Martinez-Gomez N Cecilia, Good Nathan M, Lidstrom Mary E
Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
Department of Microbiology, University of Washington, Seattle, Washington, USA.
J Bacteriol. 2015 Jun 15;197(12):2020-6. doi: 10.1128/JB.02595-14. Epub 2015 Apr 6.
During an environmental perturbation, the survival of a cell and its response to the perturbation depend on both the robustness and functionality of the metabolic network. The regulatory mechanisms that allow the facultative methylotrophic bacterium Methylobacterium extorquens AM1 to effect the metabolic transition from succinate to methanol growth are not well understood. Methenyl-dephosphotetrahydromethanopterin (methenyl-dH4MPT), an early intermediate during methanol metabolism, transiently accumulated 7- to 11-fold after addition of methanol to a succinate-limited culture. This accumulation partially inhibited the activity of the methylene-H4MPT dehydrogenase, MtdA, restricting carbon flux to the assimilation cycles. A strain overexpressing the gene (mch) encoding the enzyme that consumes methenyl-dH4MPT did not accumulate methenyl-dH4MPT and had a growth rate that was 2.7-fold lower than that of the wild type. This growth defect demonstrates the physiological relevance of this enzymatic regulatory mechanism during the acclimation period. Changes in metabolites and enzymatic activities were analyzed in the strain overexpressing mch. Under these conditions, the activity of the enzyme coupling formaldehyde with dH4MPT (Fae) remained constant, with concomitant formaldehyde accumulation. Release of methenyl-dH4MPT regulation did not affect the induction of the serine cycle enzyme activities immediately after methanol addition, but after 1 h, the activity of these enzymes decreased, likely due to the toxicity of formaldehyde accumulation. Our results support the hypothesis that in a changing environment, the transient accumulation of methenyl-dH4MPT and inhibition of MtdA activity are strategies that permit flexibility and acclimation of the metabolic network while preventing the accumulation of the toxic compound formaldehyde.
The identification and characterization of regulatory mechanisms for methylotrophy are in the early stages. We report a nontranscriptional regulatory mechanism that was found to operate as an immediate response for acclimation during changes in substrate availability. Methenyl-dH4MPT, an early intermediate during methanol oxidation, reversibly inhibits the methylene-H4MPT dehydrogenase, MtdA, when Methylobacterium extorquens is challenged to switch from succinate to methanol growth. Bypassing this regulatory mechanism causes formaldehyde to accumulate. Fae, the enzyme catalyzing the conversion of formaldehyde to methylene-dH4MPT, was also identified as another potential regulatory target using this strategy. The results herein further our understanding of the complex regulatory network in methylotrophy and will allow us to improve metabolic engineering strategies of methylotrophs for the production of value-added products.
在环境扰动期间，细胞的存活及其对扰动的反应取决于代谢网络的稳健性和功能。兼性甲基营养细菌扭脱甲基杆菌AM1实现从琥珀酸到甲醇生长代谢转变的调控机制尚不清楚。亚甲基 - 脱磷酸四氢甲烷蝶呤（methenyl-dH4MPT）是甲醇代谢过程中的早期中间体，在向琥珀酸限制培养物中添加甲醇后，其瞬时积累了7至11倍。这种积累部分抑制了亚甲基 - H4MPT脱氢酶MtdA的活性，限制了碳流向同化循环。过表达编码消耗methenyl-dH4MPT的酶的基因（mch）的菌株没有积累methenyl-dH4MPT，其生长速率比野生型低2.7倍。这种生长缺陷证明了这种酶促调节机制在适应期的生理相关性。对过表达mch的菌株中的代谢物和酶活性变化进行了分析。在这些条件下，将甲醛与dH4MPT偶联的酶（Fae）的活性保持恒定，同时伴随着甲醛积累。methenyl-dH4MPT调节的解除在添加甲醇后立即不影响丝氨酸循环酶活性的诱导，但在1小时后，这些酶的活性下降，可能是由于甲醛积累的毒性。我们的结果支持这样的假设，即在不断变化的环境中，methenyl-dH4MPT的瞬时积累和MtdA活性的抑制是允许代谢网络灵活适应同时防止有毒化合物甲醛积累的策略。
甲基营养调控机制的鉴定和表征尚处于早期阶段。我们报道了一种非转录调控机制，它被发现是在底物可用性变化期间作为适应的即时反应而发挥作用。当扭脱甲基杆菌面临从琥珀酸生长转变为甲醇生长的挑战时，甲醇氧化过程中的早期中间体methenyl-dH4MPT可逆地抑制亚甲基 - H4MPT脱氢酶MtdA。绕过这种调节机制会导致甲醛积累。使用该策略，催化甲醛转化为亚甲基 - dH4MPT的酶Fae也被鉴定为另一个潜在的调控靶点。本文的结果进一步加深了我们对甲基营养中复杂调控网络的理解，并将使我们能够改进甲基营养菌用于生产增值产品的代谢工程策略。
