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 (∼2 mmol gDW-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.
Highlights
Removing transcriptional feedback regulation of metabolic pathways is a classical approach to enhance overproduction of chemicals in microbes
One reason for the slow growth could be the deletion of the transcriptional repressor ArgR (Figure 1a), which has a regulatory function beyond arginine metabolism and controls 423 direct and indirect targets.[19]
The KO-strain, CRISPR interference (CRISPRi)#7, CRISPRi#10, and E. coli wild-type were cultivated in shake flasks on glucose minimal medium to measure growth and arginine concentration in the supernatant
Summary
Even during growth on glucose minimal medium,[20,21] it needs to be clarified if ArgR can be reduced to a level at which overproduction is sufficiently high, while cellular physiology is not affected by the missing regulation. The KO-strain, CRISPRi#7, CRISPRi#10, and E. coli wild-type were cultivated in shake flasks on glucose minimal medium to measure growth and arginine concentration in the supernatant (three independent cultures per strain) Both CRISPRi#7 and CRISPRi#10 grew much better than the complete dysregulated KO-strain, and growth rates were consistent between independent experiments that were performed on different days (Figure 4a,b). The specific arginine production of the three strains was in a similar range, showing that it was not arginine overproduction per se that caused differences in growth between the three strains One reason for these differences was that deletion of ArgR caused stronger and more global changes of amino acid enzymes than downregulation of the transcription factors. Biomass specific arginine yields and growth rates; effect of ArgO expression on growth; plasmid map of pargO; substrate specific arginine yield; growth rates with supplementation of orotate, aspartate, cytidine, and uracil; strains used in this study; plasmids used in this study; oligonucleotides used in this study (PDF)
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