Matera Agata, Dulak Kinga, Sordon Sandra, Huszcza Ewa, Popłoński Jarosław
Department of Food Chemistry and Biocatalysis, Wrocław University of Environmental and Life Sciences, C.K. Norwida 25, Wrocław, 50-375, Poland.
J Biol Eng. 2025 Feb 10;19(1):14. doi: 10.1186/s13036-025-00483-2.
Molecular and synthetic biology tools enable the design of new-to-nature biological systems, including genetically engineered microorganisms, recombinant proteins, and novel metabolic pathways. These tools simplify the development of more efficient, manageable, and tailored solutions for specific applications, biocatalysts, or biosensors that are devoid of undesirable characteristics. The key aspect of preparing these biological systems is the availability of appropriate strategies for designing novel genetic circuits. However, there remains a pressing need to explore independent and controllable systems for the co-expression of multiple genes.
In this study, we present the characterisation of a set of bacterial plasmids dedicated to recombinant expression in broadly used Escherichia coli. The set includes plasmids with four different, most commonly used bacterial expression cassettes - RhaS/RhaBAD, LacI/Trc, AraC/AraBAD, and XylS/Pm, which can be used alone or freely combined in up to three-gene monocistronic expression systems using Golden Standard Molecular Cloning kit assembly. The independent induction of each of the designed cassettes enables the autonomous expression of up to three recombinant proteins from one plasmid. The expression of a triple-enzyme cascade consisting of sucrose synthase, UDP-rhamnose synthase and flavonol-7-O-rhamnosyltransferase, confirmed that the designed system can be applied for the complex biocatalysts production.
Presented herein strategy for the multigene expression is a valuable addition to the current landscape of different co-expression approaches. The thorough characterisation of each expression cassette indicated their strengths and potential limitations, which will be useful for subsequent investigations in the field. The defined cross-talks brought a better understanding of the metabolic mechanisms that may affect the heterologous expression in the bacterial hosts.
分子生物学和合成生物学工具能够设计新型生物系统，包括基因工程微生物、重组蛋白和新型代谢途径。这些工具简化了针对特定应用、生物催化剂或无不良特性的生物传感器开发更高效、可管理且量身定制的解决方案的过程。制备这些生物系统的关键在于是否有合适的策略来设计新型基因电路。然而，迫切需要探索用于多个基因共表达的独立且可控的系统。
在本研究中，我们展示了一组专门用于在广泛使用的大肠杆菌中进行重组表达的细菌质粒的特性。该组质粒包括带有四种不同的、最常用的细菌表达盒——RhaS/RhaBAD、LacI/Trc、AraC/AraBAD和XylS/Pm的质粒，这些表达盒可单独使用，也可使用金标准分子克隆试剂盒组装，在多达三基因的单顺反子表达系统中自由组合。对每个设计的表达盒进行独立诱导，可使一个质粒自主表达多达三种重组蛋白。由蔗糖合酶、UDP-鼠李糖合酶和黄酮醇-7-O-鼠李糖基转移酶组成的三酶级联反应的表达证实，所设计的系统可用于生产复杂的生物催化剂。
本文提出的多基因表达策略是对当前不同共表达方法的有价值补充。对每个表达盒的全面表征表明了它们的优势和潜在局限性，这将有助于该领域的后续研究。明确的相互作用有助于更好地理解可能影响细菌宿主中异源表达的代谢机制。
