Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

Abstract

Background: Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive.

Results: In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH) and fumarase (RoFUM1) were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2) was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1) than the control strain FMME-001 empty vector.

Conclusions: The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner.

Figure 1

The cytosolic fumarate biosynthetic pathway in S. cerevisiae. Abbreviations of enzymes: Pyc, pyruvate carboxylase; Mdh, malate dehydrogenase; Fum, fumarase; Pdc, pyruvate decarboxylase; Adh, alcohol dehydrogenase; Ald, acetaldehyde dehydrogenase; Acs, acetyl-CoA synthetase.

Figure 2

Fermentation profile for cell growth, glucose utilization and product accumulation during aerobic batch cultures of control strain FMME-001 empty vector, engineered strains FMME-001 ↑MDH3ΔSKL and FMME-001 ↑RoMDH with 6% glucose. A: growth, B: residual glucose, C: fumaric acid and D: ethanol. Symbols: square, FMME-001 empty vector, circle, FMME-001 ↑MDH3ΔSKL, triangle, FMME-001 ↑RoMDH. Error bars indicate standard deviation (n = 3).

Figure 3

The IR spectra of fumaric acid. (a) sample, (b) the fumaric acid standard.

Figure 4

¹H NMR and ¹³C NMR spectra of sample from engineered strain and the fumaric acid standard.

Figure 5

Relative gene expression levels. (A) RoMDH and RoFUM1 in the mutant strain FMME-001↑RoMDH + ↑RoFUM1 and the parent strain FMME-001 empty vector. Relative gene expression level of (B) PYC2 in the mutant strain FMME-001↑PYC2 + ↑RoMDH and the parent strain FMME-001 empty vector. Relative transcription levels were normalized to the transcription level of the β-ACTIN gene, which was taken as 1. The presented values are averages of three independent experiments; the error bars indicate standard deviations.