Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing (R)-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes
Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing (R)-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes
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DOI：
10.1128/aem.02622-16
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发表时间：
2016-11
影响因子：
4.4
通讯作者：
M. Rohde;Sylvi Tischer;H. Harms;T. Rohwerder
中科院分区：
文献类型：
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作者：
M. Rohde;Sylvi Tischer;H. Harms;T. Rohwerder
摘要 通过细菌聚 3-羟基丁酸酯 (PHB) 溢出代谢生物技术生产甲基丙烯酸甲酯前体 2-羟基异丁酸 (2-HIBA) 需要合适的 (R)-3-羟基丁酰辅酶 A (CoA) 特异性辅酶 B12 依赖性变位酶 (RCM)。在这里，我们将来自 Bacillus Massiliosenegalensis JC6 的预测变位酶定性为嗜温 RCM，与先前在 Kyrpidia tusciae DSM 2912 中鉴定的嗜热酶密切相关（M.-T. Weichler 等人，Appl Environ Microbiol 81：4564–4572，2015， https://doi.org/10.1128/AEM.00716-15）。使用两种 RCM 变体，在重组扭扭甲基杆菌 AM1 的补料分批生物反应器实验中研究了从甲醇生产 2-HIBA。氮完全消耗后，PHB和2-HIBA同时形成，表明两组RCM基因均成功表达。然而，尽管选择了相同的载体系统和孵育条件，带有来自菌株DSM 2912的RCM基因的变体的代谢活性受到严重抑制，这可能是由于异源表达引起的负面影响。相比之下，表达 JC6 基因的变体的生物量产量接近野生型性能，并且可以证明 2.1 g litre−1 的 2-HIBA 效价。在这种情况下，高达 24% 进入溢出代谢的底物转化为变位酶产物，并且从甲醇中获得的 2-HIBA 加 PHB 的最大组合产量为 0.11 g g−1。逆转录定量PCR分析表明，代谢基因，如甲醇脱氢酶和乙酰乙酰辅酶A还原酶基因，在指数生长后被强烈下调，目前阻止了延长的溢出阶段，从而阻止菌株AM1获得更高的产物产量。重要性在这项研究中，我们对甲基营养型细菌进行了基因改造，以便将其溢出代谢的中间体引导至 C4 羧酸 2-羟基异丁酸（丙烯酸玻璃的前体）。这对生物技术具有影响，因为它表明甲醇和甲酸等 C1 底物减少，可以成为生产当今商品的替代原料。我们发现产物滴度和产量更多地取决于宿主生理学而不是引入的改变溢出代谢的异源功能的活性。此外，我们表明，当重组菌株表达不同来源的直系同源基因时，它们的适应性有很大差异。需要进一步的研究来延长甲基营养微生物的溢流生产阶段以实施生物技术过程。
ABSTRACT The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B12-dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564–4572, 2015, https://doi.org/10.1128/AEM.00716-15 ). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter−1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g−1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1. IMPORTANCE In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.