Engineered Corynebacterium glutamicum as the Platform for the Production of Aromatic Aldehydes

Abstract

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 <i>Corynebacterium glutamicum</i> 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 <i>in vivo</i> since the <i>NCgl0324</i> deletion strain HB-<i>Δ0324</i> produced 1.36 g/L of 4-HB aldehyde, that is, about 188% more than its parental strain. To demonstrate that <i>NCgl0324</i> knockout can also improve production of PC aldehyde and vanillin, first, a basal MA303 strain that produces protocatechuate was engineered from 4-hydroxybenzoate-synthesizing <i>C. glutamicum</i> APS963, followed by deletion of <i>NCgl0324</i> to generate PV-<i>Δ0324</i>. 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 <i>O</i>-methyltransferase encoded by a mutated <i>comt</i> ^m gene. In shake flask culture, the resulting <i>NCgl0324</i> deletion strains PV-I<i>Δ0324</i> and PV-IY<i>Δ0324</i> were shown to produce 1.18 g/L PC aldehyde and 0.31 g/L vanillin, respectively. Thus, modulation of the identified <i>NCgl0324</i> gene was shown to have the potential to boost production of valuable aromatic aldehydes and alcohols.