1 Cascade design and in vitro construction of the PAPS
biosynthesis pathway
To efficiently produce PAPS, a sequential enzymatic cascade composed of
a main module and an auxiliary module was designed (Figure 1A ).
In the main module, ATP and sulfate were first converted to APS and PPi
by ATPS. Thereafter, APS and ATP were converted to PAPS and ADP by APSK.
In the auxiliary module, a pyrophosphatase (PPA) was introduced to
hydrolyze PPi to Pi, and a polyphosphate kinase (PPK) was introduced to
regenerate ATP from ADP; in this way, the inhibitory effect of the
by-products (PPi and ADP) was eliminated. Using the BRENDA database,
four different microorganisms were selected as the sources of each
enzyme: ATPS from Kluyveromyces lactis (Kl ATPS), APSK fromPenicillium chrysogenum (Pc APSK), PPA from E. coli(Ec PPA), and PPK from Rhodobacter sphaeroides(Rs PPK). Following purification and in vitro evaluation,
their optimal specific activity was determined (Table 1 andTable S3 ).
To confirm the effectiveness of the main module in vitro ,Kl ATPS and Pc APSK were mixed at a 1:1 molar ratio in the
presence of 20 mM ATP. After 10 h, formation of 4.56 mM PAPS as the
final product was confirmed by nuclear magnetic resonance and mass
spectrometry analyses (Figure S1 ), demonstrating the efficacy
of using Kl ATPS and Pc APSK for converting ATP to PAPS. The
effect of varying the Kl ATPS:Pc APSK ratio on PAPS titer
was investigated with Kl ATPS activity fixed at 3.0
U·mL-1. The highest PAPS titer (6.06 mM, conversion
rate 59.6%) was achieved when Pc APSK activity was 4.5
U·mL-1 (Figure 1B ). This result indicated an
optimal in vitro Kl ATPS:Pc APSK ratio of 1:1.5.
Next, the effect of adding Ec PPA at 2.5, 3.0, 3.5, 4.0, 4.5, and
5.0 U·mL-1 on PAPS titer was examined (Figure
1C ). Addition of 4.0 U·mL-1 Ec PPA to the main
module led to a PAPS titer of 7.18 mM from 20 mM ATP, with conversion
rate of 81.8%. Furthermore, when
7.0
U·mL-1 Rs PPK was added to the in vitroconversion broth, the PAPS titer increased to 8.05 mM, with a conversion
rate of 92.5% (Figure 1D ). Taken together, the optimalKl ATPS:Pc APSK:Ec PPA:Rs PPK ratio was
approximately 3:4.5:4:7.