**6. Role of synthetic biology in engineering plastid metabolic pathways**

Though it is the beginning of plastid synthetic biology, advancements are being made to develop the essential tools regarding transgene expression control in chloroplast genome [37]. Currently, most recombinant expression in the plastids involves single-gene constructs created using conventional restriction enzyme-based cloning approaches. This limits the rate at which new transplastomic lines can be produced and tested, and in particular, how many different permutations of constructs (different promoters, coding variants, regulatory elements, etc.) can be evaluated. Currently, various synthetic biology principles are being applied to plastome engineering with the adoption of assembly standards such as Golden Gate and the creation of libraries of validated DNA parts that allow rapid one-step assembly of all the parts [38–40]. More ambitious design strategies involving extensive redesign of the plastome *in silico* are expected to be optimized and validated. Some of the foreseen strategies include removal of large tracts of nonessential DNA [41], refactoration of numerous essential endogenous genes into functional clusters [42], and synchronized engineering of multiple transgenes into different loci. The assembly and delivery of such synthetic genomes is technically feasible, as shown by O'Neill et al. [37], who demonstrated that the entire *C. reinhardtii* plastome could be assembled in yeast and transformed into *C. reinhardtii* by microparticle bombardment. Plastidic intercistronic expression elements (IEEs) can be used for the expression of synthetic operons [43]. The challenge is to develop selection strategies that allow the clean replacement of the endogenous plastome with the synthetic version without undesirable recombination events between the two, resulting in the creation of chimeric plastomes.

Another challenge is to improve the product yield significantly through the use of synthetic cis elements to drive expression. Currently, the promoter and 5′ UTR used to express transgenes are derived from endogenous photosynthetic genes. In some cases, expression levels can be improved by using the stronger promoter from the gene for the 16S ribosomal RNA fused to the 5′ UTR of a photosynthetic gene [44]. However, more often it is the performance of the 5′ UTR that is the bottleneck [45], with the efficiency of translation constrained by either the same feedback regulation that prevents the overaccumulation of individual photosynthetic subunits in the absence of their assembly partners (so-called "control by epistasy of synthesis") or by competition with the corresponding endogenous gene transcript for transacting factors that are required for transcript stability or translation but are present in limiting concentration in the chloroplast [46]. The strategies to overcome this involve either replacement of the 5′ UTR of the endogenous gene with that from another photosynthetic gene [47] or, more elegantly, the development of synthetic variants of the 5′ UTR that are no longer subject to these limitations and therefore enable improved expression of the transgene [48]. Further studies into the design of synthetic promoters and UTRs, combined with improved knowledge of codon optimization rules, will advance the ability to engineer plastid metabolic pathways for customized functionalities and production efficiencies of commercial scale [49].
