Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Abstract

Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, l-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology. Biotechnol. Bioeng. 2017;114: 1561-1569. © 2017 Wiley Periodicals, Inc.

Regulatory systems for gene expression control in cyanobacteria

“…Tight repression of PBAD in the absence of arabinose was also observed in Sy_6803. However, expression levels in this strain were rather low (Immethun et al 2017). Similar results were obtained for the rhamnose-induced and RhaS-regulated promoter PrhaBAD from E. coli.…”

“…The E. coli-derived PBAD is induced in the presence of arabinose and repressed by the transcription factor AraC. It was first used and further characterised in Sce_7942 (Cao et al 2017;Huang et al 2016) and subsequently optimised for applications in Sy_6803 (Immethun et al 2017). In Sce_7942, PBAD yielded a relatively high activity (approximately 50% of Ptrc) and low expression in the absence of the inducer, resulting in a 3500-fold dynamic range (Cao et al 2017).…”

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

“…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).…”

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

“…Notably, systems inducible by sugars, including l ‐arabinose and l ‐rhamnose, have been introduced to cyanobacteria. In addition, cyanobacteria have been engineered to respond to environmental signals including darkness, O 2 , and nitrogen sources . These environmental signal‐ and sugar‐based induction methods are nontoxic and economical, hence making them particularly industrially relevant for biofuel and biochemical production in cultivation lagoons or bioreactors.…”