CRISPRi screen for enhancing heterologous alpha-amylase yield in Bacillus subtilis
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CRISPRi screen for enhancing heterologous alpha-amylase yield in Bacillus subtilis. / Geissler, Adrian Sven; Fehler, Annaleigh Ohrt; Poulsen, Line Dahl; Gonzalez-Tortuero, Enrique; Kallehauge, Thomas Beuchert; Alkan, Ferhat; Anthon, Christian; Seemann, Stefan Ernst; Rasmussen, Michael Dolberg; Breuener, Anne; Hjort, Carsten; Vinther, Jeppe; Gorodkin, Jan.
I: Journal of Industrial Microbiology and Biotechnology, Bind 50, Nr. 1, kuac028, 2023.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - CRISPRi screen for enhancing heterologous alpha-amylase yield in Bacillus subtilis
AU - Geissler, Adrian Sven
AU - Fehler, Annaleigh Ohrt
AU - Poulsen, Line Dahl
AU - Gonzalez-Tortuero, Enrique
AU - Kallehauge, Thomas Beuchert
AU - Alkan, Ferhat
AU - Anthon, Christian
AU - Seemann, Stefan Ernst
AU - Rasmussen, Michael Dolberg
AU - Breuener, Anne
AU - Hjort, Carsten
AU - Vinther, Jeppe
AU - Gorodkin, Jan
N1 - Erratum: https://academic.oup.com/jimb/article/50/1/kuad003/7080731?login=true
PY - 2023
Y1 - 2023
N2 - Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two alpha-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 ' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.
AB - Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two alpha-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 ' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.
KW - Transcriptomics
KW - alpha-amylase
KW - fermentation
KW - screening
KW - CRISPRi
KW - SEQUENCE-SPECIFIC CONTROL
KW - PROTEIN SECRETION
KW - REGULATORY RNAS
KW - ELEMENT ICEBS1
KW - IN-VIVO
KW - IDENTIFICATION
KW - ENZYMES
KW - LEVEL
KW - GROWTH
KW - PRSA
UR - https://academic.oup.com/jimb/article/50/1/kuad003/7080731
U2 - 10.1093/jimb/kuac028
DO - 10.1093/jimb/kuac028
M3 - Journal article
C2 - 36564025
VL - 50
JO - Journal of Industrial Microbiology and Biotechnology
JF - Journal of Industrial Microbiology and Biotechnology
SN - 1367-5435
IS - 1
M1 - kuac028
ER -
ID: 335684747