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  • Engineering a Supersecreting Strain of Escherichia coli by Directed Coevolution of the Multiprotein Tat Translocation Machinery.

Engineering a Supersecreting Strain of Escherichia coli by Directed Coevolution of the Multiprotein Tat Translocation Machinery.

ACS synthetic biology (2021-11-11)
May N Taw, Mingji Li, Daniel Kim, Mark A Rocco, Dujduan Waraho-Zhmayev, Matthew P DeLisa
RESUMEN

Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed coevolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three supersecreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than those observed for wild-type TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of more efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

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Sigma-Aldrich
Anti-GroEL antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Anti-Alkaline Phosphatase Antibody, E. coli, bacterial only, ascites fluid, Chemicon®