DataSheet1_Engineered Corynebacterium glutamicum as the Platform for the Production of Aromatic Aldehydes.docx

Aromatic aldehydes, including 4-hydroxybenzaldehyde (4-HB aldehyde), protocatechuic (PC) aldehyde, and vanillin, are used as important flavors, fragrances, and pharmaceutical precursors and have several biological and therapeutic effects. Production of aromatic aldehydes in microbial hosts poses a challenge due to its rapid and endogenous reduction to alcohols. To address this hurdle, prospecting of the genome of Corynebacterium glutamicum yielded 27 candidate proteins that were used in comprehensive screening with a 4-hydroxybenzyl (4-HB) alcohol–producing strain. We identified that NCgl0324 has aromatic aldehyde reductase activity and contributed to 4-HB aldehyde reduction in vivo since the NCgl0324 deletion strain HB-Δ0324 produced 1.36 g/L of 4-HB aldehyde, that is, about 188% more than its parental strain. To demonstrate that NCgl0324 knockout can also improve production of PC aldehyde and vanillin, first, a basal MA303 strain that produces protocatechuate was engineered from 4-hydroxybenzoate-synthesizing C. glutamicum APS963, followed by deletion of NCgl0324 to generate PV-Δ0324. The PC aldehyde/alcohol or vanillin/vanillyl alcohol biosynthetic pathways, respectively, were able to be expanded from protocatechuate upon introduction of carboxylic acid reductase (CAR) and catechol O-methyltransferase encoded by a mutated comtm gene. In shake flask culture, the resulting NCgl0324 deletion strains PV-IΔ0324 and PV-IYΔ0324 were shown to produce 1.18 g/L PC aldehyde and 0.31 g/L vanillin, respectively. Thus, modulation of the identified NCgl0324 gene was shown to have the potential to boost production of valuable aromatic aldehydes and alcohols.

芳香醛类化合物，包括4-羟基苯甲醛（4-HB 醛）、原儿茶醛（PC 醛）和香草醛，作为重要的风味、香料及制药前体，具有多种生物学和治疗效果。在微生物宿主中生产芳香醛面临挑战，因为其迅速且内源性地还原为醇类。为克服这一难题，对谷氨酸棒杆菌（Corynebacterium glutamicum）的基因组进行挖掘，发现了27个候选蛋白。这些蛋白通过与产4-羟基苄醇（4-HB）的菌株进行综合筛选而被利用。研究表明，NCgl0324具有芳香醛还原酶活性，并参与体内4-HB 醛的还原过程，因为NCgl0324缺失菌株HB-Δ0324产生了1.36 g/L的4-HB 醛，比其亲本菌株高出约188%。为了证明NCgl0324敲除也能提高PC 醛和香草醛的生产，首先，从合成4-羟基苯甲酸的谷氨酸棒杆菌APS963中构建了一个基础MA303菌株，该菌株能产生原儿茶酸。随后，通过删除NCgl0324生成PV-Δ0324。在引入突变comtm基因编码的羧酸还原酶（CAR）和儿茶酚-O-甲基转移酶后，原儿茶酸/醇或香草醛/香草醛醇的生物合成途径得以分别从原儿茶酸中扩展。在摇瓶培养中，得到的NCgl0324缺失菌株PV-IΔ0324和PV-IYΔ0324分别产生了1.18 g/L的PC 醛和0.31 g/L的香草醛。因此，对所确定的NCgl0324基因的调节已被证明具有提升有价芳香醛和醇类生产的潜力。