Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

Enzymatic conversion of flavonoids using bacterial chalcone isomerase and enoate reductase.

Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction.

Biotechnological Production of Methyl-Branched Aldehydes.

A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.