Highly effective, regiospecific reduction of chalcone by cyanobacteria leads to the formation of dihydrochalcone: two steps towards natural sweetness

Abstract

Background: Chalcones are the biogenetic precursors of all known flavonoids, which play an essential role in various metabolic processes in photosynthesizing organisms. The use of whole cyanobacteria cells in a two-step, light-catalysed regioselective bio-reduction of chalcone, leading to the formation of the corresponding dihydrochalcone, is reported. The prokaryotic microalgae cyanobacteria are known to produce phenolic compounds, including flavonoids, as natural components of cells. It seems logical that organisms producing such compounds possess a suitable "enzymatic apparatus" to carry out their biotransformation. Therefore, determination of the ability of whole cells of selected cyanobacteria to carry out biocatalytic transformations of chalcone, the biogenetic precursor of all known flavonoids, was the aim of our study.

Results: Chalcone was found to be converted to dihydrochalcone by all examined cyanobacterial strains; however, the effectiveness of this process depends on the strain with biotransformation yields ranging from 3% to >99%. The most effective biocatalysts are Anabaena laxa, Aphanizomenon klebahnii, Nodularia moravica, Synechocystis aquatilis (>99% yield) and Merismopedia glauca (92% yield). The strains Anabaena sp. and Chroococcus minutus transformed chalcone in more than one way, forming a few products; however, dihydrochalcone was the dominant product. The course of biotransformation shed light on the pathway of chalcone conversion, indicating that the process proceeds through the intermediate cis-chalcone. The scaled-up process, conducted on a preparative scale and by using a mini-pilot photobioreactor, fully confirmed the high effectiveness of this bioconversion. Moreover, in the case of the mini-pilot photobioreactor batch cultures, the optimization of culturing conditions allowed the shortening of the process conducted by A. klebahnii by 50% (from 8 to 4 days), maintaining its >99% yield.

Conclusions: This is the first report related to the use of whole cells of halophilic and freshwater cyanobacteria strains in a two-step, light-catalysed regioselective bio-reduction of chalcone, leading to the formation of the corresponding dihydrochalcone. The total bioconversion of chalcone in analytical, preparative, and mini-pilot scales of this process creates the possibility of its use in the food industry for the production of natural sweeteners.

Keywords: Biotransformation; Chalcone; Cyanobacteria; Dihydrochalcone; Regioselective bio-reduction.

Fig. 1

Regioselective bioreduction of chalcone (1) that leads to the formation of dihydrochalcone (2) as the only product, as it is catalysed by Anabaena laxa, Aphanizomenon klebahnii, Nodularia moravica, Synechocystis aquatilis and Merismopedia glauca

Fig. 2

Specific transformation of chalcone catalysed by Anabaena sp.

Fig. 3

Transformation of chalcone catalysed by Chroococcus minutus

Fig. 4

GC–MS profiles of the samples collected on day 0 (a), day 1 (b), and day 8 (c) during the transformation of chalcone into dihydrochalcone by Aphanizomenon klebahnii

Fig. 5

Adequate mass spectra of substrate (a), intermediate (b) and product (c)

Fig. 6

Time-dependent transformations of chalcone in the culture of Aphanizomenon klebahnii (a) and in substrate control (b)

Fig. 7

The formation of two distinct, medium-dependent isomeric forms of chalcone

Fig. 8

The structures of the substrate—chalcone (1) and the products of biotransformation (2–6) performed by the examined cyanobacteria

Fig. 9

The changes of CO₂ content in the headspace of the photobioreactor (b) as a function of time in control and experimental cultures of Aphanizomenon klebahnii