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

Abstract

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO 2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

Global proteome response ofSynechocystis6803 to extreme copper environments applied to control the activity of the induciblepetJpromoter

“…; Ferreira et al . ). One of them is the petJ promoter (P petJ ) which can be regulated by varying concentrations of Cu 2+ ions present in the growth medium (Diaz et al .…”

“…PCC 6803 (hereafter Synechocystis) is widely used as a model organism for investigations directed to production of chemicals and biofuels by synthetic biology approaches (Phillips and Silver 2006;Hagemann and Hess 2018). Only few inducible promoters are available and utilized for production of valuable chemicals in this strain as well as in other Cyanobacteria (Heidorn et al 2010;Berla et al 2013;Englund et al 2016;Ferreira et al 2018). One of them is the petJ promoter (P petJ ) which can be regulated by varying concentrations of Cu 2+ ions present in the growth medium (Diaz et al 1994;Mitschke et al 2011;Eisenhut et al 2012;Kuchmina et al 2012;Gandini et al 2017;Pade et al 2017).…”

Comparative analysis of inducible promoters in cyanobacteria

“…The P trc promoter, for example, performs well in S. elongatus and is commonly used in many applications 14 . However, efforts to implement similar constructs in Synechocystis have mostly failed, resulting in either extremely leaky expression under non-induced conditions or little to no regulation at all 15 . For example, Camsund et al 2014 investigated sequence-specific repression patterns in Synechocystis 16 .…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Synechocystis sp PCC 6803 (Huang et al, 2010;Huang and Lindblad, 2013;Englund et al, 2016;Ferreira et al, 2018;Liu and Pakrasi, 2018) Synechococcus sp PCC 7002 (Markley et al, 2015;Ruffing et al, 2016;Zess et al, 2016) S. elongatus PCC 7942 Qiao et al, 2018) S. elongatus UTEX 2973 Nostoc sp PCC 712mix0 (Videau et al, 2016;Wegelius et al, 2018) Vectors and Sandardized Assembly…”