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Abstract:
Removing transcriptional feedback regulation of metabolic pathways is a
classical approach to enhance overproduction of chemicals in microbes.
However, disrupting transcriptional regulation can have broad
physiological consequences that decrease cellular growth and
productivity. Here, we compared downregulation and deletion of the
transcriptional repressor ArgR in arginine overproducing Escherichia
coli. Different levels of ArgR downregulation were achieved with CRISPR
interference (CRISPRi) and resulted in 2-times higher growth rates
compared to deletion of ArgR, while specific arginine production was
similar (similar to 2 mmol g(DW)(-1) h(-1)). Metabolomics and proteomics
data revealed that poor growth of the ArgR deletion strain was caused by
a limitation of pyrimidine nucleotide biosynthesis, because a 17-fold
overexpression of ornithine carbamoyltransferase (ArgI) perturbed the
arginine-pyrimidine branch point. These results demonstrate that
overexpression of enzymes in an engineered pathway can impair metabolism
of the host, especially in the case of branch-point enzymes. Thus,
balancing enzyme levels is important to optimize industrial microbes,
and CRISPRi of a transcription factor is a versatile tool for this
purpose.
