Improvement of a P450-Based Recombinant Escherichia coli Whole-Cell System for the Production of Oxygenated Sesquiterpene Derivatives

Abstract

Sesquiterpenes are common constituents of essential oil in plants. Their oxygenated derivatives often possess desirable flavor, fragrance, and pharmaceutical properties. Recently, the CYP264B1-based recombinant Escherichia coli whole-cell system has been constructed for the oxidation of sesquiterpenes. However, limiting factors of this system related to the high volatility of substrates and the suitability of the P450 redox partner need to be addressed. In this work, the improvement of the system was implemented with (+)-α-longipinene as a model substrate. By using 2-hydroxypropyl-β-cyclodextrin and an alternative ferredoxin reductase, the conversion of (+)-α-longipinene was improved 77.1%. Applying the optimized conditions, the yields of the main products were 54.2, 34.2, and 47.2 mg L, corresponding to efficiencies of 82.1, 51.8, and 71.5% for the conversion of (+)-α-longipinene, (-)-isolongifolene, and α-humulene, respectively, at a 200 mL scale. These products were characterized as 12-hydroxy-α-longipinene, isolongifolene-9-one, and 5-hydroxy-α-humulene, respectively, by nuclear magnetic resonance spectroscopy.

Artificial Self‐Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity

“…The cytc reduction activity reflects the reduction of PdX by PdR in PUPPET for the following reasons: PdX red can reduce cytc much rapidly (>2000 s À1 ) [34] compared with reduction of PdX by PdR [35] (k 5 >> k 3 ); direct reduction of cytc by PdR and ET between PUPPET molecules are negligible at the concentrations of the proteins in the experiment (Figure S7A, Supporting Information); and the lower reduction potential of D-camphorunbound P450cam (À340 mV) than of PdX (À239 mV) [42] hampers ET from PdX to P450cam in the absence of D-camphor. The consistency of our kinetic model with the acquired data (14) and (16). k red RmX1C and k red RmX1 were determined from the V max values of R m X 1 C and R m X 1 , respectively, in cytc reduction assay (Table S5, www.advancedsciencenews.com www.biotechnology-journal.com (Table S5, Supporting Information) validates that the reduction rate of PdX by PdR in PUPPETcan be estimated from V max of the cytc reduction reaction (Figure 3B,C).…”

“…[14,15] Recently, Bernhardt's group demonstrated that bacterial P450s are able to produce valuable compounds. [16][17][18] Generally, bacterial P450s accept two electrons, one at a time, from separate electron transfer (ET) proteins such as ferredoxin and flavodoxin. [10] There are also a few cases of naturally occurring fusions to redox domains.…”

“…Bacterial P450s, which are soluble proteins and generally have high catalytic turnover numbers compared with eukaryotic P450s, have been explored for chemical synthesis . Recently, Bernhardt's group demonstrated that bacterial P450s are able to produce valuable compounds …”

In-Situ Biocatalytic Production of Trehalose with Autoinduction Expression of Trehalose Synthase

“…It is well known that the use of intracellular enzymes in the form of whole-cell biocatalysts is an effective way to lower enzyme production costs, since complex separation and purification in downstream processing can be simplified or even discarded . Accordingly, an integrated bioprocess for trehalose production and separation from maltose with recombinant TreS in permeabilized cells was proposed in our previous study .…”

“…It is well known that the use of intracellular enzymes in the form of whole-cell biocatalysts is an effective way to lower enzyme production costs, since complex separation and purification in downstream processing can be simplified or even discarded. 38 Accordingly, an integrated bioprocess for trehalose production and separation from maltose with recombinant TreS in permeabilized cells was proposed in our previous study. 8 In the present study, we found that cells from autoinduction fermentation can directly produce trehalose from maltose in a simple phosphate buffer system even with no need for cell permeabilization (Figure S3).…”