CRISPR interference of nucleotide biosynthesis improves production of a single‐domain antibody in <i>Escherichia coli</i>

Landberg, Jenny Marie; Wright, Naia Risager; Wulff, Tune; Nielsen, Alex Toftgaard

doi:10.1002/bit.27536

Cited by 15 publications

(15 citation statements)

References 62 publications

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“…After downregulation of ilvE gene, a KIV titer of 0.4 g L −1 with 9.6 g L −1 of glucose consumption was achieved at 37 °C at laboratory scale in MG03 that was far greater (437-fold) as compared with the control (without KIV integration). The results are supported by a study where the use of CRISPRi has improved the production of single domain antibody in E. coli ( Landberg et al, 2020 ). The glucose consumption and KIV production was compared at 30 °C and 37 °C temperature.…”

Section: Discussionmentioning

confidence: 71%

Isobutanol production by combined in vivo and in vitro metabolic engineering

Gupta

Wong

Jawed

et al. 2022

Metabolic Engineering Communications

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Section: Discussionmentioning

confidence: 71%

Isobutanol production by combined in vivo and in vitro metabolic engineering

Gupta

Wong

Jawed

et al. 2022

Metabolic Engineering Communications

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“…However, unexpected targets can have other mechanisms for improving product titers. Targeting genes from purine and pyrimidine biosynthesis pathways for underexpression (including cmk and pyrH ) has been applied for antibody production 56 as a growth decoupling strategy to increase product formation. Inhibiting excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers 56 .…”

Section: Resultsmentioning

confidence: 99%

Optimization of chondroitin production inE. coliusing genome scale models

Couto,

Rodrigues,

Braga

et al. 2023

Preprint

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Chondroitin is a natural occurring glycosaminoglycan with applications as a nutraceutical and pharmaceutical ingredient and can be extracted from animal tissues. Microbial chondroitin-like polysaccharides emerged as a safer and more sustainable alternative source. However, chondroitin titers using either natural or recombinant microorganisms are still far from meeting the increasing demand. The use of genome-scale models and computational predictions can assist the design of microbial cell factories with possible improved titers of these value-added compounds. Genome-scale models have been used to predict genetic modifications inEscherichia coliengineered strains that would potentially lead to improved chondroitin production. Additionally, using synthetic biology approaches, a pathway for producing chondroitin has been designed and engineered inE. coli. Afterwards, the most promising mutants identified based on bioinformatics predictions were constructed and evaluated for chondroitin production in flask fermentation. This resulted in the production of 118 mg/L of extracellular chondroitin by overexpressing both superoxide dismutase (sodA) and a lytic murein transglycosylase (mltB). Then, batch and fed-batch fermentations at bioreactor scale were also evaluated, in which the mutant overexpressingmltBled to an extracellular chondroitin production of 427 mg/L and 535 mg/L, respectively. The computational approach herein described identified several potential novel targets for improved chondroitin biosynthesis, which may ultimately lead to a more efficient production of this glycosaminoglycan.

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“…This switch slowed down the growth but did not block it completely and resulted in a modest increase in protein production. Their approach was more efficient by targeting the genes pyrF 14 , pyrG and cmk 21 , leading to stabilization of culture density at a sub-maximal level. Notably, blocking of pyrF also led to elongated cells similar to those in our experiments (Figure 2c), indicating that this is a common reaction to the disruption of DNA metabolism.…”

Section: Resultsmentioning

confidence: 99%

Decoupling growth and production by removing the origin of replication from a bacterial chromosome

Kasari

Kärmas

et al. 2021

Preprint

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Efficient production of biochemicals and proteins in cell factories frequently benefits from a two-stage bioprocess in which growth and production phases are decoupled. Here we describe a novel growth switch based on the permanent removal of the origin of replication (oriC) from the Escherichia coli chromosome. Without oriC, cells cannot initiate a new round of replication and they stop growing while their metabolism remains active. Our system relies on a serine recombinase from bacteriophage phiC31 whose expression is controlled by the temperature-sensitive cI857 repressor from phage lambda. Reporter protein expression in switched cells continues after cessation of growth, leading to protein levels up to five times higher compared to non-switching cells. Switching induces a unique physiological state that is different from both normal exponential and stationary phases. Switched cells remain in this state even when not growing, retain their protein synthesis capacity, and do not induce proteins associated with the stationary phase. Our switcher technology is potentially useful for a range of products and applicable in many bacterial species for decoupling growth and production.

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CRISPR interference of nucleotide biosynthesis improves production of a single‐domain antibody in Escherichia coli

Cited by 15 publications

References 62 publications

Isobutanol production by combined in vivo and in vitro metabolic engineering

Isobutanol production by combined in vivo and in vitro metabolic engineering

Optimization of chondroitin production inE. coliusing genome scale models

Decoupling growth and production by removing the origin of replication from a bacterial chromosome

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