Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain<jats:named-content content-type="genus-species">Aquincola tertiaricarbonis</jats:named-content>L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an<jats:italic>mdpJ</jats:italic>knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (<jats:italic>R</jats:italic>)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (<jats:italic>R</jats:italic>)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (<jats:italic>S</jats:italic>)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.</jats:p>

Constitutive Expression of the Cytochrome P450 EthABCD Monooxygenase System Enables Degradation of Synthetic Dialkyl Ethers in Aquincola tertiaricarbonis L108

“…Based on this protocol, ethC resulted in the highest qPCR efficiency and was chosen as the target for qPCR and RT-qPCR experiments. As control genes, we chose mdpJ and hcmA, which encode key enzymes of TBA metabolism in strain L108, i.e., for the TBA monooxygenase (12,13), and the large subunit of the 2-hydroxyisobutyryl-CoA mutase (15), respectively, and the housekeeping 16S rRNA gene. The qPCR was processed in triplicates with 5 ng l Ϫ1 DNA for each reaction and target gene by using the SsoFast EvaGreen super mix according to the manufacturer's protocol on the CFX96 real-time system and analyzed by using the CFX Manager software (Bio-Rad).…”

“…Among them are different AlkB-type and cytochrome P450 systems (5,6). Interestingly, these monooxygenases differ not only in their substrate specificity (e.g., hydroxylation of only methoxy or methoxy and ethoxy groups of tert-alkyl ethers) but also in their isotope fractionation, and thus a critical evaluation of two-dimensional (D/H and 13 C/ 12 C) isotope analysis is needed for assessing degradation activities at contaminated sites (5). The best-studied fuel oxygenate monooxygenase for MTBE and ETBE hydroxylation is the cytochrome P450 EthABCD system found in several Gram-positive bacterial strains of the genera Rhodococcus, Gordonia, and Mycobacterium (7,8,9).…”

Biodegradation of gasoline ether oxygenates

“…2-Methyl-3-buten-2-ol and 3-methylcrotonic acid have been detected as intermediates and further steps in TAA catabolism have been proposed to overlap with the leucine catabolic pathway [43 ]. MpdJK can also transform short chain alcohols such as 2-propanol and 2-butanol [44].…”

Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction

“…Under oxic conditions, alkyl tert-butyl ethers are degraded via tert-butyl alcohol which is further oxidized to 2-methylpropane-1,2-diol (MPD) and 2-HIBA ( Figure 1A) (Zahn et al, 2019). Accordingly, the MPD and 2-HIBA pathways have been studied first in aerobic fuel oxygenate degraders, such as the betaproteobacterium Aquincola tertiaricarbonis L108 (Schäfer et al, 2011(Schäfer et al, , 2012Schuster et al, 2012) and the actinobacterium Mycolicibacterium austroafricanum IFP 2012 (Francois et al, 2002(Francois et al, , 2003Lopes Ferreira et al, 2006a). In the latter, the dehydrogenases MpdB and MpdC catalyze the oxidation of MPD and 2-hydroxyisobutyraldehyde, respectively, to 2-HIBA.…”