Biosynthesis of Medium-Chain ω-Hydroxy Fatty Acids by AlkBGT of Pseudomonas putida GPo1 With Native FadL in Engineered Escherichia coli

Abstract

Hydroxy fatty acids (HFAs) are valuable compounds that are widely used in medical, cosmetic and food fields. Production of ω-HFAs via bioconversion by engineered Escherichia coli has received a lot of attention because this process is environmentally friendly. In this study, a whole-cell bio-catalysis strategy was established to synthesize medium-chain ω-HFAs based on the AlkBGT hydroxylation system from Pseudomonas putida GPo1. The effects of blocking the β-oxidation of fatty acids (FAs) and enhancing the transportation of FAs on ω-HFAs bio-production were also investigated. When fadE and fadD were deleted, the consumption of decanoic acid decreased, and the yield of ω-hydroxydecanoic acid was enhanced remarkably. Additionally, the co-expression of the FA transporter protein, FadL, played an important role in increasing the conversion rate of ω-hydroxydecanoic acid. As a result, the concentration and yield of ω-hydroxydecanoic acid in NH03(pBGT-fadL) increased to 309 mg/L and 0.86 mol/mol, respectively. This whole-cell bio-catalysis system was further applied to the biosynthesis of ω-hydroxyoctanoic acid and ω-hydroxydodecanoic acid using octanoic acid and dodecanoic acid as substrates, respectively. The concentrations of ω-hydroxyoctanoic acid and ω-hydroxydodecanoic acid reached 275.48 and 249.03 mg/L, with yields of 0.63 and 0.56 mol/mol, respectively. This study demonstrated that the overexpression of AlkBGT coupled with native FadL is an efficient strategy to synthesize medium-chain ω-HFAs from medium-chain FAs in fadE and fadD mutant E. coli strains.

Enzymatic Hydroxylations of sp3-Carbons

“…AlkL improved the hydroxylation of n -octane and n -nonane, the natural substrates of Pp AlkB, thereby demonstrating the prominent, supportive role of AlkL in alkane degradation. Furthermore, the influence of signal peptides, promoters and other transport proteins such as FadL on the activity of AlkBs on several substrates has been investigated, leading to novel and increased activities. − Further classical enzyme engineering approaches have not taken place, but metabolic engineering has been successfully employed to increase product formation. More than 1500 AlkB sequences currently exist in the protein database belonging to more than 700 different taxonomic groups, which is a good indicator for the tremendous potential of further investigation regarding this enzyme class. , …”

Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica

“…Previously, biotransformation of fatty acids or FAMEs into their corresponding ω-hydroxy fatty acids was carried out using yeasts ( Lu et al, 2010 ; Durairaj et al, 2015 ) or E. coli ( van Nuland et al, 2016 ; He et al, 2019 ; Yoo et al, 2019 ) where overexpression of alkane monooxygenase or CYP was attempted to introduce a hydroxy group to the substrates. CYPs drew our attention for their ability to introduce a hydroxy group into many complex structures with high regioselectivity ( Urlacher and Girhard, 2019 ), and their application to the production of fatty acid derivatives in Y. lipolytica was quite promising.…”

Enhanced production of nonanedioic acid from nonanoic acid by engineered Escherichia coli

“…FadL expression also decreased to adjust the import of toxic nonanoic acid, as FadL is able to transport extracellular MCFAs into the periplasm. [ 52 ]…”