TY - JOUR

T1 - Expression, purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli

AU - Hartwig, S.

AU - Frister, T.

AU - Alemdar, S.

AU - Li, Z.

AU - Krings, U.

AU - Berger, R. G.

AU - Scheper, T.

AU - Beutel, S.

N1 - Funding information: The authors would like to thank the Helmholtz-Zentrum Braunschweig for support in determination of the peptide mass fingerprint. This work was funded by the European Union as part of the EFRE (European Regional Development Fund) project “Refinement of plant resources” (ZW 8-80130940).

PY - 2014/2/25

Y1 - 2014/2/25

N2 - Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co2+-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition.

AB - Probing a cDNA library extracted from Pogostemon cablin (Indian Patchouli) with gene specific primers, a variant of patchoulol synthase PTS (GenBank acc. No.: AY508730) was amplified, cloned, and sequenced. The amino acid sequence deduced from the cloned cDNA exhibited a sequence variation of 3.4% compared to the annotated sequence. The enzyme variant tended to form inclusion bodies when expressed in Escherichia coli. The coding sequence was fused to the T7-tag, His-tag and to thioredoxin. Constructs were expressed in three different E. coli expression strains, with several strain/construct combinations yielding soluble enzyme. By fusion to thioredoxin and careful codon optimization of the eukaryotic sequence, soluble expression could be improved on average by 42% in comparison to an unoptimized, His-tagged construct. The thioredoxin-fused protein was successfully purified using a one-step Co2+-IMAC purification procedure. Bioactivity assays using prepared farnesyl diphosphate (FDP) in milliliter-scale batch reactions, showed activity of the fused enzyme even with thioredoxin attached. The product spectrum of the enzyme was compared to patchouli oil standards by GC-MS and the main products were identified. Interestingly, the variant showed a shift in product spectrum with germacrene A being the most abundant product instead of patchouli alcohol. In silico structural modeling shows a possible chemical and structural change in the active site of the enzyme, which might be responsible for the shift in product composition.

KW - Farnesyl diphosphate

KW - Germacrene A

KW - Patchoulol

KW - Sesquiterpenes

KW - Terpene synthase

KW - Thioredoxin

UR - http://www.scopus.com/inward/record.url?scp=84896323680&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2014.02.003

DO - 10.1016/j.pep.2014.02.003

M3 - Article

C2 - 24576659

AN - SCOPUS:84896323680

VL - 97

SP - 61

EP - 71

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -