De novo biosynthesis of p-coumaric acid and caffeic acid from carboxymethyl-cellulose by microbial co-culture strategy

Abstract

Background: Aromatic compounds, such as p-coumaric acid (p-CA) and caffeic acid, are secondary metabolites of various plants, and are widely used in diet and industry for their biological activities. In addition to expensive and unsustainable methods of plant extraction and chemical synthesis, the strategy for heterologous synthesis of aromatic compounds in microorganisms has received much attention. As the most abundant renewable resource in the world, lignocellulose is an economical and environmentally friendly alternative to edible, high-cost carbon sources such as glucose.

Results: In the present study, carboxymethyl-cellulose (CMC) was utilized as the sole carbon source, and a metabolically engineered Saccharomyces cerevisiae strain SK10-3 was co-cultured with other recombinant S. cerevisiae strains to achieve the bioconversion of value-added products from CMC. By optimizing the inoculation ratio, interval time, and carbon source content, the final titer of p-CA in 30 g/L CMC medium was increased to 71.71 mg/L, which was 155.9-fold higher than that achieved in mono-culture. The de novo biosynthesis of caffeic acid in the CMC medium was also achieved through a three-strain co-cultivation. Caffeic acid production was up to 16.91 mg/L after optimizing the inoculation ratio of these strains.

Conclusion: De novo biosynthesis of p-CA and caffeic acid from lignocellulose through a co-cultivation strategy was achieved for the first time. This study provides favorable support for the biosynthesis of more high value-added products from economical substrates. In addition, the multi-strain co-culture strategy can effectively improve the final titer of the target products, which has high application potential in the field of industrial production.

Keywords: Caffeic acid; Carboxymethyl-cellulose; Co-culture; Saccharomyces cerevisiae; p-coumaric acid.

Fig. 1

Schematic illustration of the co-culture system of different metabolically engineered S. cerevisiae strains for de novo biosynthesis of p-CA and CA from CMC. In this system, CMC in medium was degraded by cellulolytic enzymes expressed in SK10-3 by the POT1-mediated δ-integration strategy. The released glucose was assimilated by SK10-3 and other co-culture strains to yield p-CA or CA. PEP: phosphoenolpyruvate; EPSP: 5-enolpyruvylshikimate-3-phosphate; Trp: tryptophan; Phe: phenylalanine; Tyr: tyrosine; p-CA: p-coumaric acid; CA: caffeic acid

Fig. 2

Phenotypes of betaxanthin and p-CA production in the co-culture systems in CMC medium. a Comparison of color phenotypes in mono-culture and co-culture systems. b Growth curves. c p-CA production in mono-culture and co-culture systems. Three replicates of each sample were used

Fig. 3

Growth curves of co-culture systems carried out according to different inoculum ratios of SK10-3 to NK-B2b and inoculation at different interval times. Three replicates of each sample were used

Fig. 4

The p-CA production of co-culture systems carried out according to different inoculum ratios of SK10-3 to NK-B2b and inoculation at different interval times. Three replicates of each sample were used

Fig. 5

Growth curves and p-CA production in the carbon source optimization experiment. a Growth curves in 20 g/L CMC medium. b Growth curves in 30 g/L CMC medium. c p-CA production after 120 h fermentation. d p-CA production after 168 h fermentation. Three replicates of each sample were used

Fig. 6

HPLC chromatogram of p-CA and caffeic acid. a Standards of p-CA and caffeic acid. b Co-culture sample of SK10-3 and NK-B2b; peak 1 was p-CA obtained from this co-culture system. c Co-culture sample of SK10-3 and NK-B2c; peak 2 was caffeic acid obtained from this co-culture system

Fig. 7

a Schematic illustration of the multi-strain co-culture system. b CA production and p-CA residue after 168 h fermentation in the multi-strain co-culture system with different inoculation ratios of NK-B2b to NK-B2d. Three replicates of each sample were used