Tuning the transcription and translation of L-amino acid deaminase in Escherichia coli improves α-ketoisocaproate production from L-leucine.
Tuning the transcription and translation of L-amino acid deaminase in Escherichia coli improves α-ketoisocaproate production from L-leucine.
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DOI：
10.1371/journal.pone.0179229
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Chen J
中科院分区：
文献类型：
--
作者：
Song Y;Li J;Shin HD;Liu L;Du G;Chen J
α-酮异己酸酯 (KIC) 广泛应用于制药和营养保健品行业。在之前的研究中，我们使用表达来自普通变形杆菌的l-氨基酸脱氨酶（l-AAD）的大肠杆菌全细胞生物催化剂，实现了从l-亮氨酸一步生物合成KIC。在此，我们报告了大肠杆菌 BL21 (DE3) 中 l-AAD 基因表达在转录和翻译水平上的微调，以提高 KIC 滴度。通过优化不同拷贝数的质粒起点、调节起始密码子下游的信使RNA结构以及设计核糖体结合位点的序列，我们将生物催化剂活性分别提高到31.77%、24.89%和30.20%，高于BL21/pet28a-lad所达到的水平。 KIC最高效价分别达到76.47 g·L-1、80.29 g·L-1和81.41 g·L-1。此外，这三种工程策略的整合实现了更高的KIC产量（86.55 g·L-1）和更高的L-亮氨酸转化率（94.25%）。本文提出的酶工程策略通常可适用于其他生物催化剂的构建。
α-Ketoisocaproate (KIC) is used widely in the pharmaceutical and nutraceutical industries. In previous studies, we achieved a one-step biosynthesis of KIC from l-leucine, using an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase (l-AAD) from Proteus vulgaris. Herein, we report the fine-tuning of l-AAD gene expression in E. coli BL21 (DE3) at the transcriptional and translational levels to improve the KIC titer. By optimizing the plasmid origin with different copy numbers, modulating messenger RNA structure downstream of the initiation codon, and designing the sequences at the ribosome binding site, we increased biocatalyst activity to 31.77%, 24.89%, and 30.20%, respectively, above that achieved with BL21/pet28a-lad. The highest KIC titers reached 76.47 g·L-1, 80.29 g·L-1, and 81.41 g·L-1, respectively. Additionally, the integration of these three engineering strategies achieved an even higher KIC production of 86.55 g·L-1 and a higher l-leucine conversion rate of 94.25%. The enzyme-engineering strategies proposed herein may be generally applicable to the construction of other biocatalysts.