Biocatalyst and bioreactor design for the production of green-note volatiles, characterization of their involvement in plant-pathogen defense and senescence

Abstract

The international market for fragrances and food flavours is worth several billion U.S.-dollars annually. The volatiles, hexanal and (3Z)-hexenal, which are key organoleptic elements of green-note, are important components of both fragrance and flavour, and find widespread consumer acceptance if naturally derived. Therefore, the industry is intensifying its efforts to produce green-note volatiles through extraction from naturally enriched plant sources.

In this dissertation, novel, alternative processes for the natural production of hexanal and (3Z)-hexenal are presented. Enzyme templates, known to be responsible for the synthesis of hexanal from linoleic acide (18:2), were isolated from naturally enriched tissues. These templates were immobilized in a natural alginate matrix and used as a biocatalyst within a packed-bed bioreactor. Product recovery was achieved on-line using a hollow-fiber ultrafiltration unit. Key parameters - namely pH, reaction temperature, and substrate and catalyst concentrations - affecting hexanal generation were identified and optimized. Utilizing these optimized conditions, hexanal production in the bioreactor over a 30 minute period proved to be 112-fold higher than endogenous steady-state levels of the volatile in a corresponding amount of tissue. In addition, due to the anti-microbial properties of hexanal, bacterial contamination in the bioreactor was not observed. However, bioreactor-based generation and recovery of (3Z)-hexenal from linolenic acid (18:3) was not achieved due to the high reactivity of (3Z)-hexenal. Therefore, Arabidopsis thaliana was genetically modified by up-recglating hydroperoxide lyase, one of the key enzymes involved in the in vivo formation of (3Z)-hexenal. Over-expression of hydroperoxide lyase cDNA in transgenic Arabidopsis plants resulted in increased levels of (3Z)-hexenal of up to 29-fold by comparison with wild-type plants, whereas hexanal levels remained unaltered. Thus, these transformed plants are suitable bioreactors for (3Z)-hexenal production in themselves.

Transgenic Arabidopsis thaliana plants with up-regulated (3Z)-hexenal levels were tested for increased resistance against virulent bacteria and for enhanced systemic required resistance. In planta bacterial counts revealed increased resistance against virulent Pseudomonas syringae by a factor of 6 and a 15-fold enhancement of systemic acquired resistance for the transgenic lines in comparison to wild-type plants. Thus, overall resistance against bacterial infection was increased by 90-fold in the transgenic plants.

The transgenic Arabidopsis thaliana plants were also more resistant than wild-type plants to the effect of anoxic stress induced by flooding. Water-flooding of the soil resulted in impaired leaf growth in wild-type plants, whereas leaf development in the transgenic plants was unaffected. In addition, levels of peroxidized lipid in membranes of the transgenic plants were reduced by ~ 1/3 in comparison to wild-type plants. These observations collectively indicate that hydroperoxide lyase plays an important role in membrane turnover. It is also apparent that up-regulation of hydroperoxide lyase reduces the effects of anoxic stress, presumably by converting membrane-bilayer destabilizing peroxides into green-note volatiles which readily diffuse out of the membranes.