Heterologous expression of cobalamin dependent class-III enzymes.
Heterologous expression of cobalamin dependent class-III enzymes.
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DOI：
10.1016/j.pep.2020.105743
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发表时间：
2021-01
影响因子：
1.6
通讯作者：
Leys D
中科院分区：
文献类型：
--
作者：
Halliwell T;Fisher K;Payne KAP;Rigby SEJ;Leys D
钴胺素III类依赖性酶家族由还原性脱卤酶（RDases）和相关的环氧肌苷还原酶组成。RDases是有机卤化物呼吸过程中能量守恒的关键。这些酶能够还原性切割存在于许多对环境有害的污染物中的碳-卤键，使它们在生物修复应用中引起了人们的极大兴趣。不幸的是，它是很难获得足够的产量纯RDase分离有机卤化物呼吸细菌的生化研究。因此，需要稳健的异源表达系统，其产生需要铁-硫簇和钴胺素掺入两者的活性全酶。我们目前的异源表达菌株巨大芽孢杆菌，大肠杆菌HMS 174（DE 3），Shimwellia blattae和产钠弧菌的商业菌株，钴胺素III类依赖酶的表达的比较研究。以太平洋硝化还原菌pht-3B还原性脱卤酶（NpRdhA）和嗜热链球菌环氧肌苷还原酶（StoQ）为模型酶。我们还分析了钴胺素转运蛋白BtuB的共表达是否支持钴胺素在E.杆菌我们的结论是，虽然在巨大芽孢杆菌中的表达导致了最高水平的辅因子掺入，但在E.在两种情况下，大肠杆菌在辅因子掺入和蛋白质产量之间呈现适当的平衡。还原性脱卤酶和环氧肌苷还原酶是钴胺素依赖性的。辅因子掺入对这些酶的异源表达提出了挑战。一系列细菌宿主被评估为这些酶的表达宿主。观察到蛋白质产量和辅因子含量之间呈负相关。与BtuB B12转运蛋白在E.大肠杆菌中获得了最好的结果。
The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases. Reductive dehalogenases and epoxyqueuosine reductases are cobalamin-dependent. Cofactor incorporation presents a challenge for heterologous expression of these enzymes. A range of bacterial hosts were assessed as expression host for these enzymes. An inverse correlation between protein yield and cofactor content was observed. Co-expression with the BtuB B12 transporter in E. coli yielded best results.
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