초록
<P><B>Background</B></P><P>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 <I>Escherichia coli</I> and <I>Saccharomyces cerevisiae</I> by co-expression of genes encoding <I>myo</I>-inositol-1-phosphate synthase (Ino1), <I>myo</I>-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from <I>Mus musculus</I> was proved to be the bottleneck in this pathway.</P><P><B>Results</B></P><P>A more stable <I>miox4</I> from <I>Arabidopsis thaliana</I> was chosen in the present study. In addition, high copy delta-sequence integration of <I>miox4</I> into the <I>S. cerevisiae</I> 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 <I>S. cerevisiae</I> strain produced 6 g/L (28.6 mM) glucaric acid from <I>myo</I>-inositol, the highest titer that had been ever reported in <I>S. cerevisiae</I>.</P><P><B>Conclusions</B></P><P>In this study, glucaric acid titer was increased to 6 g/L in <I>S. cerevisiae</I> by integrating the <I>miox4</I> gene from <I>A. thaliana</I> and the <I>udh</I> gene from <I>Pseudomonas syringae</I> 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 <I>S. cerevisiae</I>.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12934-018-0914-y) contains supplementary material, which is available to authorized users.</P>