Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production

Abstract

Aspergillus niger 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 A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA 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 A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.

Keywords: Aspergillus niger; CRISPR/Cas9; citric acid; flow cytometry; fluorescence protein; promoter.

Figure 1

Fluorescent-auxotrophic selection coupled with CRISPR/Cas9 system and flow cytometry. (A) Schematic overview of a promoter evaluation workflow based on an intuitive fluorescent-auxotrophic selection. This workflow combined the efficient CRISPR/Cas9 genetic manipulation system, the fluorescent protein-selection marker fused indicator, and flow cytometry-based analysis. CRISPR/Cas9 system was applied for integration of the reporter at the specific genome locus. Then, transformants with distinct fluorescence were picked in 24-deep-well plates based on fluorescence imaging. After genotype verification, fluorescence intensity was detected by laser scanning confocal microscope and flow cytometry. (B) Schematic diagram of mCherry-pyrG-expressing construct under the control of the PgpdAd promoter. The donor DNAs comprised the PgpdAd::mCherry-pyrG cassette, and 40-bp micro-homology arms targeted the flanking sequences of agdA gene. After being constructed, the donor DNAs were co-transformed with linear sgRNA constructs (agdA-sgRNA1 and agdA-sgRNA2) and the Cas9 expression cassette into protoplasts of the kusA and pyrG deficient chassis D353.8. Two DNA double-strand breaks (DSBs) at the flanking sequences of the agdA gene were generated by the Cas9 under the guide of two sgRNAs and then were repaired by homologous recombination (HR) with the integration of the donor DNAs, resulting in YDD1.13. (C) Representative fluorescence images in conidia and mycelial pellets of YDD1.13-expressing mCherry-pyrG with the PgpdAd promoter. The parent strain D353.8 was used as negative control. (D,E) Flow cytometry analysis of the conidia of YDD1.13. The 100,000 spores were analyzed by flow cytometry. Black boxes marking the same value of mCherry-log-height were used for direct comparison of YDD1.13 with the parent strain D353.8.

Figure 2

Fluorescence analysis of constructs expressing mCherry-pyrG controlled by six constitutive promoters. (A) Representative fluorescence images in conidia of constructs expressing mCherry-pyrG controlled by six constitutive promoters. The parent strain D353.8 was used as negative control and YDD1.13 was used as positive control. (B,C) Representative flow cytometry analysis of the conidia of constructs YDD2 to YDD7 expressing mCherry-pyrG controlled by six selected promoters. The 100,000 spores were analyzed by flow cytometry. To reduce the interference of background fluorescence from the parent strain D353.8, the black box marking the same value of mCherry-log-height (higher than 10²) was used for direct comparison of constructs expressing mCherry-pyrG controlled by different promoters. The number below the black box represents the percentage of spores with high fluorescence (mCherry-log-height higher than 10²) of each construct. A. niger YDD1.13 with the promoter of PgpdAd was used as positive control, while the parent strain D353.8 was used as negative control.

Figure 3

Characterization of essential elements of the PgpdAg promoter. (A) Multiple sequence alignment of the PgpdA promoters of various Aspergilli spp. The GenBank accession numbers of the selected gpdA genes included A. niger CBS 513.88 (An16g01830), A. nidulans FGSC A4 (ANIA_08041), A. clavatus NRRL1(ACLA_003290), A. fumigatus Af293 (AFUA _5G01970), A. flavus NRRL 3357 (AFLA_025100), and A. oryzae RIB40 (AO090003001322). The conserved motifs are highlighted with red boxes. The transcription start site and start codon are shown as red arrows. (B) The conserved motifs’ prediction and truncation test of PgpdAg promoter in A. niger. GpdA-box and five predicted conserved motifs are represented as orange bars and green bars, respectively. The truncation design of the PgpdAg promoter is displayed as gray bars. The mean mCherry fluorescence intensity of each truncation is shown as blue bars. Results are the mean of three replicates, and error bars indicate standard deviations (n = 4). Pairwise Student’s t-test were conducted between PgpdAg truncated mutant relative to the full-length PgpdAg reported strain and between the mutant only containing UTR and the parent strain, respectively. The p values are indicated as (>0.05, n.s.; * < 0.05; *** < 0.001).

Figure 4

Citric acid production of A. niger strains expressing cexA under the control of the PgpdA promoter. (A) Schematic diagram of constructs expressing cexA under the control donor DNAs containing the PgpdA promoter. The donor DNAs containing PgpdAd::CexA, PgpdAg::CexA, and PgpdAg-775::CexA expressing cassettes were co-transformed with linear sgRNA constructs (agdA-sgRNA1 and agdA-sgRNA2) and a Cas9 expression cassette into the protoplasts of A. niger D353.8. Two DNA double-strand breaks (DSBs) at the flanking sequences of agdA gene were generated by the Cas9 under the guide of two sgRNAs and then were repaired by HR with the integration of donor DNAs. (B,C) Citric acid production of the cexA overexpressed strains in A. niger. Citric acid titer (B) and normalized citric acid titer (g citric acid/g dry weight, C) were calculated for each strain. Then, 1 × 10⁵ spores/mL were inoculated in 20 mL citrate fermentation media and incubated at 34 °C for 120 h. The extracellular citric acid was determined by the method of HPLC. A. niger XMD6.1 with cexA overexpression under the control of PgpdAd was used as positive control, while the parent strain D353.8 was used as negative control. Results are the mean of three replicates, and error bars indicate standard deviations (n = 3). Pairwise Student’s t-test was conducted between cexA overexpression mutants relative to the parent strains. The p values are indicated as (>0.05, n.s.; *** < 0.001).

Figure 5

Transcription analysis of cexA-expressing constructs in submerged citric acid fermentation. Citric acid production (A) and the cexA transcript level (B) of the cexA-expressing constructs in 5 L bioreactors. Then, 1 × 10⁵ spores/mL were inoculated in submerged citric acid fermentation at 34 °C for 144 h. The extracellular citric acid was determined by the method of HPLC. Results are the mean of three replicates, and error bars indicate standard deviations (n = 3). The red arrow in (A) represents the time point of sampling for RNAseq analysis.