Improving aSynechocystis-based photoautotrophic chassis through systematic genome mapping and validation of neutral sites

Abstract

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms.

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters

“…All the generated constructions were transferred to pSEVA251 or pSEVA351 shuttle vectors (obtained from the 'Standard European Vector Architecture' repository) (34) and the construct harboring the RNA polymerase from phage T7 (P rnpB ::T7pol) was cloned into the pSN15K vector (13). The correct assembly of the generated constructs was confirmed by PCR, restriction analysis and sequencing (STAB VIDA).…”

“…These redesigned T7 promoters were assembled with the GFP generator BBa_E0240 (depicted in Figure 3a, see also Section 2.4), cloned in pSEVA351 and transformed into a Synechocystis mutant with the T7 RNA polymerase under the control of the P rnpB and RBS BBa_B0030 integrated into the chromosomal neutral site N15 (13).…”

“…Nevertheless, the vast majority of the available synthetic biology tools have been developed for heterotrophic chassis like E. coli or Saccharomyces cerevisiae (6,7), and most of the regulatory elements characterized in E. coli function rather poorly or not at all in cyanobacteria (8,9). Consequently, a considerable effort has been placed on the design and construction of efficient, predictable and easy-to-use molecular tools for cyanobacteria (8,(10)(11)(12)(13)(14). Within this context, native promoters such as P psbA2 , P nrsB or the 'super strong' P cpcG560 have been previously used to drive gene expression for specialty chemicals production (11,15,16).…”

Modelling and multiobjective optimization for simulation of cyanobacterial metabolism

“…Moreover, the fact that they are autotrophic organisms with low nutrient requirements, make them even more at-tractive as production platforms (Pinto et al, 2015), since they are able to survive and produce using sun energy, atmospheric carbon and a few inorganic nutrients. This way, cyanobacteria have been placed in the spotlight as basis organisms for the development of cell factories for industrial bioprocesses.…”

“…In particular, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 are two of the most studied species, they are naturally transformable 12 and several molecular tools have been developed for their genetic manipulation (Hess, 2011;Pinto et al, 2015;Kim et al, 2017). Altogether, these properties have heighten the interest of these species as potential cell factories and have motivated the necessity of studying their metabolic functionalities.…”