Abstract
BackgroundDeveloping sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803.MethodsWe designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target.ResultsWith the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.
Highlights
Cyanobacteria are emerging as versatile hosts for synthetic biology
We focused on three drawbacks that the majority of currently used episomal vectors and reporters for cyanobacteria have: low biosafety, low copy number
We believe that the use of an alternative reporter with a different type of signal generation should be used for verification of fluorescence measurements of biological parts of interest, and we propose cystatin as a promising reporter
Summary
Cyanobacteria are emerging as versatile hosts for synthetic biology. Starting as poorly adapted microorganisms for genetic engineering, advances in the last decade have made them attractive organisms for biosynthetic production of commodities ranging from biofuels to high added-value compounds. A book (Zhang & Song, 2018), several book chapters (e.g., Klemenčič et al, 2017; Badary & Sode, 2020; Hudson, 2021) and review articles (Khan et al, 2019; Lindblad et al, 2019; Santos-Merino, Singh & Ducat, 2019; Vavitsas et al, 2019; Ng, Keskin & Tan, 2020; Wang, Gao & Yang, 2020; Liu et al, 2021; Sproles et al, 2021) published in recent years emphasize the potential of cyanobacteria and present encouraging results obtained with these hosts To realize their full application potential as green engineered biofactories, a set of well-characterized genetic elements is necessary to provide a starting material for higher-order synthetic systems. Cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them
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