Genome-scale metabolic rewiring to improve titers, rates and yields of the non-native non-ribosomal peptide product indigoidine at scale
Banerjee D, Eng T, Lau AK, Sasaki Y, Wang B, Chen Y, Prahl J-P, Singan VR, Herbert RA, Liu Y, Tanjore D, Petzold CJ, Keasling JD, Mukhopadhyay A. Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale. Nat Commun [Internet]. 2020;11(1):5385. Available from: https://doi.org/10.1038/s41467-020-19171-4
- Organism: Pseudomonas putida KT2440
- Instrument: 6460 Triple Quadrupole LC/MS
Metabolic engineering, genome editing
- Lab head:
High titer, rates, yields (TRY) and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we took the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We computed MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From 63 computed solution-sets, our -omics guided process identified one experimentally feasible solution requiring 14 simultaneous reaction interventions. Using multiplex-CRISPRi, this is the first experimental implementation of a 14-gene MCS-based solution that shifted production from stationary to exponential phase. We achieved 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose) TRY respectively. These phenotypes were maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr® and 2-L bioreactors).
A targeted SRM method was developed to quantify relative levels of pathway proteins in samples under the various tested conditions in a 60 mL cultivation format. At the time points indicated, 1 mL of each sample was pelleted by centrifugation at 14,000g and flash frozen with liquid nitrogen at − 80 °C until ready for processing. Cells were lysed in 100 mM NaHCO3 using 0.1 mm glass beads using a beadbeater with 60 s bursts at maximum power and repeated three times.
Created on 9/18/20, 2:57 PM