Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Abstract

Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO 2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the P cpc560 or the P trc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.C yanobacteria are oxygenic photosynthetic prokaryotes that use solar energy to fix CO 2 , converting it into biomass and valuable products. Since these organisms do not require fixed carbon feedstocks, they have great potential for synthetic biology and metabolic engineering applications (1-4). Several strains of cyanobacteria have been engineered to act as microbial cellular factories for the production of fuels and chemicals, such as isobutanol, 2,3-butanediol, free fatty acids, and D-lactate (5-7). Despite these advances, none of these engineered strains has been able to achieve industrially relevant levels of productivity. A lack of effective and well-characterized tools for synthetic biology in cyanobacteria has limited progress relative to that with other established microbial chassis, such as Escherichia coli or Saccharomyces cerevisiae (1). Synechocystis sp. strain PCC 6803 is a naturally transformable cyanobacterial chassis for synthetic biology. This strain carries several endogenous plasmids in addition to its single chromosome (8-11). Since there are a limited number of self-replicating exogenous plasmids for the expression of heterologous genes in Synechocystis PCC 6803 (12) and other cyanobacteria, these endogenous plasmids may prove to be attractive parts for synthetic biology (13). Additionally, these plasmids increase their copy numbers during the transition from the exponential-growth phase to the stationary phase (14). Thus, we have hypothesized that the expression of heterologous genes from sites in these plasmids could be autoinduced by such a growth transition. This induction could be advantageous in a production system that first yields a dense culture of light-harvesting cyanobacteria and later uses those microbial cell factories to churn out a product of interest.One strat...

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

“…Synechocystis 6803 strain expressing YFP [50] were cultivated as described above. Following the B-PER and control treatments, cell pellets and medium supernatants were diluted in the ratio of 3:1 (v/v) in 4× Laemmli sample buffer and loaded in equal proportion on a 12% (v/v) Criterion™ TGX Stain-Free™ Protein Gel, which contains trihalo compounds that can react with tryptophan residues under the UV irradiation and gives rise to fluorescence (BioRad, US).…”

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts

“…Recently, a strong constitutive cpc560 promoter has been described, giving a 22‐fold increase in yield of 3‐hydroxypropionic acid relative to the widely used psbA2 promoter (Zhou et al ., ; Wang et al ., ). Heterologous genes are usually inserted into the genome at loci referred to as neutral sites, whose disruption does not affect cell integrity (Ng et al ., ; Pinto et al ., ), or cloned into customizable wide‐host‐range, self‐replicating expression vectors (Taton et al ., ). Issues such as genetic instability and extensive small RNA‐mediated transcriptional regulation also need to be considered when working with cyanobacteria (Mitschke et al ., ; Jones, ; Kopf and Hess, ).…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Pcpc560 (Huang et al, 2010;Zhou et al, 2014;Englund et al, 2016;Ruffing et al, 2016;Ferreira et al, 2018;Li et al, 2018;Liu and Pakrasi, 2018 Ptrc (Huang et al, 2010;Guerrero et al, 2012;Oliver et al, 2013;Markley et al, 2015;Ferreira et al, 2018;Li et al, 2018) TetR (aTc Inducible) L03 (Huang and Lindblad, 2013;Zess et al, 2016;Ferreira et al, 2018) efficient transformation using conjugation and electroporation (compared to homologous recombination); (2) avoidance of the need for time-consuming segregation to achieve incorporation of the transgenic sequence in all copies of the genome; and (3) increased orthogonality of the heterologous sequences, meaning no insertion in genomic loci that may not be neutral. A hybrid strategy that combines the advantages of using a shuttle vector and the stability of genomic insertion involves using native cyanobacterial plasmids, either as insertion sites (Ng et al, 2015) or as true shuttle vectors after isolation and modification (Jin et al, 2018). BioBricks and isothermal assembly techniques are adequate for the generation of a limited number of (Guerrero et al, 2012;Huang and Lindblad, 2013;Oliver et al, 2013;Camsund and Lindblad, 2014).…”