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Noda Institute for Scientific Research GRANT2013 Young Investigator Research GrantOutline of Research Result

Genetic engineering of Kluyveromyces marxianus for effectiveproduction of the rose-like odor 2-phenylethanolTakashi KoyanagiDepartment of Food Science, Ishikawa Prefectural University

Research aims2-Phenylethanol (2-PE) is found in various natural essential oils, particularly in that of rose and other related plants.
Because the compound elicits a pleasant, rose-like odor, itis commonly used as an ingredient in artificial flavoringand in perfumery. Annually, more than several thousandtons of this compound is industrially produced by chemicalsynthesis, and consumed all over the world at inexpensiveprices. However, there is currently a strong demand for natural aroma compounds that are not produced through chemical methods. Likewise, there is an increasing interest inwhite biotechnology and green chemistry. One valid approach for natural 2-PE production is direct extraction fromplants; however, this process requires vast farmlands andlong cultivation times. Another possibility is yeast fermentation, which is widely used for production of alcoholiccompounds.
Kluyveromyces marxianus is a species of budding yeastthat, owing to its vast metabolic capabilities and capacityfor assimilation, has been used in a variety of bioindustrialprocesses, such as ethanol fermentation and whey bioconversion. Kluyveromyces marxianus is also known as a potent producer of 2-PE.1, 2) In this species, 2-PE is producedvia the same biosynthesis pathways that generate the aromatic amino acids: The pentose-phosphate pathway and theshikimic acid pathway. In this cascade, an intermediateproduct, phenylpyruvate, is converted into phenylacetaldehyde by the enzyme phenylpyruvate decarboxylase (Ppd,EC 4.1.1.43). Phenylacetaldehyde is then converted into2-PE by alcohol dehydrogenase (Adh, EC 1.1.1.1) (Fig.
1),3) and 2-PE is partially converted into phenylethylacetate by alcohol acetyltransferase (Atf, EC 2.3.1.84). It hasalso been shown that 2-PE is alternatively producedthrough the Ehrlich pathway, in which the a-amino group ofphenylalanine is removed to produce phenylpyruvatethrough a bidirectionally regulated reaction, catalyzed byaromatic amino acid transaminase (EC 2.6.1.5 or 2.6.1.57).
In this study, we attempted to increase the levels of 2-PEproduction in K. marxianus NBRC1777 by genetic modification. In addition, we carried out metabolomic analyses togain detailed insights into 2-PE production in relation tocellular metabolic flow.

Fig. 1.

2PE biosynthesis pathway

MethodsConstruction of a recombinant K. marxianus strain thatoverexpresses PpdThe E. coli-yeast shuttle vector pDblet4) was used toclone the structural gene of Ppd (KmPPD). The gene wasamplified by polymerase chain reaction from genomicDNA of K. marxianus NBRC1777, and was placed underthe control of the strong constitutive promoter of the Saccharomyces cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The Ppd-expression plasmidwas then introduced into the auxotrophic strain, K. marxianus Δura3 Δtrp1 Δleu2 (NBRC1777 derivative), by electroporation, and cells were spread on plates of synthetic complete medium (SC) lacking uracil, to select for strains containing the plasmid. The production of 2-PE was measuredphotometrically using high-performance liquid chromatography (HPLC), equipped with Inertsil ODS3 (GL Science).
Metabolomic analysisFor metabolomic analysis, CE-TOFMS (cation and anionmodes) was employed to evaluate the metabolic balance inK. marxianus cells overproducing 2-PE. The analysis wasperformed by the Human Metabolome Technologies Co.
(Yamagata, Japan).
ResultsThe KmPPD gene was introduced into the K. marxianusΔura3 Δtrp1 Δleu2 strain, and the accumulation of 2-PE inthe culture supernatants was monitored (Fig. 2). The results

54way intermediates, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and dihydroxyacetonephosphate, in comparison with the control strain. A significant increase in sedoheptulose 7-phosphate accumulation,an important intermediate of pentose-phosphate pathway,was also observed. These results indicate that overexpression of Ppd may result in an increase in the metabolic flowof aromatic amino acid biosynthesis, including the shikimicacid pathway, which in turn also promotes glycolysis andthe pentose phosphate pathway.
Conclusion

Fig. 2. Accumulation of 2-PE in culture supernatants.
Upper left: Wild type (NBRC1777), upper right: Strain containing theempty vector, lower left: Strain containing plasmid-encoded KmPPD­lower right: Strain containing genome-integrated KmPPD­revealed that an increase in the copy number of KmPPDgreatly affected the production of 2-PE. This was especially true when the genome- integrated KmPPD was present (8-fold increase, compared to the wild-type strainNBRC1777).
We then carried out the metabolomic analysis to elucidate the flow of cellular metabolism in the 2-PE-overproducing cells. For these analyses, the auxotrophic strain (K.
marxianus Δura3 Δtrp1 Δleu2), the strain containing a genome-integrated copy of KmPPD (Δura3 Δtrp1 Δleu2URA4+S. pombe-KmPPD+), and the control strain, harboringonly the auxotrophic marker gene (Δura3 Δtrp1 Δleu2URA4+S. pombe), were cultivated in SC medium at 37°C withshaking (140 rpm), and cell extracts were harvested. Thestrain carrying an integrated copy of KmPPD­ exhibited amarked increase in the accumulation of the glycolysis path-The introduction of an additional copy of the KmPPDgene into K. marxianus resulted in an increase in the levelof intracellular 2PE accumulation. Furthermore, this increase in 2-PE was found to be attributable to a change inmetabolic flow, including glycolysis and the pentose-phosphate pathway, as revealed by a metabolomic analyses.
These results indicate that the level of Ppd expression is acritical component in the regulation of pathway flux andcan tip the balance towards the 2-PE specific productionpathway.
References1) Wittmann, C., Hans, M., and Bluemke, W. (2002) Metabolic physiology of aroma-producing Kluyveromyces marxianus. Yeast 19:1351–1363.
2) Etschmann, M.M.W., Bluemke, W., Sell, D., and Schrader, J. (2002)Biotechnological production of 2-phenylethanol. Appl. Microbiol.
Biotechnol. 59: 1–8.
3) Lertwattanasakul, N., Sootsuwan, K., Limtong, S., Thanonkeo, P.,and Yamada, M. (2007) Comparison of the gene expression patternsof alcohol dehydrogenase isozymes in the thermotolerant yeastKluyveromyces marxianus and their physiological functions. Biosci.
Biotechnol. Biochem. 71: 1170–1182.
4) de Souza Junior, C.G. and de Morais Junior, M.A. (2000) The use ofthe replicating pDblet plasmid as a cloning vector with enhanced stability in Kluyveromyces marxianus. Biotechnol. Lett. 22: 43–45.