Feb 24, 2020

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Deepanwita BanerjeeAindrila Mukhopadhyay


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 o...Continue Reading

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Mentioned in this Paper

Gene Knockdown Techniques
Fed-Batch Culture Techniques
Test Method
Tissue Engineering
Genetic Engineering
Pseudomonas putida

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