title: Enhanced protein and biochemical production using CRISPRi-based growth switches.
abstract: Production of proteins and biochemicals in microbial cell factories is often limited by carbon and energy spent on excess biomass formation. To address this issue, we developed several genetic growth switches based on CRISPR interference technology. We demonstrate that growth of Escherichia coli can be controlled by repressing the DNA replication machinery, by targeting dnaA and oriC, or by blocking nucleotide synthesis through pyrF or thyA. This way, total GFP-protein production could be increased by up to 2.2-fold. Single-cell dynamic tracking in microfluidic systems was used to confirm functionality of the growth switches. Decoupling of growth from production of biochemicals was demonstrated for mevalonate, a precursor for isoprenoid compounds. Mass yield of mevalonate was increased by 41%, and production was maintained for more than 45h after activation of the pyrF-based growth switch. The developed methods represent a promising approach for increasing production yield and titer for proteins and biochemicals.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs. Lyngby, Denmark.
authors.forename: Songyuan
authors.initials: S
authors.lastname: Li
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs. Lyngby, Denmark.
authors.forename: Christian Bille
authors.initials: CB
authors.lastname: Jendresen
authors.affiliation: Forschungszentrum Jülich GmbH, IBG-1: Biotechnology, 52425, Jülich, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Grünberger
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs. Lyngby, Denmark.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Ronda
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs. Lyngby, Denmark.
authors.forename: Sheila Ingemann
authors.initials: SI
authors.lastname: Jensen
authors.affiliation: Forschungszentrum Jülich GmbH, IBG-1: Biotechnology, 52425, Jülich, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Noack
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, 2800 Kgs. Lyngby, Denmark. Electronic address: atn@biosustain.dtu.dk.
authors.forename: Alex Toftgaard
authors.initials: AT
authors.lastname: Nielsen
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002244:Carbon / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005812:Genes, Switch / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D046940:Synechococcus / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002244:Carbon
keywords: CRISPRi; Growth switch; Metabolic engineering; Mevalonate production; Protein production