In Vivo Synthesis of Polyhydroxylated Compounds from a "Hidden Reservoir" of Toxic Aldehyde Species
In Vivo Synthesis of Polyhydroxylated Compounds from a "Hidden Reservoir" of Toxic Aldehyde Species
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DOI：
10.1002/cctc.201700469
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发表时间：
2017-08-09
期刊：
影响因子：
4.5
通讯作者：
Rudroff, Florian
中科院分区：
文献类型：
--
作者：
Bayer, Thomas;Milker, Sofia;Rudroff, Florian
由于内源性酶形成副产物或级联中间体的毒性，活细胞中的合成酶级联往往缺乏效率。高活性的醛物种会引发代谢应激反应，这会导致不希望发生的副反应和产量下降。由于大肠杆菌的代谢背景，醛可能被不可逆地氧化成羧酸或还原成相应的醇。在此，我们应用了一种方法来平衡体内的醛浓度。恶臭假单胞菌的酒精脱氢酶AlkJ将伯醇氧化成相应的醛。通过引入诺卡氏菌的羧酸还原酶，可以从羧酸池中回收目标化合物。通过内源性的大肠杆菌酶进一步将醛还原为醇，完成了醇、醛和羧酸之间的平衡。因此，醛的浓度保持在不可行的浓度以下。我们在几种伯醇上证明了这一概念，这些伯醇在6h内达到氧化还原平衡，并持续到24h，随后与依赖二羟丙酮的醛缩酶(FSA1-A129S，E.Coli)结合，证明反应中的醛物种是可自由获得的，并在较短的反应时间内以70%的产率得到了(3S，4R)-1，3，4-三羟基-5-苯基戊烷-2-酮。
Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli (E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone- dependent aldolase (Fsa1-A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3S, 4R)-1,3,4-trihydroxy-5-phenylpentan- 2-one, in 70% yield within short reaction times.