Bioproduction of alpha-humulene in metabolically engineered Escherichia coli and application in zerumbone synthesis

Zerumbone is a sesquiterpene ketone with potent anti-cancerogenic activities, produced in several ginger species of the Zingiberaceae familiy. We have investigated the biotechnological production of alpha-humulene, a precursor of zerumbone. By implementing a heterologous mevalonate pathway in combination with the alpha-humulene synthase expression, we effectively synthesized alpha-humulene from glucose in Escherichia coli. In this study, we developed a practical and efficient in situ separation method for alpha-humulene by comparison of extractive and adsorptive strategies. By the in situ adsorption of the product to the hydrophobic resin Amberlite (R) XAD4 we were able to increase alpha-humulene yield by 2310% to 60.2 mg/L. Furthermore we present an easy applicable, short subsequent chemical process for the conversion of alpha-humulene to zerumbone by using transition metal catalysis. To reduce process steps, the chemical reaction was carried out in the same solvent as the eluting solvent that was used to elute alpha-humulene from the adsorbent resin. By allylic oxidation of a-humulene with manganeseII chloride as a catalyst and tert.-butylhydroperoxide as an oxidizing agent we were able to synthetize zerumbone with a selectivity of 51.6%. Product and byproducts of the oxidation reaction were identified by GC-MS.