3.3 Producing the tagatose with various conditions
The lactose from WP was completely hydrolyzed into galactose and glucose
with β -galactosidase (Table S7). The tagatose production through
Pathways I or II using Ps XR and Pd PDH or the mutantPd PDHD36A/I37R was performed under various
conditions. D-tagatose was not produced under Pathway
I–Condition 1 (Figure 2A and Table 3), although 3.7 mM galactitol was
produced after 48 h. We speculated that galactose was converted into
galactitol by Ps XR; however, no tagatose was produced from
galactitol with wild-type Pd PDH without NAD+.
Under Pathway I–Condition 2 and Pathway II–Condition 1, 1.46 mM and
6.30 mM tagatose was produced after 96 h (Figures 2B–C), with a
conversion rate of 0.07 and 0.31 mol/mol lactose, respectively, though
the galactitol yields were similar. The tagatose yield of Pathway
II–Condition 1 was 4.35-fold higher than that of Pathway I–Condition
2. This result proved that tagatose production was considerably improved
with the cofactor cycle module.
The enzyme assembly strategies were applied to improve the tagatose
yield. First, a fused protein was prepared with the GS-linker betweenPs XR and Pd PDHD36A/I37R (Figure 3A,
Pathway II–Condition 3; Figures S4A and B). Subsequently, we designed
the SpyTag003/Catcher003 connection system between Ps XR andPd PDHD36A/I37R (Figure 3A: Pathway
II–Condition 4/5; Figures S4A and B). Finally, the
EutM-Ps XR-Pd PDHD36A/I37R-system was
constructed using EutM-SpyCatcher003, SpyTag003-Ps XR, and
SpyTag003-Pd PDHD36A/I37R (Figure 3A: Pathway
II–Condition 6; Figures S4C and D). ThePd PDHD36A/I37R activities did not considerably
change after ligation of SpyTag003 or SpyCatcher003 in the N-terminus or
C-terminus. However, ligation into the C-terminus (Figure S4E) impaired
the activity of Ps XR, whereas the ligation in the N-terminus did
not change its activity (Figure S4F). Therefore, we selected the
ligation of SpyTag003 or SpyCatcher003 in the N-terminus or C-terminus
with Pd PDHD36A/I37R and in the N-terminus withPs XR.
The tagatose yield with the GS-linker strategy (Pathway II–Condition 3)
was the highest among these cascade enzyme reactions (Figure 3B) and was
1.79-fold higher compared with that of the free enzyme system (Pathway
II–Condition 2) at 24 h. The tagatose yield with the
N-SpyTag003-Pd PDHD36A/I37R and
N-SpyCatcher003-Ps XR systems (Pathway II–Condition 4) increased
by 19% at 24 h compared with that of the free enzyme system. The
activities of the C-SpyTag003-Pd PDHD36A/I37Rand N-SpyCatcher003-Ps XR systems (Pathway II–Condition 5) did
not considerably change compared with that of the free enzyme system,
and the activity of the EutM-scaffold system (Pathway II–Condition 6)
was not higher than that of the free enzyme system.
Finally, the production of tagatose was improved using a fed-batch
culture strategy comprising Pd PDHD36A/I37R per
12 h based on Pathway II–Condition 3. This produced 14.5 mM tagatose
after 96 h with a conversion rate of 0.72 mol/mol lactose, an increase
of 9.28-fold compared with Pathway I–Condition 2.