# Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects
## 4 Emerging Synthetic Biology Technologies  
Several tools for the model cyanobacterium _Synechocystis_ 6803 have recently been developed [107; 108; 109; 12; 100; 112; 101; 102; 103; 104; 105; 106; 107; 108; 109]. The existing methods for distinguishing viable from non-viable cyanobacterial cells are either time-consuming (e.g., plating) or are not easy to prepare (e.g., fluorescent staining). Recently, a cell-viability fluorescence assay was developed that provides a quick and simple way of detecting viable from non-viable cells in a cyanobacterial culture [107]. In this method, red chlorophyll fluorescence and an unspecific green autofluorescence were used for the differentiation of viable and non-viable cells without the need of sample preparation. This method can be used to monitor the fitness of cultures grown for novel industrial biotechnological products [107]. Standardised cloning methods, like shotgun cloning or cDNA cloning [110], have been employed to express exogenous genes or knockout native genes in _Synechocystis_ 6803 [111; 112; 113; 114; 115; 116]. For maximum expression levels of heterologous genes, codon optimisation has been utilised in several studies with favorable effects [116; 117; 118; 116]. Additionally, the development of degradation tags used for the fine-tuning of protein levels and protein turnover times in this model organism have also been achieved [100]. Such advances aid in the generation of more complex metabolic systems that have the potential to oscillate in phase with circadian rhythms of cyanobacteria. Furthermore, recombinant DNA technology has been employed in _Synechocystis_ 6803 for the increased production of carotenoids [101]. In this study, the psbAII promoter was used to drive the transcription and overexpression of coding sequence of several genes involved in carotenoid biosynthesis. The genes cloned in _Synechocystis_ 6803 were the yeast isopentenyl diphosphate isomerase (_ipi_) and the _Synechocystis_ \(\beta\)-carotene hydroxylase (_crtR_) and the linked _Synechocystis_ genes coding for phytoene desaturase and phytoene synthase (_crtP_ and _crtB_, respectively). The expression of _ipi_, _crtR_, and _crtP_ and _crtB_ led to a large increase in the corresponding transcript levels that led to the accumulation of zeaxanthin and myxoxanthophyll in the mutant strains [101]. A study involving increased production of carotenoids from _Synechocystis_ 6803 reported a \(\beta\)-phellandrene yield reaching 1% of the dry cell weight [116]. This was accomplished by expressing \(\beta\)-phellandrene synthase as a fusion protein with the \(\beta\)-subunit of byocyanin. The expression of enzymes of the mevalonic acid (MVA) pathway and geranyl-diphosphate synthase (GPPS) increased substrate availability and overall terpenoid biosynthesis pathway utilisation [116]. Another study reported a pinene synthase mutant with an altered cofactor preference towards magnesium, which possess superior activity to the wild-type [119]. Co-expression of a less active farnesyl-diphosphate synthase (FPPS) increased production of pinene by allowing pinene synthase to outcompete FPPS for GPP [119]. A quick way to create knockins and knockouts and introducing a trans-operon in cyanobacteria was developed [108]. In this method, the open reading frames (ORF) for each desired protein are codon optimised in silico for the chosen cyanobacterial chassis. The intergenic region sequences identified in highly expressed native operons are then added to the sequences to create overlappingparts suitable for assembly where the synthesised parts are assembled in one step [108] into the transformation vector, downstream of a regulatable promoter. The resulting plasmid is then used to transform the chassis strain that contains a negative selectable marker (-ve) inserted into a neutral locus. Two homologous recombination events allow replacement of the marker with the trans-operon, with selection based on the loss of the marker, which results in a marker-less transgenic line.