Abstract
BackgroundAnthocyanins such as cyanidin 3-O-glucoside (C3G) have wide applications in industry as food colorants. Their current production heavily relies on extraction from plant tissues. Development of a sustainable method to produce anthocyanins is of considerable interest for industrial use. Previously, E. coli-based microbial production of anthocyanins has been investigated extensively. However, safety concerns on E. coli call for the adoption of a safe production host. In the present study, a GRAS bacterium, Corynebacterium glutamicum, was introduced as the host strain to synthesize C3G. We adopted stepwise metabolic engineering strategies to improve the production titer of C3G.ResultsAnthocyanidin synthase (ANS) from Petunia hybrida and 3-O-glucosyltransferase (3GT) from Arabidopsis thaliana were coexpressed in C. glutamicum ATCC 13032 to drive the conversion from catechin to C3G. Optimized expression of ANS and 3GT improved the C3G titer by 1- to 15-fold. Further process optimization and improvement of UDP-glucose availability led to ~ 40 mg/L C3G production, representing a > 100-fold titer increase compared to production in the un-engineered, un-optimized starting strain.ConclusionsFor the first time, we successfully achieved the production of the specialty anthocyanin C3G from the comparatively inexpensive flavonoid precursor catechin in C. glutamicum. This study opens up more possibility of C. glutamicum as a host microbe for the biosynthesis of useful and value-added natural compounds.
Highlights
Anthocyanins such as cyanidin 3-O-glucoside (C3G) have wide applications in industry as food colorants
A universal approach to increase the soluble expression of heterologous proteins is through the fusion of a protein or peptide tag, which is highly soluble in the host strain even at a very high expression level, such as maltose-binding protein (MBP) and small ubiquitin-like modifier (SUMO) [29, 30]
The C3G yields were below 1 mg/L (Fig. 2b) for all the constructs, it is clear that fusion of SUMO and MBP alone improved the production by 110% and 58%, respectively; codon optimization led to a 71% increase in titer for the wildtype genes, and 25% and 42% increase for SUMO and MBP fusion, respectively
Summary
Anthocyanins such as cyanidin 3-O-glucoside (C3G) have wide applications in industry as food colorants Their current production heavily relies on extraction from plant tissues. Anthocyanins are valuable flavonoids that have diverse applications in food processing, cosmetic production, and nutraceutical manufacturing [1,2,3] They are synthesized via the general flavonoid pathway, which converts tyrosine or phenylalanine to the flavonoid precursor flavanones, such as naringenin (Fig. 1). Hydroxylation of these flavanones at ring B and reduction by dihydroflavonol 4-reductase (DFR) form leucoanthocyanidins These compounds, or their reduced form flavan-3-ols through the action of leucoanthocyanidin reductase (LAR), can be further oxidized by anthocyanidin synthase (ANS) for the generation of the unstable flavylium cation anthocyanins, which are linked to a glucosyl residue at C3 in ring C to form anthocyanin-3-O-glucosides such as cyanidin-3-O-glucoside (C3G). The most commonly used chassis microbe in metabolic engineering is E. coli, which has been extensively engineered
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