Center for Applied ChemistryInstitute of Technical ChemistryCallinstr. 5 , 30167 Hannover, Germany

Institute of Technical Chemistry, Leibniz University Hannover, Callinstr. 5, 30167 Hannover, Germany

Production and characterization of a recombinant patchoulol synthase for the biocatalytic production of terpenoid structures

Kimia Ekramzadeh, Thomas Scheper and Sascha Beutel

Introduction

Figure 3: Influence of T on product selectivity by conversion of the natural sesquiterpene precursor farnesyl pyrophosphate (FPP) with PTS in MES buffer.

Figure 5: Enzyme kinetics of PTS with different FPP concentrations in MES buffer at optimal reaction conditions (pH 6.4 and 34 °C). Kinetic parameters were evaluated graphically: K =10.37 µM and v = 5.77 µM/min.M max

Enzyme characterization

Figure 4: Influence of the pH on product selectivity by conversion of the natural sesquiterpene precursor farnesyl pyrophosphate (FPP) with PTS in MES buffer.

In this work the bioactive patchoulol synthase was expressed recombinantly in E. coli and isolated over an optimized tandem two-step purification via IMAC and a desalting column. The PTS was produced with more than 90% purity. Characterization studies showed that the product ratio of bioconversion of FPP with PTS was influenced by reaction parameters such as pH and temperature. Under extreme reaction conditions (> 40 °C) only a 1,10-cyclization takes place.The enzyme showed intense substrate inhibition of the natural substrate FPP. Moreover the recombinant PTS offers great potential as a biocatalyst for production of various sesquiterpenoid structures under optimal conditions.

Summary and conclusion

AcknowledgementThe author would like to thank the Institute of Food Chemistry of Leibniz University of Hannover for its kind support with GC-MS analytics.

A codon-opt imized PTS isoenzyme was produced in an E. coli BL21 (DE3) strain with pET16b: :h is6-FXa-PTS(co) as expression system. The soluble protein was purified by a tandem two-step purification with a two-

2+step elution via an Ni -charged immobilized m e t a l i o n a f f i n i t y column (IMAC) and a d i rect ly connected d e s a l t i n g c o l u m n (SEC). This strategy was chosen to avoid high protein losses.

Enzyme production and purification

Figure 2: Chromatogram of desalting the purification fractions from second elution step via B) SEC column and SDS-PAGE analysis of the eluted fractions (E6-14).

B)

Figure 1: Chromatogram of purification of PTS via FPLC (Äkta Pure) by using A) IMAC with a two-step elution (1. 25 mM and 2. 250 mM imidazole) and SDS-PAGE analysis which shows the flowthrough of 100 mL sample (FT1-6) and the first elution step with 25 mM imidazole (E1-5).

A

116.0 kDa

66.2 kDa

45.0 kDa

35.0 kDa

25.0 kDa

18.4 kDa

FT1

FT2

FT3

FT4

FT5

FT6

M

E1

E2

E3

E4

E5

B

116.0 kDa

66.2 kDa

45.0 kDa

35.0 kDa

25.0 kDa

18.4 kDa

M

E6

E7

E8

E9

E10

E11

E12

E13

E14

0

0,1

0,2

0,3

0,4

0,5

5,6 5,8 6 6,2 6,4 6,6

Pro

duct

ratio

pH

Guaia-5,11-diene

trans-β-Caryophyllene

β-Patchoulene

α-Bulnesene

Patchoulol

0

0,1

0,2

0,3

0,4

0,5

0,6

30 32 34 36 38 40 42 44

Pro

duct

ratio

Temperature [°C]

PTS was characterized regarding pH- and T-dependence of kinetics and optimal parameters (pH = 6.4 and T = 34 °C) were found. Product composition strongly depends on pH and T.PTS shows substrate inhibition by converting with concentrat ions higher than ~40 µM FPP.

Patchouli oil with its intense musk flavour is used as a base note in . Patchouli oil is made by biotransformation aroma and fragrance industryof the natural substrate farnesyl pyrophosphate (FPP) with the multi-product enzyme patchoulol synthase (PTS) and contains over 20 different sesquiterpenes and sesquiterpenoids. Due to the wide product range of patchoulol synthase from Pogostemon cablin, the subject of the present study is the characterization of an isolated PTS isoenzyme and the development of an efficient process to convert sesquiterpene precursors.

0 40 80 120 160

0,0

1,5

3,0

4,5

6,0

7,5

v [

µM

/min

]

FPP [µM]

A)

Optimizedrecombinant

organism

•Optimization of labscale cultivation

•Enzyme purification•Enzyme characterization

Key substrate:

FPP

•Organic synthesis•Cheapterpene alcohol (farnesol)

High valuablesesqui-

terpenes

•Enzymatic conversion with FPP•Isolation by liquid-liquid extraction •Enzyme characterization