| Summary: | <i>Aspergillus niger</i> is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for <i>A. niger</i> are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on <i>mCherry</i>, <i>pyrG</i>, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in <i>A. niger</i>. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter P<i>gpdAg</i> showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter P<i>gpdAd</i> from <i>A. nidulans</i>. Six predicted conserved motifs, including the <i>gpdA</i>-box, were verified to be essential for the P<i>gpdAg</i> activity. To demonstrate its application, the promoter P<i>gpdAg</i> was used for enhancing the expression of citrate exporter <i>cexA</i> in a citric acid-producing isolate D353.8. Compared with the <i>cexA</i> controlled by P<i>gpdAd</i>, the transcription level of the <i>cexA</i> gene driven by P<i>gpdAg</i> increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following <i>cexA</i> overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of <i>A. niger</i> promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.
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