초록
<B>Abstract</B><B>Background</B><P>Bio-based production of organic acids promises to be an attractive alternative for the chemicals industry to substitute petrochemicals as building-block chemicals. In recent years, itaconic acid (IA, methylenesuccinic acid) has been established as a sustainable building-block chemical for the manufacture of various products such as synthetic resins, coatings, and biofuels. The natural IA producer <I>Aspergillus terreus</I> is currently used for industrial IA production; however, the filamentous fungus <I>Aspergillus niger</I> has been suggested to be a more suitable host for this purpose. In our previous report, we communicated the overexpression of a putative cytosolic citrate synthase <I>citB</I> in an <I>A. niger</I> strain carrying the full IA biosynthesis gene cluster from <I>A. terreus,</I> which resulted in the highest final titer reported for <I>A.</I><I>niger</I> (26.2 g/L IA). In this research, we have attempted to improve this pathway by increasing the cytosolic acetyl-CoA pool. Additionally, we have also performed fermentation optimization by varying the nitrogen source and concentration.</P><B>Results</B><P>To increase the cytosolic acetyl-CoA pool, we have overexpressed genes <I>acl1</I> and <I>acl2</I> that together encode for ATP-citrate lyase (ACL). Metabolic engineering of ACL resulted in improved IA production through an apparent increase in glycolytic flux. Strains that overexpress <I>acl12</I> show an increased yield, titer and productivity in comparison with parental strain CitB#99. Furthermore, IA fermentation conditions were improved by nitrogen supplementation, which resulted in alkalization of the medium and thereby reducing IA-induced weak-acid stress. In turn, the alkalizing effect of nitrogen supplementation enabled an elongated idiophase and allowed final titers up to 42.7 g/L to be reached at a productivity of 0.18 g/L/h and yield of 0.26 g/g in 10-L bioreactors.</P><B>Conclusion</B><P>Ultimately, this study shows that metabolic engineering of ACL in our rewired IA biosynthesis pathway leads to improved IA production in <I>A. niger</I> due to an increase in glycolytic flux. Furthermore, IA fermentation conditions were improved by nitrogen supplementation that alleviates IA induced weak-acid stress and extends the idiophase.</P>