Regio- and Stereoselective Fungal Oxyfunctionalisation of Limonenes

Abstract

Selective transformations of limonene by asco- and basidiomycetes were investigated. On the shake flask scale, Penicillium citrinum hydrated R-(+)-limonene to α-terpineol [83% regioselectivity (rs), more than 80 mg 1-1 product yield], and Gongronella butleri catalysed the terminal oxidation to yield perillyl alcohol (60% rs, 16 mg 1-1). On the laboratory bioreactor scale, Penicillium digitatum produced a peak concentration of 506 mg α-terpineol 1-1 in the fed-batch mode, equivalent to a theoretical yield of 67%, and no volatile by-products were found. Fusarium proliferatum transformed R-(+)-limonene enantiospecifically to cis-(+)- carveol (98.6% ee, more than 35 mg 1-1 product yield) and S-(-)-limonene predominantly to trans-(-)-carveol (96.3% ee). Pleurotus sapidus selectively dehydrogenised the accumulating trans-(-)-carveol to the corresponding enantiopure R-(-)-carvone. The results show that a careful selection of strain and bioprocess parameters may improve both the yield and the optical purity of a desired product.

Improved monoterpene biotransformation with Penicillium sp. by use of a closed gas loop bioreactor

“…1). For the latter biotransformation, our results represent a considerable improvement compared with data obtained in a conventional bioprocess set-up previously reported [27]. Fig.…”

“…Impact of monoterpene concentration on biotransformation and growth P. digitatum DSM 62840 had been previously shown to catalyze the conversion of limonene to -terpineol in a highly regioselective way and at concentrations of up to several hundreds of milligrams in a conventional bioreactor setup [27]. However, limonene, known for its antimicrobial activity, had not been systematically investigated regarding its potential toxicity towards P. digitatum DSM 62840.…”

“…For comparison, previous cultivations of the same strain in a conventional bioreactor setup without gas loop under otherwise identical conditions, i.e., by applying the same bioprocess parameters including the sequential limonene feeding, yielded Wnal biomass concentrations of up to 4.5 g L ¡1 and a maximum concentration of 506 mg L ¡1 after 172 h (as indicated by the dotted line in Fig. 4) [27]. The additional glucose feeding performed in biotransformation 2 did not signiWcantly inXuence productivity and Wnal product concentration, indicating that the C-source which had been depleted during the growth period was not limiting in the subsequent biotransformation period.…”

“…Previous experiments revealed that, using a conventional open bioreactor system, a signiWcant amount of the volatile monoterpene used as biotransformation precursor was stripped out through the exhaust air upon aeration of the bioreactor [27]. Furthermore, due to their hydrophobicity, terpenoids, especially the terpene hydrocarbons, tend to being absorbed by all materials made from plastics, such as conventional laboratory-scale bioreactor sealings, Wttings, and tubings.…”

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

“…In contrast, biotechnological strategies take advantage of the pronounced regio-and stereo selectivities of enzymatic reactions (usually by use of microbial cells) and the possibility of overproducing isoprenoids in tailored bioprocesses. One strategy is whole-cell biocatalysis, which is usually favored if structurally closely related precursor molecules are abundantly available, for example, the monoterpenes limonene or pinene for carvone or verbenone production, respectively [44,46], and therefore represent cheap starting materials. The target products are often obtained by selective oxyfunctionalization reactions catalyzed by only one or a few enzymes, such as P450 monooxygenases and alcohol dehydrogenases.…”