Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli

Abstract

Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from VT8 (CYP153A ; the strain is also known as VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from GPo1. Using a recombinant strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane-1-dodecanol substrate mixture. This study reports a very efficient method of producing C/C alkanols and C 1,12-alkanediol by whole-cell biotransformation. To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

CYP505E3: A Novel Self‐Sufficient ω‐7 In‐Chain Hydroxylase

“…However, these substrates are most likely not the natural substrates of CYP116B46, since uncoupling with decanoic acid was 11.5 % and the total turnover numbers (TTNs) were 242 with decanoic acid and 27 with dodecanoic acid. Terminal hydroxylation is the main focus of reports on n ‐alkane and fatty alcohol hydroxylation, with very few reports on the latter, despite the potential that not only diterminal diols but also internal alkane diols hold for the synthesis of polymers and even lactones and lactams . Recently, Sakai et al.…”

Dodecanol, metabolite of entomopathogenic fungus Conidiobolus coronatus, affects fatty acid composition and cellular immunity of Galleria mellonella and Calliphora vicina

“…Several organisms including Escherichia coli produce dodecanol 51 ; however, as synthesis from olefins requires high temperature and pressure and the use of heavy metal catalysts, industrial production is derived mainly from palm kernel oil and coconut oil. However, biocatalytic methods using recombinant E. coli strains with an introduced alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol have recently gained attention because of their high stereo- and regioselectivities and mild reaction conditions 52 . The pathway of dodecanol synthesis by the fungus C. coronatus remains unknown, but it will hopefully soon be revealed by the full sequencing of its genome 53 .…”

Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions

“…Effective substrate transport was also targeted by Lee and co-workers, who transferred the CYP153A operon from M. aquaeolei , which included genes encoding the cognate Fd and FdR, to E. coli , in combination with the AlkL alkane transporter from P. putida. ( 60 ) In a 5 L bioreactor containing 2 L of cell culture, and following addition of 200 mL of either dodecane or tetradecane, 1.5 and 2 g L –1 1-dodecanol and 1-tetradecanol, respectively, were produced in 20 h with a dissolved oxygen level of 40%.…”