2 In vivo construction of the PAPS biosynthesis pathway
To prove the effectiveness of the proposed catalytic pathway in
vivo , the genes encoding Kl ATPS and Pc APSK in the main
enzyme module and Ec PPA and Rs PPK in the auxiliary module
were inserted into pACYCDuet-1 and pETDuet-1 plasmids, respectively.
These were then used to transform E. coli BL21 (DE3), resulting
in E. coli strain 01. Co-expression of the four enzymes was
confirmed by protein gel electrophoresis of cell-free lysates. Then, the
conversion performance of strain E. coli 01 was investigated with
30 g·L-1 wet cell (treated with
1g·L-1 Tween 80) at 30°C, it was found that the PAPS
titer increased from 6.03 to 11.59 mM as the substrate ATP concentration
was increased from 20 to 60 mM (Figure 2A ). When the ATP
concentration was increased >60 mM, the PAPS titer did not
augment any further. In contrast, the conversion rate of PAPS decreased
from 60.13% to 25.62%, when the ATP concentration increased from 20 to
80 mM. This drop was due to APS, an intermediate of the enzymatic
cascade, which accumulated at increasing amounts (from 3.86 to 16.07 mM)
in the conversion broth (Figure 2A ). Intracellular enzymatic
activity of Kl ATPS, Pc APSK, Ec PPA, and Rs PPK
in E. coli 01 was 135.06, 12.75, 188.19, and 243.31
U·g-1 wet cells, respectively (the ratio was
3:0.28:4.18:5.41) (Figure 2B ). This finding revealed the need
to increase the intracellular enzymatic activity of Rs PPK andPc APSK.
Previous studies had shown that Rs PPK could easily form inclusion
bodies in cells. Therefore, four molecular chaperones (SUMO, MBP, TrxA,
and GST) involved in expression of soluble proteins were co-expressed
with Rs PPK to increase its activity in the cytoplasm. TrxA alone
caused the intracellular activity of Rs PPK to increase from
243.31 (E. coli 01) to 317.38 U·mg-1 wet cells
(E. coli 04) (Figure 2C ), while Kl ATPS,Pc APSK, and Ec PPA reached 132.61, 12.85, and 186.63
U·g-1 wet cells, respectively (corresponding to a
3:0.29:4.22:7.18 ratio) (Figure 2A ). To improve Pc APSK
intracellular activity, its expression was increased by adding six
ribosome-binding site (RBS) sequences (RBS1–RBS6) of different
intensities (Table S4 ). RBS5 produced the best results and
increased Pc APSK activity from 10.75 (E. coli 01) to 22.47
U·g-1 wet cells (E. coli 06) (Figure
2D ). However, increasing the expression of Pc APSK decreased the
transformation efficiency of the system (Figure 2D ). Moreover,
the intracellular activity of the other three enzymes to drop
significantly to 95.07 (Kl ATPS), 87.35 (Ec PPA), and 194.05
(Rs PPK) U·g-1 wet cells (Figure 2A ).
These results suggested that increasing enzyme expression alone had
limited efficacy on improving overall performance and might lead to an
imbalance in intracellular resource allocation. To overcome this
limitation, it is necessary to improve the inherent properties ofPc APSK through protein engineering.