Flagella disruption in Bacillus subtilis increases amylase production yield
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Flagella disruption in Bacillus subtilis increases amylase production yield. / Fehler, Annaleigh Ohrt; Kallehauge, Thomas Beuchert; Geissler, Adrian Sven; González-Tortuero, Enrique; Seemann, Stefan Ernst; Gorodkin, Jan; Vinther, Jeppe.
In: Microbial Cell Factories, Vol. 21, 131, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Flagella disruption in Bacillus subtilis increases amylase production yield
AU - Fehler, Annaleigh Ohrt
AU - Kallehauge, Thomas Beuchert
AU - Geissler, Adrian Sven
AU - González-Tortuero, Enrique
AU - Seemann, Stefan Ernst
AU - Gorodkin, Jan
AU - Vinther, Jeppe
N1 - Publisher Copyright: © 2022, The Author(s).
PY - 2022
Y1 - 2022
N2 - Background: Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients. Results: In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2–threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar. Conclusions: We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.
AB - Background: Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients. Results: In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2–threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar. Conclusions: We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.
KW - Bacillus subtilis
KW - CRISPR-dCas9
KW - Flagella
KW - Industrial production
KW - Motility
U2 - 10.1186/s12934-022-01861-x
DO - 10.1186/s12934-022-01861-x
M3 - Journal article
C2 - 35780132
AN - SCOPUS:85133406427
VL - 21
JO - Microbial Cell
JF - Microbial Cell
SN - 1475-2859
M1 - 131
ER -
ID: 315851373