Discussion
In this study, a new dual-enzyme catalytic system for in vivosynthesis of PAPS was developed that comprised a main enzyme module for
the conversion of ATP into PAPS and an auxiliary module for the removal
of by-product inhibition. Pc APSK was identified as the
rate-limiting enzyme in this cascade due to the delayed release of ADP.
To overcome this limitation and improve catalytic efficiency, a rational
“ADP expulsion” strategy was applied. Accordingly, the ADP-binding
affinity was weakened, and the binding channel was expanded to promote
the release of ADP, resulting in a 45.74-fold higher activity in the
APSK L7 variant. The latter was introduced in an E. colicatalytic system, whereby it successfully converted ATP to PAPS. This
study describes not only a feasible method for the industrial production
of PAPS but also a valuable strategy for engineering similar enzymes.
A synthetic and controllable catalytic system was designed for the
synthesis of PAPS. The proposed catalytic system eliminates pathway
inhibition from by-products by converting these by-products into
substrates for reuse. During the conversion of ATP to PAPS, two
by-products, PPi and ADP, inhibit the performance of ATPS and APSK (Bao
et al., 2015). To eliminate such by-product inhibition, PPA and Nudix
hydrolase have been used as PPi and ADP hydrolases, respectively (Hong
et al., 2014; W. Xu, Dunn, O, Handley, Smith, & Bessman, 2006).
Previously, a complex ATP conversion system was constructed using
3-bromopyruvic acid as a cheap substrate for PEP-K+,
which then acted as a phosphate donor for ADP (An et al., 2017). In this
study, an auxiliary module comprising PPA and PPK was designed. PPA
hydrolyzed PPi to phosphate, and PPK phosphorylated ADP to ATP through a
one-step reaction that employed low-cost short-chain polyphosphate
PolyP(6) as phosphate donor. In contrast to other
methods for producing PAPs via the one-pot process, the present
catalytic system is composed of two independent modules, whose strength
can be adjusted by controlling their intracellular expression to balance
the production process.
An effective protein engineering strategy was developed to improvePc APSK activity. To date, protein crystallization (Ian J. MacRae,
2000; Poyraz et al., 2015), site directed mutagenesis Wang D. Z et al.,
2016), and truncated mutagenesis (Ravilious et al., 2013; Sekulic,
Konrad, et al., 2007) have been used to characterize APSK. However,
these studies focused mainly on the structure and catalytic mechanism of
APSK, and no effective strategies for improving its catalytic
performance have been proposed. Here, a mechanism-guided “ADP
expulsion” strategy was developed by combining information about
conformational dynamics and crystal structure of the protein (Ian J.
MacRae, 2000; Lansdon et al., 2002). The strategy included three steps:
(i) computer- and protein structure-assisted binding energy calculation
and release channel identification; (ii) identification of six key
mutation hotspots affecting binding energy and release channels; and
(iii) construction of two mutation libraries aimed at weakening the
binding energy through looser inner interactions and an expanded release
channel. As a result, the specific activity and kcat /Km of
the optimal L7 variant were 46.39-fold and 73.27-fold higher than those
in wild-type Pc APSK, respectively. This approach is in line with
a recent trend in protein engineering that focuses on construction of
small and smart libraries while reducing the size of the mutation
library and improving evolution efficiency (Li, Qu, Sun, & Reetz, 2019;
Sun, Lonsdale, Ilie, Li, & Reetz, 2016). Compared to traditional
mutagenesis, this method is more rational as it relies on tunnel
identification (Song et al., 2020; Yuan et al., 2019), conformational
dynamics (Yang et al., 2017), specific hotspot scanning (Xu , Cen,
Singh, Fan, & Wu, 2019; J. Xu et al., 2019), and saturation
mutagenesis. Overall, this protein engineering strategy could greatly
improve the performance of enzymes with a release channel or lid
structure.
The study provides a simple and efficient method for in vivo ATP
conversion to PAPS. Efficient PAPS production has relied on the use of
either PAP or ATP as the substrate (Kang et al., 2018). In the first
case, PAP and PNPS are converted to PAPS by ASST (T. Wang, Liu, &
Voglmeir, 2020); however, this method does not allow for large-scale
PAPS production because of the high cost of PAP and the coupling with
sulfotransferases (Xiong et al., 2013). ATP is a much cheaper substrate
and, therefore, has been applied in the present study together with
ATPS, APSK, PPA, and pyruvate kinase II as catalysts. The phospho-donor
PEP-K+ was generated by organic synthesis. Using 0.05
mg·mL-1 of each purified enzyme and
PEP-K+, 5 g·L-1 of PAPS was
generated in 6 h with a productivity of 0.83
g·L-1·h-1 (An et al., 2017). In this
study, the main module and auxiliary module were assembled and
transformed into an E. coli strain, the rate-limiting enzyme and
catalytic conditions were optimized, and a stable whole-cell PAPS
catalytic synthesis system was constructed. Finally, 73.59 mM (37.32
g·L-1) PAPS was produced in 18.5 h with a conversion
rate of 98.1% and productivity of 1.75
g·L-1·h-1 at a 3-L scale. Therefore,
the biocatalytic process used in this study provides an attractive
strategy for the transformation of ATP into high‐value PAPS at a
fraction (1/5000) of the cost of commercial sources and thus may
remarkably facilitate the industrial production of PAPS.