4. Discussion
The conversion between aldose and ketose forms of sugars can be
catalyzed by isomerase in one step or by oxidoreductase in two steps.
Until 2018, many studies had focused on the isomerase reaction usingL-arabinose isomerase to produce tagatose from
galactose. Several studies attempted to increase the conversion rate
using immobilized enzymes,[16] recombinant cell
expression,[17-20] immobilized recombinant
cells,[8,21] cell
encapsulation,[22] or cell surface
display.[23,24] However, the isomerase reaction
had a lower conversion rate because of the unfavorable thermodynamic
equilibria.[8,9] Therefore, an oxidoreductive
pathway was selected for tagatose synthesis in this study.
Oxidoreductases are usually cofactor-dependent, and the imbalance of
cofactors in the cascade reaction may limit the conversion of the
substrate. Alleviating the redox imbalance has been reported to be
beneficial for improving the yield of the target production using
cofactor engineering strategies.[25-28] Cahn et
al. developed a general tool named CSR-SALAD to help reverse the
cofactor preference of oxidoreductases due to the large demand for
balancing cofactor availability.[27] Cofactor
reversal has now been realized and applied to many
enzymes.[29,30] For example, alcohol dehydrogenase
in Clostridium autoethanogenum was modified to NADH dependence,
resulting in improved growth and ethanol
production.[31] Xylitol dehydrogenase was modified
to be NADPH-dependent to reduce cofactor imbalance in xylose
assimilation, and the engineered S. cerevisiae exhibited high
xylose utilization and ethanol production
capabilities.[13,32-33] To alleviate the cofactor
imbalance in the biosynthesis of branched-chain amino acids, the
coenzyme preference of ketol-acid reductoisomerase was altered from
NADPH to NADH.[34,35] However, cofactor
engineering in the redox pathway of tagatose synthesis has not yet been
reported. Therefore, we designed and demonstrated the oxidoreductive
Pathway II and the conversion rate was greatly improved (Figure 3).
Basic amino acid residues in the
cofactor binding region are believed to favor the binding of NADPH,
whereas acidic amino acids facilitate the affinity of
NADH.[36,37] Based on this principle, we designed
100 mutants of Pd PDH (Table S5). Complete reversal of the
coenzyme specificity was achieved with those containing
D36A(G/R/H/K/I/F)/I37R (Table 2), which agrees with the results of a
previous study.13 We investigated the binding
mechanism between the mutants or the wild-type Pd PDH and the
NAD+ or NADP+ cofactors.
Interactions between protein and ligand, such as strong hydrogen
interactions, are essential for stable binding, and steric hindrance and
electrostatic repulsion may hinder their
affinity.[38,39] In the wild-type Pd PDH,
the adenosine portion of NAD+ could be stabilized by
strong hydrogen interactions between the 2′-and 3′-hydroxyl groups and
the negatively charged amino acid Asp36, as shown in Figure S5A. In the
D36A/I37R mutant, the 2′-and 3′-hydroxyl groups of
NAD+ were pushed apart by the amino group of Arg37 due
to spatial site resistance and electrostatic repulsion, and strong
hydrogen interaction was lacking between the 2′- and 3′-hydroxyl groups
of NAD+ and Ala36, resulting in less interaction with
D36A/I37R than that in the wild-type Pd PDH. For the
NADP+ cofactor, mutation of the Ile37 side chain to
Arg37 resulted in a stable and strong hydrogen interaction with the
2′-phosphate group of NADP+, as shown in Figure S5.
Therefore, the Km values of
NAD+ and NADP+ of Pd PDH were
0.7 mM and infinity, respectively, whereas those of thePd PDHD36A/I37R were infinity and 0.942 mM,
respectively.
Enzyme assembly could improve the efficiency of the reaction. Various
strategies for constructing artificial multienzyme complexes have been
widely reported and successfully applied, including fusion protein
technology with a linker,[40,41]SpyTag003/SpyCatcher003,[42] and self-assembling
scaffold-EutM.43 We constructed the GS-linker (Figure
S4B), SpyTag003/SpyCatcher003 (Figures S4A and B), and EutM-scaffold
systems (Figures S4C and D) and found that the GS-linker system played
the best-facilitating role. We speculated that this linkage maintained a
suitable distance between Ps XR andPd PDHD36A/I37R, allowing a faster reaction
transfer. The SpyTag003/Catcher003 linkage system may form a longer
distance compared with that of the GS-linker system because the
SpyTag003 and SpyCatcher003 were all fused to the N-terminus or
C-terminus of the protein with the same GS-linker (Figure 3A).
Therefore, the tagatose yield only increased by 19% at 24 h compared
with that of the free enzyme system. With the EutM-scaffold system, we
speculated that this linkage may not have brought the enzymes spatially
closer together as the activity was not considerably improved compared
with that of the free enzyme system.
The reuse of industrial waste is important for saving energy and
reducing emissions, while the conversion of waste into high-value
products is an important method for establishing a sustainable
bioeconomy. Whey, a by-product of the dairy industry, is an
environmental pollutant.[12] However, whey has
lactose and abundant nitrogen sources. Thus, whey is a suitable material
for microbial fermentation. Engineered E. coli strain was used to
produce tagatose from cheese whey.[18,28] However,
nongenetically modified organism (non-GMO) tagatose production
technology is being explored to obtain an increased tagatose yield from
lactose. Herein, we demonstrated an enzyme cascade pathway for producingD-tagatose from 1 kg of WP, including 81% lactose, and
the tagatose yield from lactose and WP reached 0.32 and 0.27 g/g,
respectively. In this reaction, we found that galactose was entirely
consumed; by contrast, there was no change in glucose concentration
(Figure 5). These conversion efficiencies are similar to those of
microbial cell factories.[11,28] However, this
study constructed a more robust non-GMO technology, which avoids
consumer anxiety about genetically modified technology. Furthermore,
yeast fermentation in the enzyme reaction solution was performed without
tagatose separation. We compared the ethanol production with and without
adding nitrogen source after tagatose production, and no significant
change in ethanol yields was observed (Figure 5). This suggested the
presence of an abundant nitrogen source and trace elements in whey for
yeast growth and ethanol production under anaerobic conditions.
Moreover, no change in tagatose concentration in the broth was observed,
suggesting that natural S. cerevisiae cannot utilize tagatose.
Finally, we obtained 371.3 g of ethanol and 215.4 g of dry yeast cells
from 1 kg of WP, and the protein content in the dry yeast was
approximately 38% (w/w) (Table S9).
Bioethanol
is an important fuel for mitigating
global
warming and conserving fossil fuels.[43]Microbial protein, or single-cell protein, has been regarded as an
important protein reservoir for future nutritional
needs.[44] Microbial proteins have been used as a
feed or feed supplement.[45] Microbial proteins
have good environmental benefits as an alternative to ruminant
meat.[46] S. cerevisiae is considered a
generally recognized as safe
(GRAS) strain and is therefore a good candidate for microbial
protein.[14,47,48]