Resveratrol production of a recombinant Scheffersomyces stipitis strain from molasses

Abstract

Resveratrol is a plant-derived aromatic compound with beneficial properties and it is required to develop a resveratrol production process from inexpensive biomass feedstocks. Here, we investigated the potential of Scheffersomyces stipitis, a non-conventional yeast with the capacity to utilize a wide range of sugars, to produce resveratrol from molasses, which is a by-product of sugar refineries. The S. stipitis strain metabolically engineered for resveratrol production produced resveratrol from 60 g/L mixed sugar (sucrose, glucose, and fructose), while its resveratrol titer decreased as the proportions of glucose and fructose increased. Sucrose consumption of the S. stipitis strain was clearly suppressed by the coexistence of glucose, fructose, and even ethanol. Quantitative analysis of intracellular metabolites involved in resveratrol biosynthesis using capillary electrophoresis time-of-flight mass spectrometry revealed that the composition of these sugars has a significant effect on the intracellular accumulation of glycolytic metabolites and AMP, which is an important factor involved in some cellular metabolic responses. Furthermore, the S. stipitis strain produced 1076 ± 167 mg/L of resveratrol in the fermentation with commercial sugarcane molasses (120 g/L of total sugars) as the substrate. To our knowledge, this is the first report on carbon catabolite repression in S. stipitis caused by the coexistence of sucrose, glucose, and fructose and resveratrol production from molasses. These results indicate great potential of the cost-effective resveratrol production process from molasses substrates using recombinant S. stipitis.

Keywords: Carbon catabolite repression; Intracellular metabolite analysis; Molasses; Non-conventional yeast; Resveratrol; Scheffersomyces stipitis.

Fig. 1

Schematic representation of resveratrol biosynthesis in the Ss-T4V-aro7m strain using sucrose, glucose, and fructose as carbon sources. Abbreviations: 1,3-PG, 1,3-bisphosphoglycerate; 2-PG, 2-phosphoglycerate; 3-PG, 3-phosphoglycerate; 6-PGA, 6-phosphogluconate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F1,6BP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; NAD⁺, nicotinamide adenine dinucleotide; NADP⁺, nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvate; PPi, pyrophosphate ion; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; Xu5P, xylulose-5-phosphate.

Fig. 2

Fermentation with resveratrol-producing S. stipitis in mixed sugar media comprising sucrose, glucose, and fructose in different proportions. Fermentation tests were performed in media containing (A) 60 g/L sucrose (S60), (B) 40 g/L sucrose, 10 g/L glucose, and 10 g/L fructose (S40G10F10), (C) 30 g/L sucrose, 15 g/L glucose, and 15 g/L fructose (S30G15F15), (D) 20 g/L sucrose, 20 g/L glucose, and 20 g/L fructose (S20G20F20), and (E) 30 g/L glucose and 30 g/L fructose (G30F30), respectively. The data represent the mean ± standard deviation of three independent experiments.

Fig. 3

Time-course changes in the concentration of intracellular metabolites during fermentation in different mixed sugar media. Intracellular metabolite concentrations (nmol/mg DCW) of the Ss-T4V-aro7m strain grown in three different media, containing 60 g/L sucrose (S60 medium); 40 g/L sucrose, 10 g/L glucose, and 10 g/L fructose (S40G10F10 medium); and 30 g/L glucose and 30 g/L fructose (G30F30 medium); respectively. Metabolites written in blue were not analyzed in this study. Data are presented as the mean ± standard deviation (n = 3).

Fig. 4

Molasses fermentation by the Ss-T4V-aro7m strain. Fermentation tests were performed in YP media with molasses composed of (A) 60 g/L and (B) 120 g/L of total sugars. The data represent the mean ± standard deviation of three independent experiments.