Kim Hyun-Song, Choi Jung-A, Kim Bu-Yeon, Ferrer Lenny, Choi Jung-Min, Wendisch Volker F, Lee Jin-Ho
Department of Food Science and Biotechnology, Kyungsung University, Busan, South Korea.
Genetics of Prokaryotes, Faculty of Biology and Center for Biotechnology, Bielefeld University, Bielefeld, Germany.
Front Bioeng Biotechnol. 2022 May 12;10:880277. doi: 10.3389/fbioe.2022.880277. eCollection 2022.
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 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 since the deletion strain HB- produced 1.36 g/L of 4-HB aldehyde, that is, about 188% more than its parental strain. To demonstrate that knockout can also improve production of PC aldehyde and vanillin, first, a basal MA303 strain that produces protocatechuate was engineered from 4-hydroxybenzoate-synthesizing APS963, followed by deletion of to generate PV-. 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 -methyltransferase encoded by a mutated gene. In shake flask culture, the resulting deletion strains PV-I and PV-IY were shown to produce 1.18 g/L PC aldehyde and 0.31 g/L vanillin, respectively. Thus, modulation of the identified gene was shown to have the potential to boost production of valuable aromatic aldehydes and alcohols.
包括4-羟基苯甲醛（4-HB醛）、原儿茶酸（PC）醛和香草醛在内的芳香醛被用作重要的香料、香精和药物前体，并且具有多种生物学和治疗作用。由于其会迅速内源性还原为醇类，因此在微生物宿主中生产芳香醛具有挑战性。为了解决这一障碍，对[未提及具体微生物名称]的基因组进行勘探，得到了27种候选蛋白，并将其用于与一株产4-羟基苄醇（4-HB醇）的菌株进行全面筛选。我们发现NCgl0324具有芳香醛还原酶活性，并导致4-HB醛减少，因为NCgl0324缺失菌株HB-产生了1.36 g/L的4-HB醛，即比其亲本菌株多约188%。为了证明敲除NCgl0324也能提高PC醛和香草醛的产量，首先，从合成4-羟基苯甲酸的APS963构建了一株产生原儿茶酸的基础MA303菌株，随后敲除NCgl0324以产生PV-。在引入由突变的[未提及具体基因名称]基因编码的羧酸还原酶（CAR）和儿茶酚-O-甲基转移酶后，分别能够从原儿茶酸扩展PC醛/醇或香草醛/香草醇生物合成途径。在摇瓶培养中，所得的NCgl0324缺失菌株PV-I和PV-IY分别显示能产生1.18 g/L的PC醛和0.31 g/L的香草醛。因此，对鉴定出的NCgl0324基因进行调控显示出有提高有价值的芳香醛和醇产量的潜力。
