Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi

Yu, Yang; Yin, Lei; Liu, Dandan; Guo, Wu; Wang, Lixian; Xing-ji, Wang; Lv, Hexin; Yang, Yang; Liu, Qian; Tian, Chaoguang

doi:10.1007/s00253-021-11733-w

Cited by 9 publications

(11 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Consistent with the results in T. reesei [ 27 , 42 , 43 ], the performance of the overall 100,000-conidia populations allowed us to quantitatively distinguish the difference of various promoters, overcome the tedious procedures of biomass measurement, and reduce the operating error ( Figure 2 ). Recently, flow cytometry was applied to directly sort germinated conidia [ 44 ] or transformed protoplasts without plating [ 45 ], demonstrating the potential of high-throughput screening with mCherry-PyrG fusion by fluorescence-activated cell sorting (FACS). Moreover, coupled with droplet-based microfluidics [ 44 ], this fluorescent-auxotrophic selection workflow could be applied for screening inducible promoters under various conditions.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, flow cytometry was applied to directly sort germinated conidia [ 44 ] or transformed protoplasts without plating [ 45 ], demonstrating the potential of high-throughput screening with mCherry-PyrG fusion by fluorescence-activated cell sorting (FACS). Moreover, coupled with droplet-based microfluidics [ 44 ], this fluorescent-auxotrophic selection workflow could be applied for screening inducible promoters under various conditions.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Zheng

Wang

et al. 2022

JoF

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Zheng

Wang

et al. 2022

JoF

View full text Add to dashboard Cite

show abstract

“…While this manuscript was in revision, two papers regarding the strain engineering of A. niger N1 [ 71 ] and O1 [ 72 ] using the protoplast transformation procedure described in M. thermophila were published. In A. niger O1, Guo et al described that the four overexpression and four knockout transformants of the acid α-amylase gene were simply obtained after protoplast transformation, while four overexpression and two knockout transformants of the neutral α-amylase gene were generated [ 72 ].…”

Section: Discussionmentioning

confidence: 99%

“…In A. niger O1, Guo et al described that the four overexpression and four knockout transformants of the acid α-amylase gene were simply obtained after protoplast transformation, while four overexpression and two knockout transformants of the neutral α-amylase gene were generated [ 72 ]. In A. niger N1, 6 out of 52 transformants were triple-gene mutants, with a gene editing efficiency of 11.5% [ 71 ]. Compared to previous studies [ 71 , 72 ], the PMT method we developed here is relatively simple.…”

Section: Discussionmentioning

confidence: 99%

“…In A. niger N1, 6 out of 52 transformants were triple-gene mutants, with a gene editing efficiency of 11.5% [ 71 ]. Compared to previous studies [ 71 , 72 ], the PMT method we developed here is relatively simple. First, in previous studies, the young mycelia were washed twice with 1 mol/L MgSO 4 before lysing with enzymatic hydrolysate [ 71 , 72 ], while in our study, enough protoplasts could be obtained without carrying out these washing steps; therefore, the PMT method that we developed is relatively simple and saves time.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains

Liu

Guo

et al. 2022

Biology

View full text Add to dashboard Cite

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.

show abstract

Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora

Wang

et al. 2022

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi

Cited by 9 publications

References 60 publications

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

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

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains

Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora

Contact Info

Product

Resources

About