PT  - JOURNAL ARTICLE
AU  - Deepanwita Banerjee
AU  - Thomas Eng
AU  - Andrew K. Lau
AU  - Brenda Wang
AU  - Yusuke Sasaki
AU  - Robin A. Herbert
AU  - Yan Chen
AU  - Yuzhong Liu
AU  - Jan-Philip Prahl
AU  - Vasanth R. Singan
AU  - Deepti Tanjore
AU  - Christopher J. Petzold
AU  - Jay D. Keasling
AU  - Aindrila Mukhopadhyay
TI  - Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale
AID  - 10.1101/2020.02.21.954792
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.02.21.954792
4099  - http://biorxiv.org/content/early/2020/02/24/2020.02.21.954792.short
4100  - http://biorxiv.org/content/early/2020/02/24/2020.02.21.954792.full
AB  - Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the inherent trade-off between cellular use of carbon towards growth vs. target metabolite production. We hypothesized that being able to strongly couple product formation with growth may lead to improvements across both metrics. In this study, we use elementary mode analysis to predict metabolic reactions that could be targeted to couple the production of indigoidine, a sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting engineered P. putida strain could achieve high TRY. The engineered pairing of product formation with carbon use also shifted production from stationary to exponential phase and the high TRY phenotype was maintained across scale. In one design cycle, we constructed an engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in exponential phase. These desirable phenotypes were maintained from batch to fed-batch cultivation mode, and from 100ml shake flasks to 250 mL ambr® and 2 L bioreactors.