Manipulation of the carbon storage regulator system for metabolite remodeling and biofuel production in Escherichia coli.
BACKGROUND
Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.


RESULTS
We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO(2).


CONCLUSIONS
We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.
Joint BioEnergy Institute, Emeryville, CA, USA.
Adrienne E
AE
McKee
Becky J
BJ
Rutherford
Dylan C
DC
Chivian
Edward K
EK
Baidoo
Darmawi
D
Juminaga
Dwight
D
Kuo
Peter I
PI
Benke
Jeffrey A
JA
Dietrich
Suzanne M
SM
Ma
Adam P
AP
Arkin
Christopher J
CJ
Petzold
Paul D
PD
Adams
Jay D
JD
Keasling
Swapnil R
SR
Chhabra
D020001:1-Butanol / Q000378:metabolism; D056804:Biofuels / Q000032:analysis / Q000382:microbiology*; D002244:Carbon / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D060847:Metabolic Engineering; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism
D056804:Biofuels; C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D002244:Carbon; D020001:1-Butanol