Center of Applied ChemistryInstitute of Technical Chemistry Callinstr. 5, 30167 Hannover, Germany

Increasing heterologous expression of plant sesquiterpene synthases in E. coli by use of fusion strategies

S. Hartwig1, T. Frister1, T. Scheper1, S. Beutel1 1 Institute of Technical Chemistry, Leibniz University of Hannover, Germany

Discussion and OutlookThis study shows, how important the choice of strain and fusion tag strategy is when expressing eukaryotic genes in a prokaryotic host. Fusing a modified ubiquitin moeity to the sesquiterpene synthase significantly increased the yield of soluble enzyme while retaining its activity. In addition, use of a cspA controlled induction at very low temperatures was able to increase expressi-on levels. An IMAC-based Ni2+ chromatography step, exploiting a N-terminal His-Tag epitope, produced high amounts of purified (> 90 %) enzyme. In GC-FID analysis of batch bioactivity assays, (+)-zizaene could be identified as the only product resulting from cyclization of FPP. Further studies will be undertaken to characterize kinetic parameters of the novel synthase.

AcknowledgementsThe authors would like to thank Prof. Berger and Dr. Krings (Institute of Food Chemistry, University Han-nover) for assistance with terpene analytics. 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).

IntroductionPlant essential oils consist mainly of terpenoids, which are used extensively by the fragrance industry in everyday personal care products and costly perfumes. The extraction process of the relevant plant source material is often laborious, unreliable and cost demanding. Biotechnology enables new approaches to interesting terpene compounds. We over-expressed a plant enzyme catalyzing the synthesis of (+)-zizaene, an interesting and valuable precursor to a-vetivone, in a recombinant E. coli host. As the class of plant sesquiterpene synthases are considered hard-to -express in a soluble form, different solubility enhancing strategies were evaluated in this study. The recombinant enzyme catalyzed the production of (+)-zizaene from farnesyl pyrophosphate.

Cloning of protein expression plasmidsThe plant Vetiveria zizanoides grows natively in Madagascar and is reknown for its pleasant vetiver oil, consisting mainly of khusimol and a-/b-vetivone. Although no terpene cyclase catalyzing the formation of these two compo-nents is known to date, a cDNA sequence coding for their precursor (+)-zi-

zaene (GenBank HI931360) was identified earlier [2], but no further data describing the enzyme was published up to date. The sequence was careful-ly codon optimized, synthesized as two indepent double-stranded DNA strings, and cloned into ex-pression vectors using a modified Gibson-assem-bly [3] approach. Constructs were transformed into E. coli BL21(DE3) competent cells to utilize the T7-Promoter and cspA-Promoter (cold shock protein A) driven protein expression.

Literature[1] Roy et al., Nat. Protoc. (2010), 725-738[2] Schalk et al., Patent US 2012/0021475A1[3] Gibson et al., Nat. Methods (2009), 343-345[4] Marblestone et al., Protein Sci. (2006), 182-189

Expression and purification of the recombinant synthaseExpression experiments using the His-tagged construct (pET16b) yielded no detectable soluble protein production, even during low temperature cultivations. In contrast, both the cspA-Promoter driven induction as well as fusion to a ubiquitin-modifier moeity resulted in strong and effici-ent soluble expression of the plant enzyme in E. coli. The relatively low cultivation temperatures (15 °C) needed for cspA induction slow down the protein translation machi-

nery in the organism, so that proper folding is pos-sible. The SUMO domain is highly soluble in E. coli and was shown previ-ously to enable and enhance the solubility of fusion partners attached to the N-terminus [4]. Purification was performed on a sepharo-se column decorated with Ni2+ (GE Healthcare HiTrap™ IMAC FF 5 ml), using a two elution step method. Both enzymes were successfully purified as shown by SDS-PAGE analysis and western blots of the corresponding fractions.

Innovation clusterRefinement of plant resources

Fig. 2 Chemical structure of the sesquiterpene (+)-zizaeneFig. 1 Putative structure of recombinant zizaene synthase, modelled by use of the I-TASSER algorithm [1]

Fig. 8 Western Blot using His-epitope antibody, (A) elution fraction of purified pColdI::Ziz(co) raw extract. (B) elution fraction of purified pETSUMO::Ziz(co) raw extract.

Fig. 3 Root systems of two cultivars of V. zizanoides, grown and harvested in Madagascar.

Fig. 7 SDS-PAGE showing purification steps of zizaene synthase by use of Ni2+ based IMAC method. Using (A) pColdI-construct (theor. MW = 66 kDa). (B) pETSUMO-construct (theor. MW = 77.4 kDa). RE = raw extract, FT = flow through frac., WF = wash fractions, EF = elution fractions, UF = after dialysis/ultrafiltration using a MWCO of 10,000 Da

Fig. 5 Plasmid map and schematic representation of the construct utilizing the cspA promoter for cold shock induction.

Fig. 6 Plasmid map and schematic representation of the SUMO fusion construct used in this study.

Fig. 4 Plasmid map and schematic representation of the pET vector construct utilizing T7 promoter for protein expression.

Enzymatic production of (+)-zizaeneBioconversions of the substrate farnesyl pyrophosphate (FPP) were carried out in ml-scale batch reactions using purified zizaene synthase enzyme (elution frac-tions). The liquid phase consisting of buf-fer, enzyme, and substrate were overlaid by isooctane to yield a two-phase system. Optimal reaction conditions were pH 7.0, 1h @ 30 °C. After a short extraction pro-cess, the upper organic phase was analy-zed by GC-FID and compared to standard sesquiterpene compounds. Both recom-binant enzymes were active and produ-ced (+)-zizaene from FPP.

Fig. 9 GC-FID chromatograms showing bioactivity assays of the two different zizaene synthase constructs.

retention time [min]

E. coli BL21(DE3)pColdI::Ziza(co)50 µM FPP1 h @ 30 °C

E. coli BL21(DE3)pETSUMO::Ziza(co)50 µM FPP1 h @ 30 °C

E. coli BL21(DE3)no insert control50 µM FPP1 h @ 30 °C

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