Enhancement of glucaric acid production in Saccharomyces cerevisiae by expressing Vitreoscilla hemoglobin.

OBJECTIVE: To enhance the glucaric acid (GA) production in Saccharomyces cerevisiae, the Vitreoscilla hemoglobin was employed to reinforce cellular oxygen supplement. Additionally, the pH-free fermentation strategy was engaged to lower the cost brought by base feeding during the acid-accumulated and long-period glucaric acid production. RESULTS: Recombinant yeast Bga-4 was constructed harboring Vitreoscilla hemoglobin on the basis of previous Bga-3. Higher glucose uptake rate, growth rate, and ethanol reuse rate were achieved in Bga-4 in shake-flask fermentation than those in Bga-3. Furthermore, the fed-batch fermentation in a 5-L bioreactor was performed without pH control, resulting in a final glucaric acid titer of 6.38 g/L. CONCLUSIONS: Both the GA titer and biomass were enhanced along with the efficiency of ethanol re-utilization in the presence of VHb. Moreover, the absence of base feeding for long-period fermentation reduced production cost, which is meaningful for industrial applications.

Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer.

BACKGROUND: Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. RESULTS: A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae. CONCLUSIONS: In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae.