Cell physiology rather than enzyme kinetics can determine the efficiency of cytochrome P450-catalyzed C–H-oxyfunctionalization

Abstract

Cell physiology is a critical factor determining the efficiency of reactions performed by microbial biocatalysts. In order to develop an efficient biotransformation procedure for the hydroxylation of (S)-limonene to (S)-perillyl alcohol by recombinant Pseudomonas putida cells harboring the cytochrome P450 monooxygenase CYP153A6, physiological parameters were optimized. The previously reported synthesis of (S)-perillyl alcohol by P. putida GPo12 was based on complex and sensitive octane feeding strategies (van Beilen et al. in Appl Environ Microbiol 71:1737-1744, 2005), indicating the pivotal role of cell physiology. In contrast to previous findings, the screening of different carbon sources showed that glycerol and citrate are suitable alternatives to octane allowing high specific limonene hydroxylation activities. The use of P. putida KT2440 as an alternative host strain and citrate as the carbon source improved practical handling and allowed a 7.5-fold increase of the specific activity (to 22.6 U g (CDW) (-1) ). In two-liquid-phase biotransformations, 4.3 g of (S)-perillyl alcohol L (tot) (-1) were produced in 24 h, representing a sixfold improvement in productivity compared to previously reported results. It is concluded that, for selective cytochrome P450-based hydrocarbon oxyfunctionalizations by means of living microbial cells, the relationship between cell physiology and the target biotransformation is crucial, and that understanding the relationship should guide biocatalyst and bioprocess design.

Catalytic Pseudomonas taiwanensis VLB120ΔC Biofilms Thrive in a Continuous Pure Styrene Generated by Multiphasic Segmented Flow in a Capillary Microreactor

“…However, by recycling the organic styrene phase, the overall conversion was increased up to 20-fold (starting from 0.04% to 0.8%) and 70 mM (8.2 g/L) of (S)-styrene oxide was produced. Product titers (8.2 g/L org ) and productivity (~10 g/ L tube /day) achieved in this work are well comparable with other oxygenase-based biocatalytic processes [18][19][20]. To improve the productivity, the capillary length was shortened to 0.5 m, and the flow rates were kept constant in order to reduce the residence time.…”

Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli

“…Different approaches have been described to overcome uptake limitations in whole-cell biotransformations. The two-liquid-phase concept applied in stirred-tank reactors can increase mass transfer by ensuring maximal substrate availability and typically allows in situ product extraction (13,20,33,37,40,47,53,72). Next to that, the addition of rhamnolipids, synthetic surfactants, and cosolvents has been described as enhancing biotransformation rates.…”

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

“…Usually a number of organic solvents with appropriate log P Oct values are compiled and experiments conducted to determine biocompatibility, partition coefficients for substrate, and/or product and biodegradability [6,22,37,56,80]. To test for biocompatibility an activity profile can be created by plotting an organic solvent log P Oct against biocatalyst activity.…”

“…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.…”