Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris.
Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris.
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DOI：
10.3389/fbioe.2022.800734
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发表时间：
2022
影响因子：
5.7
通讯作者：
Saha R
中科院分区：
文献类型：
--
作者：
Immethun CM;Kathol M;Changa T;Saha R
利用非模式微生物的独特生物化学能力将扩大生物制造基质、工艺条件和产品的范围。存在固定氮和二氧化碳、从光获得能量、分解代谢甲烷和木质素衍生的芳香族化合物、耐受生理化学应激和恶劣环境条件、大量储存脂质并产生氢的非模式微生物。模型微生物通常只分解单糖，需要低应力条件，但它们已被设计用于可持续制造许多产品，如香水，药品，化妆品，表面活性剂和特种化学品，通常使用合成生物学的工具。转移复杂的途径已被证明是极其困难的，因为该途径发挥功能所需的辅因子、细胞条件和能量来源可能不存在于宿主生物体中。利用独特的生物化学能力也可以通过改造宿主来实现;尽管为模型微生物开发的合成生物学工具通常不像在其他微生物中设计的那样发挥作用。代谢多功能的Rhodopropylenpalustris CGA 009是一种紫色非硫细菌，在有氧和厌氧条件下分解代谢木质素衍生的芳香族化合物，并可以使用光，无机和有机化合物作为其能量来源。R.沼泽藻利用三种固氮酶同工酶来满足其氮需求，同时还产生氢。此外，细菌响应于二氧化碳/碳酸氢盐的可用性产生两种形式的RuBisCo。虽然这种潜在的底盘具有许多有益的性状，但由于其对许多抗生素的内在抗性以及缺乏在这种微生物中研究的合成生物学部分，稳定的异源基因表达一直存在问题。为了解决这些问题，我们对不同选择标记的基因表达和质粒维持进行了表征，启动了一个专门针对光合细菌的合成生物学工具箱。palustris，包括复制起点、荧光报告子、终止子和5′非翻译区，并利用微生物的内源质粒生产外源蛋白。这项工作为R. palustris的许多独特的生物化学过程，并帮助定义了通过可应用于其他非模式微生物的方法在这种有前途的微生物中表达异源基因的原则。
Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology. Transferring complex pathways has proven to be exceedingly difficult, as the cofactors, cellular conditions, and energy sources necessary for this pathway to function may not be present in the host organism. Utilization of unique biochemical capabilities could also be achieved by engineering the host; although, synthetic biology tools developed for model microbes often do not perform as designed in other microorganisms. The metabolically versatile Rhodopseudomonas palustris CGA009, a purple non-sulfur bacterium, catabolizes aromatic compounds derived from lignin in both aerobic and anaerobic conditions and can use light, inorganic, and organic compounds for its source of energy. R. palustris utilizes three nitrogenase isozymes to fulfill its nitrogen requirements while also generating hydrogen. Furthermore, the bacterium produces two forms of RuBisCo in response to carbon dioxide/bicarbonate availability. While this potential chassis harbors many beneficial traits, stable heterologous gene expression has been problematic due to its intrinsic resistance to many antibiotics and the lack of synthetic biology parts investigated in this microbe. To address these problems, we have characterized gene expression and plasmid maintenance for different selection markers, started a synthetic biology toolbox specifically for the photosynthetic R. palustris, including origins of replication, fluorescent reporters, terminators, and 5′ untranslated regions, and employed the microbe’s endogenous plasmid for exogenous protein production. This work provides essential synthetic biology tools for engineering R. palustris’ many unique biochemical processes and has helped define the principles for expressing heterologous genes in this promising microbe through a methodology that could be applied to other non-model microorganisms.