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\begin{document}

\title{Production of \textsubscript{D}-tagatose, bioethanol, and microbial
protein from the dairy industry by-product whey powder using an
integrated bioprocess}



\author[1]{wei Ma}%
\author[1]{Fengyi Li}%
\author[1]{Longyue Li}%
\author[2]{Bin Li}%
\author[1]{Kangle Niu}%
\author[2]{Qinghua Liu}%
\author[3]{Laichuang Han}%
\author[1]{Lijuan Han}%
\author[1]{Xu Fang}%
\affil[1]{Shandong University}%
\affil[2]{Shandong Henglu Biotechnology Co., Ltd.}%
\affil[3]{Jiangnan University}%


\vspace{-1em}



  
  \date{June 18, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
We designed and constructed a green, sustainable, and nongenetically
modified organism (non-GMO) bioprocess to efficiently coproduce
\textsubscript{D}-tagatose, bioethanol, and microbial protein from whey
powder. First, a one-pot biosynthesis process involving lactose
hydrolysis and \textsubscript{D}-galactose redox reactions for
\textsubscript{D}-tagatose production was established in vitro via a
three-enzyme cascade. Second, a nicotinamide adenine dinucleotide
phosphate-dependent galactitol dehydrogenase mutant, D36A/I37R, based on
the nicotinamide adenine dinucleotide-dependent polyol dehydrogenase
from Paracoccus denitrificans was created through rational design and
screening. Moreover, an NADPH recycling module was created in the
oxidoreductive pathway, and the tagatose yield increased by 3.35-fold
compared with that achieved through the pathway without the cofactor
cycle. The reaction process was accelerated using an enzyme assembly
with a glycine--serine linker, and the tagatose production rate was
9.28-fold higher than the initial yield. Finally, Saccharomyces
cerevisiae was introduced into the reaction solution, and 266.5 g of
\textsubscript{D}-tagatose, 371.3 g of bioethanol, and 215.4 g of dry
yeast (including 38\% protein) were obtained from 1 kg of whey powder
(including 810 g lactose). This study provides a promising non-GMO
process for functional food (\textsubscript{D}-tagatose) production.
Moreover, this process fully utilized whey powder, demonstrating good
atom economy.%
\end{abstract}%



\sloppy


Production of\textsubscript{D}-tagatose, bioethanol, and microbial
protein from the dairy industry by-product whey powder using an
integrated bioprocess

Wei Ma \textsuperscript{1}, Fengyi Li \textsuperscript{1}, Longyue Li
\textsuperscript{1} Bin Li\textsuperscript{2}, Kangle Niu
\textsuperscript{1}, Qinghua Liu\textsuperscript{2}, Laichuang Han
\textsuperscript{3}, Lijuan Han\textsuperscript{1}* and Xu Fang
\textsuperscript{1, 4} *

1. State Key Laboratory of Microbial Technology, Shandong University,
Qingdao, Shandong, 266237, China

2. Shandong Henglu Biotechnology Co., Ltd., Jinan, Shandong, 250013,
China

3. School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, 214122,
China

4. Rongcheng Huihai Chuangda Biotechnology Co., Ltd., Weihai, Shandong
264300, China

\textbf{*} Correspondence:

Xu Fang, E-mail:\emph{fangxu@sdu.edu.cn},\emph{Tel:(+86})-532-5863-1507;

Lijuan Han, E-mail:\emph{201820286@mail.sdu.edu.cn},\emph{Tel:}
17860821513.

\textbf{Keywords:} \textsubscript{D}-tagatose, oxidoreductive pathway,
whey powder, cofactor balance, NADPH cycling, computer-aided screening

\textbf{Abbreviations}

GDH, galactitol dehydrogenase; GRAS, generally recognized as safe;
GS-linker, glycine--serine linker; IPTG,
isopropyl-beta-D-thiogalactopyranoside; NAD(H), nicotinamide adenine
dinucleotide; NADP(H), nicotinamide adenine dinucleotide phosphate;
non-GMO, nongenetically modified organism; \emph{Pd} PDH, polyol
dehydrogenase from \emph{Paracoccus denitrificans} ; WP, whey powder;
XR, aldose reductase.

\textbf{Abstract:}

We designed and constructed a green, sustainable, and nongenetically
modified organism (non-GMO) bioprocess to efficiently
coproduce\textsubscript{D}-tagatose, bioethanol, and microbial protein
from whey powder. First, a one-pot biosynthesis process involving
lactose hydrolysis and \textsubscript{D}-galactose redox reactions
for\textsubscript{D}-tagatose production was established \emph{in
vitro}via a three-enzyme cascade. Second, a nicotinamide adenine
dinucleotide phosphate-dependent galactitol dehydrogenase mutant,
D36A/I37R, based on the nicotinamide adenine dinucleotide-dependent
polyol dehydrogenase from \emph{Paracoccus denitrificans} was created
through rational design and screening. Moreover, an NADPH recycling
module was created in the oxidoreductive pathway, and the tagatose yield
increased by 3.35-fold compared with that achieved through the pathway
without the cofactor cycle. The reaction process was accelerated using
an enzyme assembly with a glycine--serine linker, and the tagatose
production rate was 9.28-fold higher than the initial yield. Finally,
\emph{Saccharomyces cerevisiae}was introduced into the reaction
solution, and 266.5 g of\textsubscript{D}-tagatose, 371.3 g of
bioethanol, and 215.4 g of dry yeast (including 38\% protein) were
obtained from 1 kg of whey powder (including 810 g lactose). This study
provides a promising non-GMO process for functional food
(\textsubscript{D}-tagatose) production. Moreover, this process fully
utilized whey powder, demonstrating good atom economy.

\textbf{1. Introduction}

\textsubscript{D}-tagatose is widely used in the food and pharmaceutical
industry and is receiving increasing attention for its disease-modifying
properties in controlling diabetes and obesity, as a blood metabolism
regulator, and for its health-promoting
effects.\textsuperscript{{[}1--3{]}} \textsubscript{D}-tagatose is an
ideal substitute for sucrose owing to its high sweetness and low
calorific value (92\% as sweet and 30\% of the energy of an equivalent
amount of sucrose).\textsuperscript{{[}3{]}} It is an epimer
of\textsubscript{D}-fructose and a ketose isomer
of\textsubscript{D}-galactose that is classified as a ``rare sugar''
because of its limited occurrence in nature.\textsuperscript{{[}1{]}}

\textsubscript{D}-tagatose can be chemically synthesized using calcium
catalysts and strong acids. The process is energy-intensive and requires
complex purification steps.\textsuperscript{{[}1,4{]}}Therefore,
environmentally friendly biosynthesis technology for producing tagatose
should be developed. \textsubscript{D}-tagatose has been produced using
\textsubscript{L}-arabinose isomerase \emph{in vitro}
.\textsuperscript{{[}5--7{]}} However, the theoretical conversion rate
of the isomerase reaction is less than 0.5 mol/mol galactose at room
temperature because of the unfavorable thermodynamic equilibrium
(\selectlanguage{greek}\emph{ΔG}\selectlanguage{english} \textsuperscript{o} = 4.96 kJ mol\textsuperscript{--1} at
298.15 K).\textsuperscript{{[}8,9{]}}

Recently, Sha et al. reported that the conversion rate of tagatose from
galactitol reached 91\% through oxidation.\textsuperscript{{[}10{]}} Liu
et al. constructed an oxidoreductive reaction with aldose reductase (XR)
and galactitol dehydrogenase (GDH) in \emph{Saccharomyces cerevisiae}for
producing tagatose from lactose.\textsuperscript{{[}11{]}} The
theoretical conversion rate of the oxidoreductive pathway is higher than
that of the isomerase reaction because of the lack of a reverse reaction
in the oxidoreductive pathway for tagatose production. However, XR and
GDH exhibit coenzyme dependence on NADP\textsuperscript{+} and
NAD\textsuperscript{+}, respectively; this difference in the coenzyme
dependence of XR and GDH limited the target production yield.

Moreover, improving the economics of the production process in
industrial production is important. Many scientists are looking for
cheap materials as an alternative to galactose or galactitol. Whey, a
by-product of the dairy industry, is abundant and inexpensive. Global
production is estimated to be approximately 160 million tons per
year.\textsuperscript{{[}12{]}} Whey is an ideal substrate
for\textsubscript{D}-tagatose production because it is lactose-rich.
Simultaneously, whey contains proteins, fats, trace elements, and other
nutrients that facilitate microbial fermentation. Thus, an efficient
bioprocess should be developed to produce \textsubscript{D}-tagatose and
utilize the nutrients in whey powder (WP).

Herein, \textsubscript{D}-tagatose was synthesized using lactose from WP
through oxidoreductive pathways. Subsequently, the coenzyme dependence
of polyol dehydrogenase from \emph{Paracoccus denitrificans}(\emph{Pd}
PDH) in the oxidoreductive pathways was modified from
NAD\textsuperscript{+} to NADP\textsuperscript{+} using rational design
for improving the tagatose yield. Moreover, the reaction process was
accelerated using an enzyme assembly with a glycine--serine linker
(GS-linker). Furthermore, the efficient coproduction
of\textsubscript{D}-tagatose, bioethanol, and microbial protein from WP
was achieved. This study provides a promising application
for\textsubscript{D}-tagatose biosynthesis while improving the economics
of WP processing.

\textbf{2. Materials and methods}

\textbf{2.1 Chemicals, plasmids, and culture media}

Lactose, galactitol, NADH, NADPH, NAD\textsuperscript{+}, and
NADP\textsuperscript{+} were purchased from Coolaber Co. Ltd. (Beijing,
China). The five types of WP were obtained from Qianwei Biotech
(Shanghai, China), Miaolingziwei Biotech (Henan, China), Wanbang Biotech
(Henan, China), Gushuo Biotech (Henan, China), and Fengtai Biotech
(Shandong, China). The \selectlanguage{greek}\emph{β} -\selectlanguage{english}galactosidase with \textasciitilde{}1.2
\selectlanguage{ngerman}× 10\textsuperscript{5} U was purchased from Weilan Biotech (Shandong,
China). The KOD-Plus-Mutagenesis Kit was purchased from Toyobo Co., Ltd.
(Osaka, Japan). ABclonal MultiF Seamless Assembly was purchased from
ABclonal Technology Co., Ltd. (Wuhan, China). pET-32a was purchased from
Invitrogen (Carlsbad, CA, USA). Ampicillin and
isopropyl-beta-D-thiogalactopyranoside (IPTG) were purchased from
Gen-View Scientific Inc. (El Monte, CA, USA).\emph{Escherichia coli}
DH5\selectlanguage{greek}α \selectlanguage{english}and \emph{E. coli} BL21 (DE3) were purchased from Tsingke
Biotechnology Co., Ltd. and TransGen Biotech (Beijing, China),
respectively. Primers were synthesized by Ruibiotech Co., Ltd. (Beijing,
China). Phanta\selectlanguage{ngerman}®Max Super-Fidelity DNA Polymerase was purchased from
Vazyme Biotech Co., Ltd. (Nanjing, China). Any other chemicals used in
this study were analytical grade and obtained commercially unless
otherwise noted.

\textbf{2.2 Genetic constructs and site-directed mutagenesis}

The gene coding for \emph{Ps} XR (GenBank ID: XP\_001385181) was
amplified from the genome of \emph{Pichia stipites.} Genes coding
for\emph{Pd} PDH from \emph{Paracoccus denitrificans} (GenBank ID:
WP\_011751060), \emph{Rl} GDH from \emph{Rhizobium
leguminosarum}(GenBank ID: WP\_011650422.1), EutM from \emph{Salmonella
enterica}(GenBank ID: NP\_461400.1), and fbaB (SpyTag-Catcher)
from\emph{Streptococcus pyogenes} (GenBank ID: NZ\_LS483338.1) were
synthesized by BGI Genomics Co., Ltd. (Shenzhen, China). All the above
genes were amplified with primers harboring a homologous arm and ligated
into pET-32a to obtain the recombinant vectors pET-32a-\emph{Ps} XR,
pET-32a-\emph{Pd} PDH, pET-32a-\emph{Rl} GDH, and pET-32a-EutM. All
mutants were constructed using pET-32a-\emph{Pd} PDH as a template for
the KOD-Plus-Mutagenesis Kit. Strains and plasmids used in this study
are listed in Table S1, and primers are summarized in Table S2.

\textbf{2.3 Expression and purification}

Recombinant plasmids, including pET-32a-\emph{Ps} XR, pET-32a-\emph{Rl}
GDH, pET-32a-\emph{Pd} PDH, and mutant \emph{Pd} PDH, were transformed
into \emph{E. coli} BL21 (DE3) cells. Recombinant strains were
precultured in 20-mL LB media with 10 \selectlanguage{greek}μ\selectlanguage{english}g mL\textsuperscript{-1}
ampicillin at 37 \selectlanguage{ngerman}°C and 220 rpm for 24 h. Subsequently, a 20-mL
preculture was inoculated into 1-L LB medium (with 10 \selectlanguage{greek}μ\selectlanguage{english}g
mL\textsuperscript{-1} ampicillin) at 37 \selectlanguage{ngerman}°C and 200 rpm until the
optical density at 600 nm reached 0.6--0.8. Then, 0.1 M IPTG was added,
and incubation continued at 16 °C with shaking at 180 rpm for 20 h for
enzyme expression. Sodium dodecyl-sulfate polyacrylamide gel
electrophoresis analysis and His-tagged protein purification were
performed as previously described.\textsuperscript{{[}13{]}}

\textbf{2.4 Activity assays and kinetic parameters}

The activity was assayed by measuring the reduction of NAD(P) or the
oxidation of NAD(P)H at 340 nm (\selectlanguage{english}e = 6.22
mM\textsuperscript{-1}cm\textsuperscript{-1}) at 30 \selectlanguage{ngerman}°C using a
spectrophotometer (AMR-100 Microplate Reader, Hangzhou,
China).\textsuperscript{{[}10{]}} The standard assay mixture for
\emph{Ps} XR contained 50 mM galactose in 100 mM Tris-HCl (pH 8.0)
buffer. The standard assay mixture for\emph{Rl} GDH, \emph{Pd} PDH, and
its mutants contained 50 mM galactitol in 100 mM Tris-HCl (pH 8.0)
buffer. Reactions were started by adding 0.25 mL of a 10 mM NAD(P) or
NADPH solution to a final volume of 1.0 mL. One unit is a protein that
produces or consumes 1 \selectlanguage{greek}µ\selectlanguage{english}M of NADH/NADPH per second. The
\emph{K\textsubscript{m}} and \emph{k\textsubscript{cat}}values were
calculated using the Michaelis--Menten equation by nonlinear fitting
using GraphPad Prism 9. The results are showed in Table 1 and Table 2.

\textbf{2.5 Hydrolysis enzymatic synthesis and fermentation}

Enzyme reactions were performed based on the conditions in Table 3.
Furthermore, reaction conditions were investigated, including
temperature, pH value, buffer type, concentration, and the \emph{Ps} XR
and \emph{Pd} PDH\textsuperscript{D36A/I37R} ratio, as shown in Figure
S1. WP was hydrolyzed in citrate buffer at pH 5.5, 55 \selectlanguage{ngerman}°C with a suitable
amount of \selectlanguage{greek}\emph{β} -\selectlanguage{english}galactosidase. Enzymatic hydrolysis was performed in
a conical flask containing 122 g/L WP or 100 g/L lactose, 50 mM citrate
buffer (pH 5.5, 55 \selectlanguage{ngerman}°C), and a suitable amount of \selectlanguage{greek}\emph{β} -\selectlanguage{english}galactosidase
for 3 h. Enzymatic synthesis and fermentation were performed in a
conical flask containing previously obtained enzymatic hydrolysates, 100
mM Tris-HCl buffer (pH 8.0, 30 \selectlanguage{ngerman}°C), suitable amounts of\emph{Pd}
PDH-\emph{Ps} XR complex, cofactor NADPH, and yeast cells with a final
A\textsubscript{600} = 1. For the fermentation of enzymatic hydrolysates
derived from lactose, 2\% tryptone was required. The protein content of
the WP solution and yeast cells was determined using a BCA Protein Assay
Kit, as previously reported.\textsuperscript{{[}14{]}}

\textbf{2.6 Analytical methods}

The reaction was stopped by placing it in a boiling bath, and the
reaction solution was centrifuged at 16,200 × \emph{g} for 2 min, and
the supernatant was \selectlanguage{english}filtered with a 0.22-\selectlanguage{greek}µ\selectlanguage{english}m organic phase membrane. The
concentrations of galactose, galactitol, ethanol, and tagatose were
determined using high-performance liquid chromatography equipped with a
refractive index detector (RI detector L2490, HITACHI, Japan) using a
Shodex SP-0810 column (300 \selectlanguage{ngerman}× 8 mm, Showa Denko K.K., Tokyo, Japan). The
mobile phase was double distilled water, and the samples were separated
at 70 °C at a flow rate of 1 mL min\selectlanguage{english}\textsuperscript{-1}. The retention
times of galactose, galactitol, and tagatose were 9.91, 21.83, and 14.29
min, respectively. The formation of scaffolds was confirmed by negative
stain transmission electron microscopy, as previously
described.\textsuperscript{{[}15{]}}

\textbf{2.7 Interaction analysis and substrate docking}

The docking results and interaction analysis were shown using LigPlot
software. The docking between \emph{Pd} PDH (PDBID: 7E6O) and the
cofactors NADP\textsuperscript{+} (PubChem CID: 5886) and
NAD\textsuperscript{+} (PubChem CID: 5892) was performed using the
Schrodinger Suites 2021-4.

\textbf{3. Results}

\textbf{3.1 Designing the oxidoreductive pathways
for\textsubscript{D}-tagatose production}

We constructed the oxidoreductive pathways for producing tagatose from
lactose. The synthesis pathway was composed of three modules, as shown
in Figure 1: (1) lactose hydrolysis module (with\selectlanguage{greek}\emph{β}
-\selectlanguage{english}galactosidase); (2) galactose reduction module (with aldose reductase,
EC: 1.1.1.307); and (3) galactitol oxidation module (with galactitol
dehydrogenase, EC: 1.1.1.14). A three-enzyme cascade converting lactose
into tagatose (Pathway I) was designed using\selectlanguage{greek}\emph{β} -\selectlanguage{english}galactosidase,
NADPH-preferring \emph{Ps} XR from \emph{P. stipites} (GenBank ID:
XP\_001385181), NAD\textsuperscript{+}-dependent\emph{Pd} PDH from
\emph{P. denitrificans} (GenBank ID: WP\_011751060), or the
NAD\textsuperscript{+}-dependent \emph{Rl} GDH from \emph{R.
leguminosarum} (GenBank ID: WP\_011650422.1) (Figure 1A). The coenzyme
dependence of \emph{Ps} XR and \emph{Rl} GDH or \emph{Pd} PDH is
different. Furthermore, we designed a novel Pathway II for constructing
a cofactor cycle between the reduction and oxidation modules (Figure
1B).

\par\null

\textbf{3.2 Constructing the NADPH cycle with the modification
of\emph{Pd}PDH}

There is no NADP\textsuperscript{+}-dependent galactitol dehydrogenase
in the ENZYME database, and it is difficult to mine the
NADP\textsuperscript{+}-dependent galactitol dehydrogenase in nature.
Therefore, we must develop a rational design for
NADP\textsuperscript{+}-dependent galactitol dehydrogenase. We
mined\emph{Pd} PDH and \emph{Rl} GDH from the enzyme bank, and their
dehydrogenase activities were compared (Figure S2A). The activity
of\emph{Pd} PDH with respect to galactitol dehydrogenation was 17.29
times greater than that of \emph{Rl} GDH (25.42 and 1.47 U
mg\textsuperscript{-1}, respectively) (Figure S2B). Therefore,\emph{Pd}
PDH was selected as the template for the rational design.

We docked \emph{Pd} PDH (PDBID: 7E6O) with the cofactor
NAD\textsuperscript{+} (PubChem CID: 5892) using the Schrodinger
software, and the docking results are shown using LigPlot software
(Figure S3A). We selected 22 amino acids within 5 \selectlanguage{ngerman}Å of
NAD\textsuperscript{+} that interact with the cofactor based on hydrogen
bond or hydrophobic interaction. The \emph{Pd}
PDH-NAD\textsuperscript{+}combination model was predicted using the
CSR-SALAD website (Table S3 and Table S4). According to the predicted
results, we designed 100 mutants of \emph{Pd} PDH (Table S5) based on 13
amino acid residues (Figure S3B). Subsequently, a point mutation of
\emph{Pd} PDH was simulated using Schrodinger software, and the mutants
were docked with the cofactor NADP\textsuperscript{+}. Then, the docking
binding energies of the mutants were counted (Table S5). A total of 14
mutants were selected according to their lower binding energies,
indicating that they had a higher affinity for NADP\textsuperscript{+}
than the other mutants. Furthermore, their specific activities for
NAD\textsuperscript{+} and NADP\textsuperscript{+} were measured. As
shown in Table 1, nine mutants completely reversed the cofactor
preference and exhibited improved specific activity toward
NADP\textsuperscript{+}. Six mutants had NADP\textsuperscript{+}
activities of more than 15 U mg\selectlanguage{english}\textsuperscript{-1}; particularly, the
NADP\textsuperscript{+}activity of the D36A/I37R mutant reached 19.4 U
mg\selectlanguage{english}\textsuperscript{-1}. However, the triple mutants (A14T/D36A/I37R and
A14S/D36A/I37R) did not improve their activities further than those of
the double mutants. The single mutants (D36A, D36G, and I37R) exhibited
no activity against NADP\textsuperscript{+}. The screening accuracy was
increased sevenfold the computer-aided screening (9/14) compared with
the original screening (9/100).

The kinetic parameters of \emph{Pd} PDH mutants and the wild-type are
shown in Table 2. The\emph{K\textsubscript{m}} value of
NADP\textsuperscript{+} with the D36A/I37R mutant was 0.942 mM and was
lower than that of the wild-type enzyme (infinite), proving that there
was a substantial increase in the affinity of the D36A/I37R mutant for
NADP\textsuperscript{+}. Moreover, the specificity constant
(\emph{k\textsubscript{cat}/K\textsubscript{m}} ) of the D36A/I37R
mutant toward NADP\textsuperscript{+} reached 0.322
mM\selectlanguage{english}\textsuperscript{-1}s\selectlanguage{english}\textsuperscript{-1}, whereas that of the
wild-type was 0 mM\selectlanguage{english}\textsuperscript{-1}s\selectlanguage{english}\textsuperscript{-1}. Conversely,
the specificity constant (\emph{k\textsubscript{cat}/K\textsubscript{m}}
) of the wild-type toward NAD\textsuperscript{+} was 0.4872
mM\selectlanguage{english}\textsuperscript{-1}s\selectlanguage{english}\textsuperscript{-1}, whereas that of the
D36A/I37R mutant was 0 mM\selectlanguage{english}\textsuperscript{-1}s\selectlanguage{english}\textsuperscript{-1}.
Furthermore, we identified the binding free energy of each cofactor with
the wild-type and its mutant D36A/I37R (Table S6). The binding free
energy of the D36A/I37R mutant toward NADP\textsuperscript{+} decreased
by 54\% (from \selectlanguage{english}-8.011 to \selectlanguage{english}-12.335 kcal mol\selectlanguage{english}\textsuperscript{-1}), whereas
that toward NAD\textsuperscript{+} increased by 46\% (from \selectlanguage{english}-13.614 to
\selectlanguage{english}-7.325 kcal mol\selectlanguage{english}\textsuperscript{-1}). These results suggested that the
binding affinity of the D36A/I37R mutant toward NADP\textsuperscript{+}
was higher than that toward NAD\textsuperscript{+}. Therefore, we
obtained an NADP\textsuperscript{+}-dependent\emph{Pd}
PDH\textsuperscript{D36A/I37R} for performing the NADPH cycle during
tagatose production.

\textbf{3.3 Producing the tagatose with various conditions}

The lactose from WP was completely hydrolyzed~into galactose and glucose
with \selectlanguage{greek}\emph{β} -\selectlanguage{english}galactosidase (Table S7). The tagatose production through
Pathways I or II using \emph{Ps} XR and \emph{Pd} PDH or the
mutant\emph{Pd} PDH\textsuperscript{D36A/I37R} was performed under
various conditions. \textsubscript{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 \emph{Ps} XR; however, no tagatose was
produced from galactitol with wild-type \emph{Pd} PDH without
NAD\textsuperscript{+}. 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
between\emph{Ps} XR and \emph{Pd} PDH\textsuperscript{D36A/I37R} (Figure
3A, Pathway II--Condition 3; Figures S4A and B). Subsequently, we
designed the SpyTag003/Catcher003 connection system between \emph{Ps} XR
and\emph{Pd} PDH\textsuperscript{D36A/I37R} (Figure 3A: Pathway
II--Condition 4/5; Figures S4A and B). Finally, the EutM-\emph{Ps}
XR-\emph{Pd} PDH\textsuperscript{D36A/I37R}-system was constructed using
EutM-SpyCatcher003, SpyTag003-\emph{Ps} XR, and SpyTag003-\emph{Pd}
PDH\textsuperscript{D36A/I37R} (Figure 3A: Pathway II--Condition 6;
Figures S4C and D). The\emph{Pd} PDH\textsuperscript{D36A/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 \emph{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 \emph{Pd}
PDH\textsuperscript{D36A/I37R} and in the N-terminus with\emph{Ps} 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-\emph{Pd} PDH\textsuperscript{D36A/I37R} and
N-SpyCatcher003-\emph{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-\emph{Pd}
PDH\textsuperscript{D36A/I37R}and N-SpyCatcher003-\emph{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 \emph{Pd} PDH\textsuperscript{D36A/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.

\textbf{3.4 Coproduction of \textsubscript{D}-tagatose, bioethanol,
and~microbial protein from WP}

WP is a by-product of the dairy industry during the cheese production
process. We analyzed the chemical composition of five different
commercial sources of WP and found that the lactose content was
70\%--80\%, and the soluble protein content was 0.2\%--0.7\% (Table
S8).\selectlanguage{greek}\emph{β} -\selectlanguage{english}Galactosidase hydrolyzed lactose into glucose and
galactose. Galactose is used to synthesize tagatose, while glucose and
other nutrients can be used for yeast growth and ethanol production.
Therefore, a scheme workflow was designed to illustrate the process for
producing tagatose, bioethanol, and microbial protein from WP (Figure
4).

The WP solution contained 100-g/L lactose, which was hydrolyzed
by\selectlanguage{greek}\emph{β} -\selectlanguage{english}galactosidase to yield 52.4-g/L glucose and galactose. Then,
galactose in the enzymatic hydrolysate was converted into 32.9 g/L
tagatose based on Pathway II--condition 3 (Figure 5A, Table 4); however,
the glucose in the enzymatic hydrolysate was not consumed. Subsequently,
0.8 g \emph{S. cerevisiae} was added into the enzymatic hydrolysate, and
the glucose was entirely consumed in 12 h, whereas there was no obvious
change in \textsubscript{D}-tagatose concentration (Figure 5B). First,
the yeast cell was separated using solid/liquid separation, and 26.6 g/L
yeast cells (dry weight, including 38\% protein) was obtained. Second,
the ethanol was distilled from the broth using reduced pressure
distillation, and 45.8 g/L ethanol under anaerobic conditions was
produced. Third, tagatose was purified through concentration and
crystallization.

\textbf{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
using\textsubscript{L}-arabinose isomerase to produce tagatose from
galactose. Several studies attempted to increase the conversion rate
using immobilized enzymes,\textsuperscript{{[}16{]}} recombinant cell
expression,\textsuperscript{{[}17-20{]}} immobilized recombinant
cells,\textsuperscript{{[}8,21{]}} cell
encapsulation,\textsuperscript{{[}22{]}} or cell surface
display.\textsuperscript{{[}23,24{]}} However, the isomerase reaction
had a lower conversion rate because of the unfavorable thermodynamic
equilibria.\textsuperscript{{[}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.\textsuperscript{{[}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.\textsuperscript{{[}27{]}} Cofactor
reversal has now been realized and applied to many
enzymes.\textsuperscript{{[}29,30{]}} For example, alcohol dehydrogenase
in \emph{Clostridium autoethanogenum} was modified to NADH dependence,
resulting in improved growth and ethanol
production.\textsuperscript{{[}31{]}} Xylitol dehydrogenase was modified
to be NADPH-dependent to reduce cofactor imbalance in xylose
assimilation, and the engineered \emph{S. cerevisiae} exhibited high
xylose utilization and ethanol production
capabilities.\textsuperscript{{[}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.\textsuperscript{{[}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.\textsuperscript{{[}36,37{]}} Based on this principle,
we designed 100 mutants of \emph{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.\textsuperscript{13} We investigated the binding
mechanism between the mutants or the wild-type \emph{Pd} PDH and the
NAD\textsuperscript{+} or NADP\textsuperscript{+} 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.\textsuperscript{{[}38,39{]}} In the wild-type \emph{Pd} PDH,
the adenosine portion of NAD\textsuperscript{+} 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\textsuperscript{+} 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\textsuperscript{+} and Ala36, resulting in less interaction with
D36A/I37R than that in the wild-type \emph{Pd} PDH. For the
NADP\textsuperscript{+} cofactor, mutation of the Ile37 side chain to
Arg37 resulted in a stable and strong hydrogen interaction with the
2'-phosphate group of NADP\textsuperscript{+,} as shown in Figure S5.
Therefore, the \emph{K\textsubscript{m}} values of
NAD\textsuperscript{+} and NADP\textsuperscript{+} of \emph{Pd} PDH were
0.7 mM and infinity, respectively, whereas those of the\emph{Pd}
PDH\textsuperscript{D36A/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,\textsuperscript{{[}40,41{]}}SpyTag003/SpyCatcher003,\textsuperscript{{[}42{]}}
and self-assembling scaffold-EutM.\textsuperscript{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 \emph{Ps} XR
and\emph{Pd} PDH\textsuperscript{D36A/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.\textsuperscript{{[}12{]}} However, whey has
lactose and abundant nitrogen sources. Thus, whey is a suitable material
for microbial fermentation. Engineered \emph{E. coli} strain was used to
produce tagatose from cheese whey.\textsuperscript{{[}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
producing\textsubscript{D}-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.\textsuperscript{{[}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 \emph{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.\textsuperscript{{[}43{]}}Microbial protein, or single-cell
protein, has been regarded as an important protein reservoir for future
nutritional needs.\textsuperscript{{[}44{]}} Microbial proteins have
been used as a feed or feed supplement.\textsuperscript{{[}45{]}}
Microbial proteins have good environmental benefits as an alternative to
ruminant meat.\textsuperscript{{[}46{]}} \emph{S. cerevisiae} is
considered a generally recognized as safe (GRAS) strain and is therefore
a good candidate for microbial protein.\textsuperscript{{[}14,47,48{]}}

\textbf{5. Conclusion}

Herein, we successfully created the
NADP\textsuperscript{+}-dependent\emph{Pd}
PDH\textsuperscript{D36A/I37R} and constructed the NADPH recycling
module. By employing the constructed GS-linker system \emph{in vitro} to
assemble the oxidoreductases, the \textsubscript{D}-tagatose yield from
lactose (0.378 g/g) was 9.28-fold higher than the initial yield.
Furthermore, whey, a by-product in cheese production, was used as the
sole material for producing \textsubscript{D}-tagatose, bioethanol, and
microbial protein with an integrated bioprocess combined with enzyme
cascade and anaerobic fermentation. Finally, 266.5
g\textsubscript{D}-tagatose, 371.3 g bioethanol, and 215.5 g dry yeast
(including 38\% protein) were obtained from 1 kg WP (including 810 g
lactose). The economic feasibility was improved owing to the expensive
price of \textsubscript{D}-tagatose, compared with those of bioethanol
and microbial protein. Moreover, the risk of food safety was avoided
owing to non-GMO technology. This bioprocess in which the dairy waste
was entirely utilized helps reduce the net emission of carbon dioxide
and drives sustainable development.

Acknowledgements

The authors would like to thank Dr. Sen Wang from State Key Laboratory
of Microbial Technology of Shandong University for help and guidance in
transmission electron microscopy. This work was supported by National
Key R\&D Program of China (No. 2018YFA0901700), National Natural Science
Foundation of China (No. 32271526).

\textbf{Declaration of Competing Interest}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{Author Contributions}

All authors contributed to the background research and writing of the
article, as well as the editing. In addition, all authors have read and
approved the final version of this manuscript.

\par\null

\textbf{Research Data Policy}

The original/source data and resources are available from the
correspondence author Xu Fang (fangxu@sdu.edu.cn) on request.

\textbf{Chemical compounds studied in this article}

NAD\textsuperscript{+} (PubChem CID: 5892);
NADP\textsuperscript{+}(PubChem CID: 5886); \textsubscript{D}-tagatose
(PubChem CID:439312);\textsubscript{D}-Galactose (PubChem CID:6036);
Galactitol (PubChem CID:11850).

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\textbf{Tables}

\textbf{Table 1.} Specific activity of\emph{Pd}
PDH\emph{\textsuperscript{a}} and mutants toward galactitol using either
NAD\textsuperscript{+} or NADP\textsuperscript{+}.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Enzyme & Specific activity (U mg\textsuperscript{-1})
\emph{\textsuperscript{b}} & Specific activity (U
mg\textsuperscript{-1}) \emph{\textsuperscript{b}}\tabularnewline
\midrule
\endhead
& NAD\textsuperscript{+} & NADP\textsuperscript{+}\tabularnewline
WT & 25.42 \selectlanguage{ngerman}± 1.37 & n.a.\emph{\textsuperscript{c}}\tabularnewline
D36A & 20.31 \selectlanguage{ngerman}± 0.85 & n.a.\tabularnewline
D36G & 24.46 \selectlanguage{ngerman}± 0.65 & n.a.\tabularnewline
I37R & 22.03 \selectlanguage{ngerman}± 0.86 & n.a.\tabularnewline
D36A/I37R & n.a. & 19.40 \selectlanguage{ngerman}± 0.99\tabularnewline
D36G/I37R & n.a. & 18.23 \selectlanguage{ngerman}± 0.95\tabularnewline
D36R/I37R & n.a. & 11.36 \selectlanguage{ngerman}± 0.30\tabularnewline
D36H/I37R & n.a. & 18.02 \selectlanguage{ngerman}± 1.30\tabularnewline
D36K/I37R & n.a. & 17.58 \selectlanguage{ngerman}± 0.52\tabularnewline
D36I/I37R & n.a. & 6.62 \selectlanguage{ngerman}± 1.26\tabularnewline
D36F/I37R & n.a. & 12.36 \selectlanguage{ngerman}± 0.26\tabularnewline
A14T & 13.55 \selectlanguage{ngerman}± 0.75 & n.a.\tabularnewline
A14S & 13.58 \selectlanguage{ngerman}± 0.74 & n.a.\tabularnewline
A14T/D36A/I37R & n.a. & 18.02 \selectlanguage{ngerman}± 0.34\tabularnewline
A14S/D36A/I37R & n.a. & 17.65 \selectlanguage{ngerman}± 0.07\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textsuperscript{\emph{a} )} \emph{Pd} PDH, polyol dehydrogenase
from\emph{Paracoccus denitrificans} ; \textsuperscript{\emph{b} )} One
unit is defined as the amount of protein that produces or consumes 1 \selectlanguage{greek}µ\selectlanguage{english}M
of NAD(H)/NADP(H) per second; \textsuperscript{\emph{c} )} n.a. = no
measurable activity.

\textbf{Table 2.} Kinetic parameters of \emph{Pd} PDH mutants determined
with NAD\textsuperscript{+} or NADP\textsuperscript{+} using galactitol
as substrate in excess.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Enzyme\emph{\textsuperscript{a}} & \emph{K\textsubscript{m}} (mM) &
\emph{K\textsubscript{m}} (mM) & \emph{k\textsubscript{cat}}
(s\textsuperscript{-1}) & \emph{k\textsubscript{cat}}
(s\textsuperscript{-1}) & \emph{k\textsubscript{cat}/K\textsubscript{m}}
(mM\textsuperscript{-1} s\textsuperscript{-1}) &
\emph{k\textsubscript{cat}/K\textsubscript{m}} (mM\textsuperscript{-1}
s\textsuperscript{-1}) & Vmax (\selectlanguage{greek}μ\selectlanguage{english}mol min\textsuperscript{-1}
mg\textsuperscript{-1}) & Vmax (\selectlanguage{greek}μ\selectlanguage{english}mol min\textsuperscript{-1}
mg\textsuperscript{-1})\tabularnewline
\midrule
\endhead
& NAD\textsuperscript{+} & NADP\textsuperscript{+} &
NAD\textsuperscript{+} & NADP\textsuperscript{+} &
NAD\textsuperscript{+} & NADP\textsuperscript{+} &
NAD\textsuperscript{+} & NADP\textsuperscript{+}\tabularnewline
WT & 0.7723 & i.g.\emph{\textsuperscript{b}} & 0.3763 &
n.a.\emph{\textsuperscript{c}} & 0.4872 & n.a. & 1.028 &
n.a.\tabularnewline
D36A/I37R & i.g. & 0.942 & n.a. & 0.3040 & n.a. & 0.3224 & n.a. &
0.7845\tabularnewline
D36G/I37R & i.g. & 1.299 & n.a. & 0.2831 & n.a. & 0.2179 & n.a. &
0.7372\tabularnewline
D36H/I37R & i.g. & 1.305 & n.a. & 0.2673 & n.a. & 0.2048 & n.a. &
0.7287\tabularnewline
D36K/I37R & i.g. & 1.355 & n.a. & 0.1103 & n.a. & 0.0814 & n.a. &
0.7077\tabularnewline
D36I/I37R & i.g. & 1.801 & n.a. & 0.1411 & n.a. & 0.0783 & n.a. &
0.2677\tabularnewline
D36F/I37R & i.g. & 1.378 & n.a. & 0.0776 & n.a. & 0.0563 & n.a. &
0.4982\tabularnewline
D36R/I37R & i.g. & 1.603 & n.a. & 0.0673 & n.a. & 0.0420 & n.a. &
0.4590\tabularnewline
A14T/D36A/I37R & i.g. & 1.259 & n.a. & 0.1084 & n.a. & 0.0861 & n.a. &
0.7287\tabularnewline
A14S/D36A/I37R & i.g. & 1.308 & n.a. & 0.1071 & n.a. & 0.0819 & n.a. &
0.7137\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{\emph{a} )} All enzymes were purified before
characterization. For determining kinetic parameters, the initial rate
of the enzyme was measured in 100 mM Tris-HCl pH 8.0 with
NAD\textsuperscript{+} or NADP\textsuperscript{+} (0.2 mM) in the
presence of saturating concentrations of substrate galactitol (50 mM) at
30 \selectlanguage{ngerman}°C; \textsuperscript{\emph{b} )} i.g. = infinitely
great;\textsuperscript{\emph{c} )} n.a. = no measurable activity.

\textbf{Table 3.} Information on the pathway conditions.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway I\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway I\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway I\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway II\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway II\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway II\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway II\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway II\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Pathway II\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Condition 6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Ps}XR (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\emph{\textsuperscript{a}}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Pd}PDH (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Pd}PDH\textsuperscript{D36A/I37R} (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
36\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\emph{Ps}XR-Linker-\emph{Pd}PDH\textsuperscript{D36A/I37R} (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
N\emph{\textsuperscript{b}}-SpyTag003-\emph{Pd}PDH\textsuperscript{D36A/I37R}
(mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
C-SpyTag003-\emph{Pd}PDH\textsuperscript{D36A/I37R} (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
N-SpyCatcher003-\emph{Ps}XR (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
N-SpyTag003-\emph{Ps}XR (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
18\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
EutM-C-SpyCatcher003 (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
36\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
lactose (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NADPH (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NAD\textsuperscript{+} (mM)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{\emph{a} )} Dash indicates no
addition;\textsuperscript{\emph{b} )} N or C means that SpyTag003 or
SpyCatcher003 fused to an N-terminus or C-terminus of \emph{Ps} XR
or\emph{Pd} PDH\textsuperscript{D36A/I37R}.

\textbf{Table 4.} \textsubscript{D}-tagatose production with lactose and
whey powder.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Materials & \textsubscript{D}-tagatose concentration (g/L) &
\textsubscript{D}-tagatose yield (g/g lactose) &
\textsubscript{D}-tagatose yield (g/g whey powder) & Conversion rate
(mol/mol lactose)\tabularnewline
\midrule
\endhead
Lactose \emph{\textsuperscript{a}} & 35.7 & 0.357 & - &
0.678\tabularnewline
Whey powder \emph{\textsuperscript{b}} & 32.9 & 0.329 & 0.269 &
0.625\tabularnewline
\bottomrule
\end{longtable}

\emph{\textsuperscript{a}} 100g/L lactose in the
solution.\emph{\textsuperscript{b}} 100g/L lactose in cheese whey
powder.



\textbf{Figures and legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Schematic diagram of the production of tagatose from
lactose via the oxidoreductive pathways. A: Pathway I without cofactor
cycle; B: Pathway II with NADPH cycle.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Validation of \textsubscript{D}-tagatose synthetic
pathways. All reactions were performed with 20 mM lactose, 100 mM
Tris-HCl (pH 8.0) at 30 \selectlanguage{ngerman}°C. The information on pathway conditions is
shown in Table 3. The yields of tagatose and galactitol in (A) Pathway
I--Condition 1, (B) Pathway I--Condition 2, and (C) Pathway
II--Condition 1 are shown. Experiments were performed in triplicates,
and error bars represent the standard deviation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} \textsubscript{D}-tagatose yield with different
enzyme cascade strategies.

(A) Schematic diagram of different enzyme cascade strategies.
(B)\textsubscript{D}-tagatose yields at 24, 48, 72, and 96 h of reaction
in 100 mM Tris-HCl (pH 8.0) at 30 \selectlanguage{ngerman}°C. Markers correspond to those in
Figure 3A. The information on pathway conditions is shown in Table 3.
Experiments were performed in triplicates, and error bars represent the
standard deviation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} . A scheme of the overall process for coproduction
of\textsubscript{D}-tagatose, bioethanol, and microbial protein from
whey powder.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5} . Time course of \textsubscript{D}-tagatose production
from 100 g/L lactose and 122 g/L cheese whey powder (including 100 g/L
lactose) (A). Fermentation of mixed sugar syrup derived from lactose and
cheese whey powder by \emph{S. cerevisiae} (B). Solid dots represent
lactose as substrate, and hollow dots represent whey powder as
substrate. Experiments were performed in triplicates, and error bars
represent the standard deviation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Synopsis:}

One-pot integrated bioprocess for the coproduction
of\textsubscript{D}-tagatose, bioethanol, and microbial protein from
whey powder.

\selectlanguage{english}
\FloatBarrier
\end{document}

