Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10-15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0-6.8 and resulted in production of 127-140 g l(-1) CA with a yield Y-CA of 0.75-0.82 g g(-1), whereas at pH 5.0, 87 g l(-1) with a yield Y (CA) of 0.51 gg(-1) were produced. The CA-productivity Q (CA) increased from 0.40 g l(-1) h(-1) at pH 5.0 up to 0.73 g l(-1) h(-1) at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene-dose-dependent high isocitrate lyase expression resulting in strong reduction (< 5%) of isocitric acid, a by-product during CA production.