Gillane Rosemary, Daygon Dara, Khalil Zeinab G, Marcellin Esteban
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia.
ARC Centre of Excellence in Synthetic Biology, University of Queensland, Brisbane, QLD, Australia.
Front Bioeng Biotechnol. 2024 Oct 9;12:1468974. doi: 10.3389/fbioe.2024.1468974. eCollection 2024.
Non-proteinogenic amino acids (npAAs) are valuable building blocks for the development of advanced pharmaceuticals and agrochemicals. The surge in interest in their synthesis is primarily due to the potential to enhance and diversify existing bioactive molecules. This can be achieved by altering these bioactive molecules to improve their effectiveness, reducing resistance compared to their natural counterparts or generating molecules with novel functions. Traditional production of npAAs in native hosts requires specialized conditions and complex cultivation media. Furthermore, these compounds are often found in organisms that challenge genetic manipulation. Thus, the recombinant production of these npAAs in a model organism like paves the way for groundbreaking advancements in synthetic biology. Two synthetic operons, comprising of five heterologous proteins were genomically integrated into for the synthesis of npAAs β-methylphenylalanine (BmePhe), β-hydroxyenduracididine (BhEnd), and enduracididine (End). Proteomic and metabolomic analysis confirmed production of these compounds in E. coli for the first time. Interestingly, we discovered that the exogenous addition of pathway precursors to the system enhanced the yield of BmePhe by 2.5 times, whereas it concurrently attenuated the production of BhEnd and End, signifying a selective precursor-dependent yield enhancement. The synthetic biology landscape is broadened in this study by expanding the repertoire of amino acids beyond the conventional set of 22 standard proteinogenic amino acids. The biosynthesized npAAs, End, BhEnd, and BmePhe hold promise for engineering proteins with modified functions, integrating into novel metabolites and/or enhancing biological stability and activity. Additionally, these amino acids' biological production and subsequent purification present an alternative to traditional chemical synthesis methods, paving a direct pathway for pharmacological evaluation.
非蛋白质氨基酸（npAAs）是先进药物和农用化学品开发的重要组成部分。对其合成兴趣激增主要是因为有潜力增强现有生物活性分子并使其多样化。这可以通过改变这些生物活性分子来提高其有效性、降低与天然对应物相比的抗性或产生具有新功能的分子来实现。在天然宿主中传统生产npAAs需要特殊条件和复杂的培养基。此外，这些化合物通常存在于对基因操作有挑战的生物体中。因此，在诸如大肠杆菌这样的模式生物中重组生产这些npAAs为合成生物学的突破性进展铺平了道路。两个由五个异源蛋白组成的合成操纵子被基因组整合到大肠杆菌中以合成npAAsβ-甲基苯丙氨酸（BmePhe）、β-羟基持久霉素（BhEnd）和持久霉素（End）。蛋白质组学和代谢组学分析首次证实了这些化合物在大肠杆菌中的产生。有趣的是，我们发现向大肠杆菌系统中外源添加途径前体可使BmePhe的产量提高2.5倍，而同时会减弱BhEnd和End的产生，这表明产量提高具有选择性的前体依赖性。通过将氨基酸种类扩展到传统的22种标准蛋白质氨基酸之外，本研究拓宽了合成生物学的领域。生物合成的npAAs，End、BhEnd和BmePhe有望用于工程改造具有修饰功能的蛋白质、整合到新型代谢物中以及/或增强生物稳定性和活性。此外，这些氨基酸的生物生产及随后的纯化提供了一种替代传统化学合成方法的途径，为药理学评估铺平了直接道路。
