In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)
In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)
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DOI：
10.3390/catal10080935
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发表时间：
2020-08
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影响因子：
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通讯作者：
Sarah Maxel;Linyue Zhang;Edward King;Ana Paula Acosta;R. Luo;Han Li
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作者：
Sarah Maxel;Linyue Zhang;Edward King;Ana Paula Acosta;R. Luo;Han Li
来自不动杆菌属的环己酮单加氧酶 (CHMO)。 NCIMB 9871 的特点是具有广泛的底物通用性，可将（环）酮生物氧化成具有高立体特异性的酯和内酯。尽管具有工业潜力，但 CHMO 的使用受到热稳定性差的限制。有限的高通量筛选工具和合理工程热稳定性方面的挑战阻碍了 CHMO 工程工作。我们展示了需氧、高通量生长选择平台在大肠杆菌（MX203 菌株）中的应用，以发现 CHMO 的热稳定性增强突变。该选择利用生长来轻松读取体内 CHMO 活性，需要烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 消耗酶来恢复细胞氧化还原平衡。在天然底物环己酮存在的情况下，从 42°C 筛选的随机诱变文库中发现了变体 CHMO GV (A245G-A288V)。与野生型相比，该变体保留了天然活性，30°C 孵育后残留活性提高了约 4.4 倍，并且在 37°C 下环己酮转化率提高了约 5 倍。分子模型表明 CHMO GV 具有更有利的残基堆积并支持额外的主链氢键。进一步合理的设计使得 CHMO A245G-A288V-T415C 在 45 °C 下具有更高的热稳定性。我们的加氧酶进化平台能够快速工程化蛋白质稳定性，这对于工业可扩展性至关重要。
Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.