Zorn, HolgerZhang, YanyanKanter, Jean-PhilippeJean-PhilippeKanter2024-10-172024-10-172024https://jlupub.ub.uni-giessen.de/handle/jlupub/19676https://doi.org/10.22029/jlupub-19034Due to rising consumers’ environmental and health consciousness and therewith an increasing trend towards natural flavour ingredients, the industry constantly seeks for alternative production methods to fulfil these demands. Biocatalytic strategies are hereby playing an increasingly prominent role, as they enable ecologically and economically sustainable production of flavour chemicals in line with the principles of ‘Green Chemistry’ and meet consumers’ demand for products of natural origin. Fatty aldehydes represent a class of highly demanded odour-active compounds contributing to the smell of many foods including fruits, herbs, and meat products, and are therefore important ingredients for respective flavourings. Moreover, they are applied in a variety of cosmetic products including highly valuable fragrances. In nature, fatty aldehydes are biosynthesised mainly from fatty acids via enzymatic cascade reactions. These involve lipoxygenases together with hydroperoxide lyases, carboxylic acid reductases or α-dioxygenases, which are key enzymes of the plant α-oxidation pathway. Recently, a cyanobacterial α-dioxygenase from Crocosphaera subtropica (Csα-DOX) has been identified to be a highly promising candidate as biocatalyst. It enables the production of unusual odd-chain numbered, mono- or polyunsaturated fatty aldehydes from naturally abundant fatty acids in the presence of molecular oxygen as sole cofactor. In the present study, a recombinant, cyanobacterial fatty aldehyde dehydrogenase from Vibrio harveyi (VhFALDH) has been jointly applied with Csα-DOX in a coupled-enzyme reaction to produce a series of carbon chain shortened fatty aldehydes. The biocatalytic approach was systematically established and optimised to increase the production efficiency towards the desired Cn-x aldehydes. It was subsequently used to convert various single fatty acids as well as hydrolysed lipid extracts with unusual fatty acid profiles obtained from plant and fungal sources. The resulting aldehydes were quantified and characterised by means of gas chromatography coupled with mass spectrometry and olfactometry alongside sensory evaluations of the aldehyde mixtures. Thereby, odours of numerous (Z)-unsaturated fatty aldehydes have been described for the first time. Apart from the generation of natural odourants, the field of non-volatile flavour compounds is equally in the spotlight. With the growing trend of sugar reduced foods and those enriched with protein or amino acids, artificial sweeteners are usually applied to compensate for the lack of sugar and to mask off-flavours. However, with lately accumulating studies on health concerns regarding such sweeteners, alternatives are highly demanded. A number of O-methylated flavonoids, such as neohesperidin dihydrochalcone, homoeriodictyol, and hesperetin have been described as taste-active, with sweetening, bitter-masking or sweet-enhancing properties. Direct catecholic precursors of such flavonoids e.g. eriodictyol are abundantly found in nature. Thus, a suitable O-methyltransferase activity was sought to efficiently produce the corresponding products. The mycelia of several fungi from the phylum Basidiomycota were screened for the targeted enzyme activity, and Lentinula edodes (syn. shiitake) was identified as the most promising candidate with the targeted biocatalytic activity against eriodictyol [dihydrochalcone], resulting in considerable product concentrations of homoeriodictyol [dihydrochalcone] and hesperetin [dihydrochalcone]. In addition, other catecholic compounds were enzymatically converted into the corresponding O-methylated analogues, including industrially highly relevant flavouring substances such as vanillin. By means of a bottom-up proteomics approach, genes of L. edodes encoding for the responsible O-methyltransferases were successfully identified and functionally expressed in Escherichia coli. The purified enzymes were systematically characterised including reaction conditions, enzyme kinetic parameters, and activity against numerous phenolic substrates. Thereby, high product yields of up to 100% were achieved. The present work demonstrates the successful enzymatic production of odour-active fatty aldehydes and flavour-active O-methylated flavonoids from natural sources. In contrast to chemical synthetic methods and the elaborate sourcing of naturally scarce substances, this represents a sustainable and environmentally friendly production strategy and is therefore of major relevance to the flavours & fragrances industry.enIn CopyrightFlavourAromaBiocatalysisFatty aldehydesFlavonoidsFungiddc:000ddc:333.7ddc:500ddc:540ddc:570