Weichler Maria-Teresa, Kurteva-Yaneva Nadya, Przybylski Denise, Schuster Judith, Müller Roland H, Harms Hauke, Rohwerder Thore
Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Leipzig, Germany.
Helmholtz Centre for Environmental Research-UFZ, Department of Environmental Microbiology, Leipzig, Germany
Appl Environ Microbiol. 2015 Jul;81(14):4564-72. doi: 10.1128/AEM.00716-15. Epub 2015 Apr 24.
The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed.
最近在嗜温细菌Aquincola tertiaricarbonis L108中发现了一种依赖辅酶B12的酰基辅酶A（acyl-CoA）变位酶，可使3-羟基丁酰-CoA和2-羟基异丁酰-CoA发生异构化反应（N. Yaneva、J. Schuster、F. Schäfer、V. Lede、D. Przybylski、T. Paproth、H. Harms、R. H. Müller和T. Rohwerder，《生物化学杂志》287:15502 - 15511，2012年，http://dx.doi.org/10.1074/jbc.M111.314690），这可能为从可再生碳源构建化学物质2-羟基异丁酸的完整生物合成途径铺平道路。然而，该酶仅以合理的速率催化立体异构体（S）-3-羟基丁酰-CoA的转化，这严重阻碍了变位酶与成熟的细菌聚（R）-3-羟基丁酸（PHB）溢流代谢的有效结合。在此，我们对嗜热嗜氢细菌Kyrpidia tusciae DSM 2912中发现的一种新的2-羟基异丁酰-CoA变位酶进行了表征。重组的变位酶亚基在55°C时显示出最高活性。令人惊讶的是，在30°C时，（R）-3-羟基丁酰-CoA的异构化效率就比L108菌株的变位酶高出约7000倍。这两种变位酶之间最显著的结构差异，可能决定了立体特异性，是L108菌株中位于117位的活性位点残基天冬氨酸被DSM 2912菌株的酶中的缬氨酸取代，导致该结合位点的极性反转。整体序列比较表明，这两种酶均源自不同的原核嗜热甲基丙二酰-CoA变位酶。在37°C下于葡萄糖酸培养基中培养的大肠杆菌JM109和BL21菌株中，将提供（R）-3-羟基丁酰-CoA的PHB酶（来自食酸戴尔福特菌的β-酮硫解酶PhaA和乙酰乙酰-CoA还原酶PhaB）与新的变位酶共表达，可产生最高滴度为0.7 mM的2-羟基异丁酸。文中还讨论了提高大肠杆菌中产量的措施，如伴侣蛋白MeaH的共表达和硫酯酶II的抑制。
