# Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects
## 4 Emerging Synthetic Biology Technologies
The aim for using intergenic operon sequences taken directly from well-understood and highly expressed operons within the host was to avoid internal transcriptional terminator sequences and to prevent any self-hybridisation or secondary structure in mRNA transcripts. Expression of the trans-operon is induced by a transcriptional activator encoded by a \"regulator\" gene in the chassis strain such that inducer X (e.g., a metal ion) triggers production of the activator (Figure 2). Since the level and strength of gene expression is key for higher product titers, which is of industrial biotechnology interest, a lot of attention has been directed to find ways to increase expression of foreign genes in cyanobacteria. Current methods for the regulation of gene expression in cyanobacteria include native and heterologous promoters that can be regulated by different inputs [26] and the use of strong inducible promoters. The use of different types of inducible promoters in cyanobacteria has been described in detail [29]. A list of inducible promoters that can be used in cyanobacteria can be found in [29] and thus will not be discussed here. Recently, however, efficient heterologous protein production from cyanobacteria was accomplished by utilizing a newly discovered strong promoter (P\({}_{\text{cp560}}\)) [109]. The promoter consists of two predicted promoters from the cpcB gene (sll1577) and 14 predicted transcription factor binding sites (TFBSs) that are crucial for its promotor strength. It was found that using this promoter to express two genes, encoding enzymes involved in metabolism in _Syntechocystis_ 6803 resulted in the buildup of functional proteins at a level of up to 15% of total soluble protein in the cyanobacterium. This level is comparable to that obtained using the _E. coli_ expression system, which demonstrated the strength and efficiency of P\({}_{\text{cp560}}\). These findings demonstrated the potential of utilizing P\({}_{\text{cp560}}\) for the efficient production of recombinant proteins in cyanobacteria. Figure 2: Generation of marker-less mutants through homologous recombination using a codon optimised trans-operon. Yellow boxes are the open reading frame (ORF). The green box is a gene used as a negative marker (-ve). The purple circle is the activator that is produced by addition of inducer \(X\). New methods for the precise regulation of gene expression became also available. For example, theophylline-responsive riboswitches were utilised for the effective regulation of gene expression in four diverse cyanobacterial species [12]. The riboswitches were evaluated for the expression of a yellow fluorescent protein reporter in _Synechococcus elongatus_ PCC 7942, _Leptolyngbya_ sp. strain BL0902, _Anabaena_ sp. strain PCC 7120, and _Synechocystis_ sp. strain WHSyn. The study demonstrated that the riboswitches were effective and superior in regulating gene expression than the commonly used isopropyl-\(\beta\)-d-thiogalactopyranoside induction of a lacI\({}^{\text{q}}\)-P\({}_{\text{trc}}\) promoter system. It has also been shown that the new system can be used to regulate toxic genes (_sacB_), demonstrating the potential of utilizing riboswitches to regulate proteins that are detrimental to biomass accumulation. Such advancement in the field of synthetic biology and genetic engineering in diverse cyanobacteria facilitate the development of microalgae bioengineering. Another tool that has a particularly important industrial application is the development of gene replacement methods that allow for the creation of antibiotic-free genetically modified cyanobacteria (marker-less mutants) [101]. Such strains can be created using the toxic levanusucrase gene (_sacB_) [120] that is illustrated in Figure 3. In addition to the _sacB_ method, a more recent method of creating marker-less mutants has been developed for and demonstrated in the model cyanobacterium _Synechocystis_. In this method, a _mazF_ expression under the nickel-inducible, _nrsB_, promoter was used [121].