Biosynthesis of apigenin glucosides in engineered Corynebacterium glutamicum

Abstract

Background; Glucosylation is a well-known approach to improve the solubility, pharmacological and biological properties of flavonoids. In recent years, efforts such as enzymatic synthesis have been developed to enhance the production of flavonoid glucosides. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of these systems for large-scale synthesis for human needs. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid. Results; For the production of apigenin glucosides, a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis was successfully expressed in C. glutamicum. Additionally, the endogenous C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase genes involved in the synthesis of UDP-glucose were overexpressed to create a C. glutamicum cell factory system capable of efficiently glucosylating apigenin with a high yield of glucosides in a comparatively short time. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin 4’-O-β-glucoside), 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin 4’,7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin 4’,5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin. Conclusion; The developed C. glutamicum cell factory system highly glucosylated apigenin with higher efficiency and the high substrate susceptibility of C. glutamicum makes it the best alternative for large-scale biosynthesis of flavonoid glucosides. The developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars, thus making a cost-effective system.

Enhancing astaxanthin biosynthesis and pathway expansion towards glycosylated C40 carotenoids by Corynebacterium glutamicum

“…In Yarrowia lipolytica this challenge of complexity was tackled by the establishment of an alternative plant-derived astaxanthin biosynthesis from Adonis aestivalis 20 resulting in astaxanthin as the exclusive hydroxylated carotenoid due to the sequential activity of carotenoid 4-hydroxy-β-ring 4-dehydrogenase (HBFD) and carotenoid β-ring 4-dehydrogenase (CBFD). It was recently demonstrated that also the UDP-glucose pool can be enhanced by overexpression of the endogenous UDPd-glucose pathway genes, phosphoglucomutase (pgm) and UDP-d-glucose pyrophosphorylase (galU1) together with the heterologous expression of ydhE from Bacillus licheniformis 65 and this strategy might also improve the biosynthesis of glycosylated carotenoids.…”

Metabolic Engineering of Corynebacterium glutamicum for the Production of Flavonoids and Stilbenoids

“…Although various types of diversified flavonoids have been synthesized in E. coli and S. cerevisiae, glycosylation of flavonoids has been reported as only modification of flavonoid in engineered C. glutamicum [57,58]. Glycosylation is catalyzed by glycosyltransferase, which transfers sugar molecules into the oxygen, carbon, nitrogen, and sulfur atoms of aglycon.…”