Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol.

Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18 mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65 g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.

Chemical transformations of characteristic hop secondary metabolites in relation to beer properties and the brewing process: a review.

The annual production of hops (Humulus lupulus L.) exceeds 100,000 mt and is almost exclusively consumed by the brewing industry. The value of hops is attributed to their characteristic secondary metabolites; these metabolites are precursors which are transformed during the brewing process into important bittering, aromatising and preservative components with rather low efficiency. By selectively transforming these components off-line, both their utilisation efficiency and functionality can be significantly improved. Therefore, the chemical transformations of these secondary metabolites will be considered with special attention to recent advances in the field. The considered components are the hop alpha-acids, hop beta-acids and xanthohumol, which are components unique to hops, and alpha-humulene and beta-caryophyllene, sesquiterpenes which are highly characteristic of hops.

Cytotoxic evaluation of essential oil from Zanthoxylum rhoifolium Lam. leaves

Zanthoxylum rhoifolium Lam is a plant popularly used as antimicrobial, for malaria and inflammatory treatment. The essential oil of Z. rhoifolium was extracted and its cytotoxic effects against HeLa (human cervical carcinoma), A-549 (human lung carcinoma), HT-29 (human colon adenocarcinoma), Vero (monkey kidney) cell lines and mice macrophages were evaluated. Some of the terpenes of its essential oil (ß-caryophyllene, alpha-humulene, alpha -pinene, myrcene and linalool) were also tested to verify their possible influence in the oil cytotoxic activity. The results obtained permitted to confirm that the essential oil is cytotoxic against tumoral cells (CD50 = 82.3, 90.7 and 113.6 μ/ml for A-549, HeLa e HT-29 cell lines, respectively), while it did not show cytotoxicity against non-tumoral cells (Vero and mice macrophages). Thus, the essential oil from Z. rhoifolium leaves seems to present a possible therapeuthic role due to its selective cytotoxic activity against tumoral cell lines.

O Zanthoxylum rhoifolium Lam. é uma planta popularmente utilizada como antimicrobianos, no tratamento da malária e de inflamações. O óleo volátil do Z. rhoifolium foi extraído e posteriormente foi avaliada a sua citotoxicidade contra células HeLa (carcinoma cervical humano), A-549 (carcinoma de pulmão humano), HT-29 (adenocarcinoma de cólon humano), Vero (rim de macaco) e macrófagos de camundongos. Alguns terpenos constituintes do óleo volátil (beta-cariofileno, alfa -humuleno, alfa -pineno, mirceno e linalool) também foram testados para verificar as possíveis influências sobre a citotoxicidade do óleo. Os resultados obtidos permitiram verificar que o óleo volátil é citotóxico contra células as tumorais (CD50 = 82.3, 90.7 e 113.6 μg/ml para A-549, HeLa e HT-29 cell lines, respectivamente), mas não apresenta citotoxicidade contra as células não tumorais (Vero e macrófagos de camundongos). Desta forma o óleo volátil das folhas do Z. rhoifolium demonstra possuir uma possível ação terapêutica em função de sua citotoxicidade contra células tumorais.