With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl-CoA synthase (ACS), enabled C. acetobutylicum to catalyze both CO2 reduction and CO oxidation. Importantly, CODH exhibited activity in both the presence and absence of ACS. 13C-tracer studies confirmed that the engineered C. acetobutylicum strains can reduce CO2 to CO and oxidize CO during growth on glucose.

译文

随着合成生物学的最新进展,假设电子的可用性,CO2可以被天然或工程生物用作碳原料。自养CO2固定中使用的两种关键酶是CO脱氢酶 (CODH) 和乙酰辅酶a (乙酰辅酶a) 合酶 (ACS),它们形成了双功能的异四聚体复合物。CODH/ACS复合物可以可逆地将CO2催化为CO,从而在环境温度和压力下有效地实现了生物水煤气变换反应。CODH/ACS复合物是乙酰原菌用来固定CO2的Wood-Ljungdahl途径 (WLP) 的一部分,并且在天然宿主中已得到很好的表征。到目前为止,在异源宿主中仅表达了少数重组CODH/ACS复合物,但均未证明体内CO2还原。在这里,羧基梭菌CODH/ACS复合物的功能性表达在丙酮丁醇梭菌中得到证实,该梭菌经工程设计可单独或与ACS一起表达CODH。两种菌株均表现出CO2还原和CO氧化活性。使用同位素标记对CODH反应进行了询问,从而验证了CO是CO2还原的直接产物,反之亦然。CODH显然使用天然的丙酮丁基铁氧还蛋白作为减少CO2的电子载体。异源CODH活性取决于活跃生长的细胞,需要添加镍,镍被插入CODH中,而不需要表达天然的Ni插入酶蛋白。气相中CO浓度的增加会抑制CODH活性并改变表达CODH的细胞的代谢物谱。这项工作为在非本地宿主生物体中设计完整且功能性的WLP奠定了基础。重要的是,来自碳衣梭菌的CO脱氢酶 (CODH) 的功能表达在丙酮丁基梭菌中得到证明,该菌本身无法固定CO2。CODH单独或与C. carboxidivorans乙酰辅酶a合酶 (ACS) 一起表达,使C.Acetutylicum能够催化CO2还原和CO氧化。重要的是,CODH在存在和不存在ACS时均表现出活性。13c-示踪剂研究证实,工程改造的丙酮丁醇C菌株可以将CO2还原为CO,并在葡萄糖上生长过程中氧化CO。

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