4 One-pot production of PAPS at a 3-L scale
To demonstrate the in vivo applicability of the proposed
strategy, the L7 variant was introduced in a whole‐cell catalytic
system, resulting in strainE. coli 11. Finally, the
intracellular activity of Kl ATPS, Pc APSK, Ec PPA andRs PPK could be controlled to 166.32, 253.08, 225.91, and 403.26
U·g-1 wet cells, respectively (Figure 5A ).
Optimal operational conditions were determined as pH 8.0 and 35 °C. As
shown in Figure S2 , increasing substrate concentration from 100
to 150 mM achieved a conversion rate >97%, but a further
increase to 200 mM caused the conversion rate to drop to 80.3%.
Therefore, 150 mM substrate was considered optimal for conversion. The
strainsE. coli 04 (L0) and 11 (L7) were each grown in a 3‐L bioreactor
under optimal conditions, with air flow of 4.5 L·min-1and shaking speed of 210 r·min-1 (Figure 5B ).Escherichia coli 11 produced 73.59 mM (37.32
g·L-1) PAPS after incubation for 18.5 h,
corresponding to a 98.1% conversion rate and space‐time yield of 1.8
g·L-1·h-1. In comparison, E.
coli 04 could produce only 37.94 mM (19.25 g·L-1)
PAPS after 20.5 h, corresponding to a 50.6% conversion efficiency and a
space‐time yield of 0.94 g·L-1·h-1.
In addition, almost no APS could be detected in the reaction solution ofE. coli 11, whereas 22.35 mM APS was found in the reaction
solution of E. coli 04. These results showed that protein
engineering of Pc APSK in the dual-enzyme catalytic cascade had
successfully eliminated the rate-limiting step in PAPS production.