Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells
Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells
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DOI：
10.1016/j.ymben.2023.04.014
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发表时间：
2023-05-02
影响因子：
8.4
通讯作者：
Kunjapur, Aditya M.
中科院分区：
文献类型：
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作者：
Butler, Neil D.;Anderson, Shelby R.;Kunjapur, Aditya M.
醛是有吸引力的化学目标，既作为香料和香料工业中的最终产品，又作为合成中间体，这是由于它们倾向于形成C-C键。在这里，我们确定和解决的芳香醛，包括许多来自生物质降解的模型集合的意外氧化。在E.在需氧条件下生长的大肠杆菌细胞中，如预期的，它们被野生型MG 1655菌株还原或被经改造用于还原芳香醛还原的菌株（E. coliRARE菌株）。令人惊讶的是，当这些相同的醛被补充到E.大肠杆菌菌株，在许多条件下，我们观察到大量的氧化。通过对E.大肠杆菌基因组使用多路自动化基因组工程（法师），我们证明，这种氧化可以大大减缓，与大于50%的保留，8醛时，测定4小时后，他们的添加。鉴于我们的新工程菌株表现出减少芳香醛的氧化和还原，我们将其命名为E。coliROAR菌株。我们将新菌株应用于静息细胞生物催化两种反应-2-糠酸还原为糠醛和3-羟基苯甲醛与甘氨酸缩合形成非标准β-羟基-α-氨基酸。在每种情况下，我们观察到反应开始后20小时产物滴度的显著改善（分别为9倍和10倍）。展望未来，使用该菌株产生静息细胞应允许醛产物分离、进一步酶促转化或在更好地适应醛毒性的细胞环境下的化学反应性。
Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C-C bond formation. Here, we identify and address unex-pected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as ex-pected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supple-mented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic alde-hydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions - the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard beta-hydroxy-alpha-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.