Whole‐cell‐based CYP153A6‐catalyzed (S)‐limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL

Abstract

Living microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)-limonene to (S)-perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8-PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate-limited in the two-liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products. In contrast to P. putida KT2440, E. coli W3110 was found to catalyze perillyl aldehyde reduction to the alcohol and no oxidation to the acid. Furthermore, E. coli W3110 harboring CYP153A6 showed high limonene hydroxylation activities (7.1 U g CDW-1). The outer membrane protein AlkL was found to enhance hydroxylation activities of E. coli twofold in aqueous single-phase and fivefold in two-liquid phase biotransformations. In the latter system, E. coli harboring CYP153A6 and AlkL produced up to 39.2 mmol (S)-perillyl alcohol L tot-1 within 26 h, whereas no perillic acid and minor amounts of perillyl aldehyde (8% of the total products) were formed. In conclusion, undesired perillyl alcohol oxidation was reduced by choosing E. coli's enzymatic background as a reaction environment and co-expression of the alkL gene in E. coli represents a promising strategy to enhance terpene bioconversion rates.

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

“…Even so, the whole-cell catalyzed reduction of ␣,␤-unsaturated carbonyl compounds can be complex and hampered by competing enzymatic reactions (8). Thus, in the asymmetric bioreduction of citral to the ␣,␤-saturated aldehyde citronellal, the undesirable by-products nerol, geraniol, and citronellol were formed due to the action of competing alcohol dehydrogenases and citral lyase activity (9); in stereoselective biocatalytic reduction of ␣-ionone by Glomerella cingulata, (6S,9R)-␣-ionol was produced, some of which was subsequently hydrogenated by enoate reductase to form (6S,9R)-7,8-dyhidro-␣-ionol (10); and with use of ␣,␤-unsaturated aldehydes as the substrate, the aldehyde oxidation catalyzed by aldehyde dehydrogenase usually led to the formation of undesirable ␣,␤-unsaturated carboxylic acids (11,12). Therefore, novel biocatalysts with high chemoselectivity are needed for synthesis of ␣,␤-unsaturated alcohols.…”

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

“…Internal SFPR STR extractive phase hexadecane (log P Oct = 8.9) Fontanille and Larroche [34] Internal SFPR STR extractive phase sunflower oil Bicas et al [9] Internal SFPR STR extractive phase bis (2-ethylhexyl) phthalate (log P Oct = 7.5) [21] (continued)…”

“…For instance, limonene is completely removed from an aqueous solution of 7.5 mg/L in a 3-L reactor, which is stirred at 1000 rpm and aerated with 0.9 L/min, within only 30 min [33]. This makes it comprehensible, that even when applying certain ISPR methods substrate and/or product loss may occur in significant amounts [15,21,64]. The following chapters give examples of application of ISPR techniques in bioprocess engineering for synthesis and conversion of isoprenoids using the categories depicted in Fig.…”

“…Also, the plant cell culture based biosynthesis of precursors of the anticancer blockbuster drug Taxol [51] represents an important industrially applied isoprenoid biotechnology. Finally, in the area of microbial isoprenoid conversion, a number of high-yielding approaches have been published as well, for example, for the biotransformations of limonene to perillyl alcohol [21,22], to perillic acid [54], and to α-terpineol [9] or of α-pinene oxide to isonovalal [34], to name only a few. The reader is kindly referred to the respective chapters in the same volume series addressing the biotechnology of specific isoprenoid products.…”

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

“…The products included the ω-alcohol, aldehyde and acid. AlkL has been shown in several reports to facilitate transport of hydrophobic molecules with a logPo/w > 4 58,59,79 .…”