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 -