Abstract
α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.
Highlights
Cytochrome P450 monooxygenases (CYPs) are oxidoreductases that catalyze the insertion of an oxygen atom into diverse substrates, with excellent regio-/stereo-selectivity (Park et al, 2020b); CYP consists of heme-thiolate structures in its catalytic core
Putidaredoxin camB and putidatedoxin reductase camA from Pseudomonas putida were overexpressed as the redox proteins for CYP catalysis, and the long-chain fatty acid transporter fadL from Escherichia coli was overexpressed in E. coli BW25113(DE3)ΔfadD (Bae et al, 2014; Park and Choi, 2020)
The CYP153A33-encoding gene was co-expressed with CamAB redox proteins for the whole-cell biotransformation of 1dodecanol. 10 mM of 1-Dodecanol (A) was used as the substrate, and the whole-cell bioconversion resulted in diverse production profiles, including α,ω-dodecanediol (B), dodecanoic acid (C), ω-hydroxydodecanoic acid (D), and α,ω-dodecanedioic acid (E) (Figure 1)
Summary
Cytochrome P450 monooxygenases (CYPs) are oxidoreductases that catalyze the insertion of an oxygen atom into diverse substrates, with excellent regio-/stereo-selectivity (Park et al, 2020b); CYP consists of heme-thiolate structures in its catalytic core. Α,ω-alkanediols are versatile chemicals that can be obtained via the consecutive oxidation of alkanes/1alkanol or the reduction of diacids by carboxylic acid reductase (Kirillova et al, 2009; Olmedo et al, 2016; Hsieh et al, 2018). They are widely used as monomer precursors for polyesters, polyamides, and polyurethane, through cascade oxidation, amination, and polymerization reactions (Ahsan et al, 2018). Α,ω-alkanediols are widely used as building blocks for polyester synthesis through direct esterification reactions (Dai et al, 2017)
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