Thermosynechococcus elongatus (BP-1) on a self-replicating RSF1010-based plasmid. The results indicated that heterologous PC subunits (Te-PC) were properly expressed and functional, and improved the growth phenotype of the Olive mutant. Further analyses revealed that Te-PC was expressed at comparable levels to native PC in WT Synechocystis, while maintaining thermostability properties similar to that of the PC from T. elongatus. I then demonstrated a pilot scale (120 L total) batch production, extraction and purification process for Te-PC from the complemented Olive mutant to investigate whether the engineered strain could be used as a platform for commercial production of thermostable PC. Absence of kanamycin had no negative impact on growth rates or Te-PC content. Efficient biomass flocculation of Synechocystis cells was achieved using chitosan, and the conditions for high-pressure homogenisation were optimised to maximise Te-PC extraction and minimise release of contaminants such as chlorophyll. Subsequent Te-PC purification was performed using a novel two-step ammonium sulfate and a heat treatment approach to yield near analytical Te-PC purity levels and an 84 ± 12% recovery of Te-PC. Finally, I attempted to express two PC operons in the Olive mutant from one of the most hyperthermophilic cyanobacterium yet identified. Preliminary evidence suggests that transconjugant Olive strains expressing either of the PC operons showed reduced growth phenotype compared to Olive mutant. Analysis of the soluble cell extract confirmed that novel PC subunits were expressed and at least partially matured. However, the levels of PC expression were significantly reduced compared to the WT Synechocystis. In addition, the upstream putative promoter sequences from both PC operons were analysed and compared with the strong native PC operon promoter in Synechocystis using an eYFP expression cassette. In white and red light conditions, the strength of the new promoters was high and comparable to the native PC operon promoter. In blue light the strength of the new promoters remained high, while the native PC operon promoter was inactive. In green light the expression was low in all promoters analysed. The discovery and characterisation of the two new strong promoters will be a valuable addition to the rapidly developing field of cyanobacterial synthetic biology. However, additional work is required to further understand and improve the expression of the PC from hyperthermophilic cyanobacteria in Synechocystis

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community