Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in EngineeredE. coli

Abstract

We report improved strain and bioprocess robustness as a result of the dynamic deregulation of central metabolism using two-stage dynamic control. Dynamic control is implemented using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes in the context of a standardized two-stage bioprocesses. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network. The deregulated network is more robust to environmental conditions improving process robustness, which in turn leads to predictable scalability from high throughput small scale screens to fully instrumented bioreactors as well as to pilot scale production. Additionally, as these two-stage bioprocesses are standardized, a need for traditional process optimization is minimized. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology. In this work we demonstrate that the improved robustness of E. coli strains engineered for the improved scalability of the important industrial chemicals alanine, citramalate and xylitol, from microtiter plates to pilot reactors.

The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

“…Importantly, this methodology enables predictability across scales from microtiter plates to instrumented reactors. 22,24 This enables reliable scale up of a strain or strain variant identified using the microfermentation protocol.…”

“…This is not to say that the autoinduction protocol cannot be leveraged in metabolic engineering programs, but we have recently demonstrated that for small molecules, performance of the 2-stage wash protocol in microtiter plates is predictive of performance in larger minimal media bioreactors, again a least for a subset of our engineered E. coli . 21,22…”

“…We have recently reported the development of a methodology enabling 2-stage production of both proteins and small molecules. [21][22][23] This approach leverages engineered strains of E. coli and standardized (product independent) methods for 2-stage production in instrumented bioreactors as well as microtiter plates. Importantly, this methodology enables predictability across scales from microtiter plates to instrumented reactors.…”

Decoupling growth and production by removing the origin of replication from a bacterial chromosome

“…Lynch and colleagues combined phosphate limitation with a metabolic valve approach to limit cell growth and adjust its metabolism respectively. This led to the increased production of both small molecules 12 and proteins 13 .…”

Construction of a Convenient Screening Method for P-Hydroxybenzoate Hydroxylase To Enable Efficient Gallic Acid and Pyrogallol Biosynthesis

“…In recent years, microbial-based biosynthesis has been attempted to produce many value-added compounds from simple carbon sources [22][23][24][25][26][27], including GA and pyrogallol [28,29]. GA and pyrogallol biosynthetic pathways generally extended from 4-hydroxybenzoic acid (4-HBA), which was an Escherichia coli (E. coli) endogenous compound generated from the shikimate pathway [28].…”