Juteršek Mojca, Dolinar Marko
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
Current Affiliation: National Institute of Biology, Ljubljana, Slovenia.
PeerJ. 2021 Nov 3;9:e12199. doi: 10.7717/peerj.12199. eCollection 2021.
Developing 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 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 sp. PCC 6803.
We 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 and 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.
With the new vector we observed a significantly higher GFP expression in and 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 and 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.
开发可持续的自养细胞工厂在很大程度上依赖于强大且特征明确的生物元件。对于蓝细菌而言，这些元件仍落后于更先进的工具包。在之前使用蓝细菌进行蛋白质表达实验的过程中，我们在使用当前基于RSF1010的穿梭质粒时遇到了不便，特别是由于它们的生物安全性低以及重组蛋白产量低。我们还认识到常用荧光报告基因的一些缺点，因为定量可能会受到蓝细菌固有荧光的影响。为了克服这些缺点，我们设想了一种可用于蓝细菌合成生物学的新型嵌合载体和一种替代报告基因，并在模式蓝细菌集胞藻PCC 6803中对它们进行了测试。
我们基于广泛宿主范围的RSFmob-I复制子设计了pMJc01穿梭质粒。按照RFC10合成生物学标准，使用标准克隆技术进行载体构建。在集胞藻PCC 6803中用选定的调控元件测试了pMJC01用于已确立的绿色荧光蛋白（GFP）报告基因和一种新的报告蛋白胱抑素生物合成的行为。使用木瓜蛋白酶作为同源靶标测定胱抑素活性。
与常用的基于RSF1010的pPMQAK1相比，使用新载体我们在集胞藻PCC 6803中观察到了显著更高的GFP表达。胱抑素是一种半胱氨酸蛋白酶抑制剂，使用新载体在集胞藻PCC 6803中均成功表达。其表达水平使得定量与标准使用的荧光报告基因GFPmut3b相当。新载体的一个重要优点是由于不存在编码接合转移成分的质粒区域，其生物安全性得到了改善。广泛宿主范围的载体pMJc01因其相对较小的尺寸、稳定性和易用性，可在蓝细菌的合成生物学和生物技术中找到应用。此外，胱抑素在所有不含有木瓜蛋白酶型蛋白酶和抑制剂的细胞系统（如蓝细菌）中可能是一种有用的报告基因，并可作为荧光报告基因的替代品或对其进行补充。
