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Abstract：
The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.
摘要：
精氨酸和赖氨酸生物合成途径之间的进化关系已经在细菌和超嗜热古细菌中得到了很好的建立，但在卤古细菌中仍然未知。这里,利用内源性CRISPR-Cas系统编辑了卤代古菌NatrinemagariJ7-2中精氨酸和赖氨酸生物合成相关基因。ΔargW，ΔargX,ΔargB,和ΔargD突变菌株显示精氨酸营养缺陷型表型，而ΔdapB突变体显示赖氨酸营养缺陷型表型，表明菌株J7-2利用ArgW介导的途径和二氨基庚二酸（DAP）途径合成精氨酸和赖氨酸，分别。与大肠杆菌中的ArgD在精氨酸生物合成途径和DAP途径中作为双功能转氨酶不同，菌株J7-2中的ArgD仅参与精氨酸的生物合成。同时,在菌株J7-2中，argB的功能不能通过DAP途径中的进化对应物ask来补偿。此外，菌株J7-2不能利用α-氨基己二酸（AAA）通过ArgW介导的途径合成赖氨酸，与使用双功能LysW介导的途径从谷氨酸和AAA合成精氨酸（或鸟氨酸）和赖氨酸的超嗜热古细菌相反，分别。此外,用其超嗜热古细菌同源物替换J7-2ArgX菌株的底物特异性的5个氨基酸特征基序不能赋予ΔdapB突变体从AAA生物合成赖氨酸的能力。体外分析显示菌株J7-2ArgX作用于谷氨酸而不是AAA。这些结果表明，菌株J7-2的精氨酸和赖氨酸生物合成途径在进化过程中高度专业化。重要性由于它们在氨基酸代谢和密切的进化关系中的作用，精氨酸和赖氨酸生物合成途径代表了探索代谢途径功能专业化的有趣模型。与细菌和超嗜热古细菌相比，目前有关卤代古细菌的精氨酸和赖氨酸生物合成的知识有限。我们的结果表明，卤代古菌NatrinemagariJ7-2采用ArgW介导的途径和DAP途径进行精氨酸和赖氨酸生物合成，分别,两种途径在功能上相互独立；同时，ArgX是菌株J7-2中ArgW介导途径的底物特异性的关键决定因素。这项研究提供了有关盐生古细菌氨基酸代谢的新线索，并证实了基于内源性CRISPR-Cas系统的基因组编辑在盐生古细菌中的便利性和效率。
