Improving citric acid production in Aspergillus niger by overexpression of citrate synthase with strong promoter

Abstract

Aspergillus niger CGMCC10142, a strain widely used for industrial citric acid (CA) production, encodes four citrate synthase genes (cs1-cs4), yet their specific roles in CA biosynthesis remain poorly understood. We found that the transcript levels of the genes encoding mitochondrial citrate synthases, cs3 or cs4 were significantly higher than those encoding cytoplasmic citrate synthases cs1 and cs2 with 94.26–766.0 folds or 219.33–1782.25 folds, from 27.8 (cs2) to 49546.6 (cs4) at 24 h during CA fermentation. Given the naturally low transcription levels and cytoplasmic localization of cs1 and cs2, we hypothesized that their overexpression would enhance CA production by increasing the CA available for export. Transformants overexpressing cytoplasmic cs1 (T-c1–19) and cs2 (T-c2–81) showed increases in CA production of 5.38 % and 18.39 %, respectively. In contrast, no significant change was observed in transformant overexpressing mitochondrial cs3 (T-c3–11). The results showed a 6.8 % increase in acid production by strain T-c2–81, reaching 181.2 g/L at 56 h in 30 L bioreactors, compared to the starting strain of 169.6.g/L. qRT-PCR analysis of revealed that the relative expression levels of cs2 in the T-c2–81 strain was 8.6 times higher than in the wild-type strain at 24 h. In addition, the expression levels of key genes pfk, pc and the citrate transporter cexA were significantly increased, ranging from 1.3 to 67 times than the control levels. This 6.8 % improvement of CA yield not only reduces unit fermentation costs but also represents a significant breakthrough in regulating intracellular metabolic flow.