posted on 2021-11-10, 19:34authored byMay N. Taw, Mingji Li, Daniel Kim, Mark A. Rocco, Dujduan Waraho-Zhmayev, Matthew P. DeLisa
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.