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\DOI{10.5802/crchim.376}
\datereceived{2024-11-14}
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\dateposted{2025-04-07}
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\CDRsetmeta{articletype}{review}

\title{Extending the toolbox for enzymatic carboligation: synthesis of
\tralicstex{Î±}{$\upalpha$}-hydroxyketones catalyzed by thermostable transketolase from
\textit{Geobacillus stearothermophilus}}

\alttitle{Extension des m\'{e}thodes de carboligation enzymatique :
synth\`{e}se d'\tralicstex{Î±}{$\upalpha$}-hydroxyc\'{e}tones catalys\'{e}e par la
transc\'{e}tolase thermostable  de \textit{Geobacillus
stearothermophilus}}

\author{\firstname{Franck} \lastname{Charmantray}\CDRorcid{0000-0001-8638-8349}}
\address{Universit\'{e} Clermont Auvergne, CNRS, Clermont Auvergne INP, 
Institut de Chimie de Clermont-Ferrand (ICCF), F-63000 Clermont-Ferrand, France}

\author{\firstname{Laurence} \lastname{Hecquet}\CDRorcid{0000-0003-2971-5686}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Clermont Auvergne, CNRS, Clermont Auvergne INP, 
Institut de Chimie de Clermont-Ferrand (ICCF), F-63000 Clermont-Ferrand, France}
\email[L. Hecquet]{laurence.hecquet@uca.fr}

\keywords{\kwd{Biocatalysis}
\kwd{Thiamine-dependent enzyme}
\kwd{C--C bond formation}
\kwd{Transketolase}
\kwd{Mutagenesis}
\kwd{\tralicstex{Î±}{$\upalpha$}-hydroxyketones}}

\altkeywords{\kwd{Biocatalyse}
\kwd{Enzyme d\'{e}pendante de la thiamine diphosphate}
\kwd{Formation de liaison C--C}
\kwd{Transc\'{e}tolase}
\kwd{Mutagen\`{e}se}
\kwd{\tralicstex{Î±}{$\upalpha$}-hydroxyc\'{e}tones}}

\thanks{Fonds R\'{e}gional Innovation Laboratoire (grant
DOS00494484/00), Pack Ambition Recherche ID 1701105201-61617, Agence
Nationale de la Recherche (grants ANR-09-BLAN-0424-CSD3 and
ANR-22-CE07-0038-01), ERA CoBioTech TRALAMINOL---ID: 64, and
MSCA-ITN-ETN-2020 CC-TOP---ID: 956931}

\begin{abstract}
Enzymatic 
carbon--carbon (C--C) bond formation represents an efficient
asymmetric alternative for the preparation of multifunctional products.
This paper presents an overview of the advances made by
engineering a thermostable thiamine-dependent carboligase,
transketolase from \textit{Geobacillus stearothermophilus}
(TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$}), for the synthesis of various
\tralicstex{Î±}{$\upalpha$}-hydroxyketones (aliphatic, hydroxylated, and aromatic). While
TKs in cells exclusively transfer a ketol unit from a ketose phosphate
to an aldose phosphate Cn leading to a Cn\tralicstex{+}{${+}$}2 ketose phosphate yielding
a reversible reaction, the results reported in this paper showed the
wide range of non-phosphorylated substrates accepted by the selected
TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$} variants, particularly towards \tralicstex{Î±}{$\upalpha$}-ketoacids
used as nucleophiles, render the reaction irreversible due to the
release of carbon dioxide. To enhance TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$} activity
towards the targeted nucleophiles, analogues of pyruvate, and 
electrophiles such as aliphatic, polyhydroxylated, and aromatic
aldehydes, the best variants were selected from libraries created by
rational design. As the main hurdle for biocatalytic application is the
instability/cost of  \tralicstex{Î±}{$\upalpha$}-ketoacids, one-pot strategies were
performed for in situ generation of \tralicstex{Î±}{$\upalpha$}-ketoacids from
corresponding amino acids with transaminase or amino acid oxidase. A
novel promising promiscuous TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$} reaction based on 
selective cross-acyloin condensation of two aldehydes, one playing the
role of the nucleophile in place of the \tralicstex{Î±}{$\upalpha$}-ketoacid and the other
aldehyde acting as an electrophile, was also investigated. This original
TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$}
 catalyzed reaction provides atom economy while
avoiding carbon dioxide release and achieving similar efficiency compared
to the usual pathway. 
\end{abstract}

\begin{altabstract}
La formation de liaison carbone--carbone par voie enzymatique
repr\'{e}sente une alternative efficace pour la synth\`{e}se
\'{e}nantios\'{e}lective de compos\'{e}s multifonctionnels. Cet article
donne un aper\c{c}u des avanc\'{e}es obtenues par ing\'{e}nierie d'une
carboligase thermostable d\'{e}pendante de la thiamine, la
transc\'{e}tolase de \textit{Geobacillus stearothermophilus}
(TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$}
), pour la synth\`{e}se
d'\tralicstex{Î±}{$\upalpha$}-hydroxyc\'{e}tones chirales (aliphatiques,
hydroxyl\'{e}es, aromatiques). Alors que dans les cellules, les TKs
transf\`{e}rent une unit\'{e} c\'{e}tol d'un c\'{e}tose phosphate
donneur sur un aldose phosphate (Cn) accepteur conduisant \`{a} un
c\'{e}tose phosphate (Cn\tralicstex{+}{${+}$}2) selon une r\'{e}action r\'{e}versible,
les r\'{e}sultats rapport\'{e}s ici montrent l'extension du spectre de
substrats en particulier vers des \tralicstex{Î±}{$\upalpha$}-c\'{e}toacides
utilis\'{e}s comme nucl\'{e}ophiles, rendant la r\'{e}action
irr\'{e}versible en raison de la lib\'{e}ration de dioxyde de carbone.
Pour am\'{e}liorer l'activit\'{e} de la TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$}
vis-\`{a}-vis des substrats nucl\'{e}ophiles, analogues du pyruvate et
les \'{e}lectrophiles tels que des ald\'{e}hydes aliphatiques,
polyhydroxyl\'{e}s ou aromatiques, des enzymes mut\'{e}es efficaces ont
\'{e}t\'{e} s\'{e}lectionn\'{e}es dans des banques d'enzymes mut\'{e}es
con\c{c}ues de fa\c{c}on rationnelle. Le principal obstacle pour des
applications synth\'{e}tiques \'{e}tant l'instabilit\'{e}/le co\^{u}t
des \tralicstex{Î±}{$\upalpha$}-c\'{e}toacides, des strat\'{e}gies one-pot ont
\'{e}t\'{e} d\'{e}velopp\'{e}es pour les g\'{e}n\'{e}rer in
situ \`{a} partir d'acides amin\'{e}s avec une transaminase ou une
aminoacide oxydase. Une autre voie prometteuse catalys\'{e}e par la
TK\tralicstex{\textsubscript{gst}}{$_{\mathrm{gst}}$} a \'{e}t\'{e} envisag\'{e}e. Elle est bas\'{e}e sur
une r\'{e}action promiscuitaire de type condensation acyloine
crois\'{e}e entre deux ald\'{e}hydes, l'un jouant le r\^{o}le de
nucl\'{e}ophile \`{a} la place de l'\tralicstex{Î±}{$\upalpha$}-c\'{e}tocide, l'autre
ald\'{e}hyde agissant comme \'{e}lectrophile. Cette strat\'{e}gie est
aussi efficace que la voie habituelle et \'{e}vite la lib\'{e}ration de
dioxyde de carbone conduisant \`{a} une \'{e}conomie d'atomes.
\end{altabstract}

\maketitle

\twocolumngrid

\end{noXML}

\let\rmmu\upmu

\section{Introduction}\label{sec1}
The creation of carbon--carbon (C--C) bonds with tight control of
chemo-, regio-, and 
\mbox{stereoselectivity} is the key reaction to construct
the carbon framework of organic molecules~\cite{1a,1b}. Consequently,
sophisticated methods for asymmetric synthesis as well as
protection/deprotection strategies have to be considered together with
eco-friendly conditions. In this context, enzymatic biocatalytic C--C
coupling represents an efficient (asymmetric) 
\mbox{alternative} for the
preparation of multifunctional products~\cite{2a,2b,2c}. In the last few decades,
several carboligases have been reported, each of them leading to a
specific functional group or motif such as aldolases,
Pictet--Spenglerases, oxidases, prenyltransferases, squalene/hopene
cyclases, engineered hemoproteins, and thiamine diphosphate
(ThDP) dependent enzymes. The last group is recognized as a powerful
tool for the preparation by C--C bond formation of enantiopure
$\rmalpha$-hydroxyketones~\cite{3a,3b,3c,3d}, a structural motif present in a wide
range of highly valuable compounds such as pharmaceuticals~\cite{4a,4b,4c,4d,4e,4f},
flavors~\cite{5}, sweetners~\cite{6a,6b}, surfactants~\cite{7}, and
important synthetic intermediates for the preparation of diols or
aminoalcohols. 

ThDP-dependent enzymes display the formation of a C--C bond between two
carbonylated compounds (aldehyde or ketone) according to a common
mechanism involving activated ThDP \textbf{I}  (Figure~\ref{fig1})
to produce a highly reactive intermediate (\textbf{III)} via polarity
reversal (umpolung reaction) characterized by two mesomeric forms
(carbanion--enamine). The carbanion subsequently attacks the carbonyl
group of a more electrophile (acceptor) substrate, leading to the
formation of a C--C bond (\textbf{IV}) and to the release of the
product  (Figure~\ref{fig2}). The stereocontrol of the new asymmetric
carbon depends on the type of ThDP enzyme and the structure of 
substrates.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Activation of thiamine diphosphate.}
\end{figure*}

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Catalytic cycle of ThDP-dependent carboligases.}
\end{figure*}

Among ThDP enzymes, transketolase (TK, EC 2.2.1.1) has been largely
studied and used for the synthesis of various enantiopure
$\rmalpha$-hydroxyketones considering its large scope of substrates.
TKs are ubiquitous enzymes found in the cytoplasm of animal, plant, and
microbial cells {and} in vivo catalyze the reversible
transfer of a two-carbon ketol unit from \textsc{d}-xylulose
5-phosphate to \textsc{d}-ribose 5-phosphate or
\textsc{d}-erythrose-4-phosphate to
generate \textsc{d}-sedoheptulose-7-phosphate or
\textsc{d}-fructose-6-phos\-phate 
and \textsc{d}-glyceraldehyde-3-phosphate 
{(Figure~\ref{fig3}).} \looseness=-1

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}In vivo transketolase reaction.}
\end{figure*}

With TK providing a link between the glycolysis and pentose phosphate
pathway, this enzyme plays a key role in metabolic regulation  in all
living  cells~\cite{8}. Particularly in humans, TK has been reported to
be involved in neurodegenerative diseases Wernicke--Korsakoff syndrome
and Alzheimer's disease, and  diabetes and  cancers~\cite{9a,9b,9c}.

The first in vitro  biocatalytic applications of this enzyme
were described by Whitesides et~al.\ in 1992~\cite{10}.  The
reaction becomes more generally useful for synthetic purposes by using
hydroxypyruvic acid (HPA) as a C2-ketol group donor, rendering the
reaction almost irreversible due to the concomitant release of carbon
dioxide as a by-product  (Figure~\ref{fig4}). The lithium salt of HPA
is preferred as it is the commercially available cheaper form  of this
compound, and its synthesis is also  described~\cite{11}.

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Irreversible reaction catalyzed by TKs in the presence
of HPA.}
\end{figure*}

Wild-type transketolases from spinach, \textit{S.~cerevisiae}, and
\textit{E.~coli} were first used for the synthesis of
$\rmalpha$-hydroxyketones mostly polyhydroxylated such as natural and
unnatural ketoses and analogues obtained from Li-HPA as donors and
preferentially $(R)$-hydroxylated aldehydes as  acceptors~\cite{12a,12b,12c,12d,12e,12f,12g}. To
extend the substrate spectra, the engineering of TKs by mutagenesis was
developed together with appropriate screening assays providing
efficient variants towards new $\rmalpha$-ketoacids as donors (analogues
of pyruvate) and aldehydes as acceptors (polyhydroxylated or not,
arylated), leading to enantiopure $\rmalpha$-hydroxyketones~\cite{13a,13b,13c}.

Another criterion to make biocatalytic applications more competitive is
related to the thermostability of enzymes. Indeed, from the industrial
point of view, enzymatic processes at elevated temperature offer many
advantages such as higher reaction rate, improved solubility of
organic substrates, and higher tolerance towards non-conventional media.
In addition, for improvement by mutagenesis, thermostable enzymes also
display better resistance against protein destabilizing 
factors~\cite{14}. For these reasons, this paper presents an overview
of the advances obtained with a thermostable transketolase from
\textit{Geobacillus stearothermophilus} (TK$_{\mathrm{gst}}$) for the
synthesis of various $\rmalpha$-hydroxyketones in the context of other
thermostable TKs having been isolated and used in biocatalysis but at  
a faster rate~\cite{15a,15b}.

\section{Results and discussion}\label{sec2}

\subsection{Production of TK$_{\mathrm{gst}}$}\label{sec2.1} 
We have chosen \textit{Geobacillus stearothermophilus} (formerly
\textit{Bacillus stearothermophilus}) as a potential source of a
thermally stable TK  enzyme~\cite{16}. This Gram-positive thermophilic
bacterium is widely distributed in soil, hot springs, and ocean
sediments, and grows in the temperature range of 30--758~\textdegree C. Many
heat-stable enzymes, such as  xylanase~\cite{17}, \mbox{\textsc{l}-arabinose}
isomerase~\cite{18},  glycosidases~\cite{19}, and
$\rmalpha$-amylases~\cite{20}, have been isolated from this
thermophilic bacterium. Before our discovery in 2013 in collaboration
with  de Berardinis et~al.~\cite{16}, no complete genome sequence
was available in public databases for any \textit{G.\ 
stearothermophilus} strain and no transketolase enzyme had hitherto
been described. TK$_{\mathrm{gst}}$ was expressed with a His6-tag at
the N-terminus in \textit{E.~coli} BL21(DE3) and was 
{purified} using
immobilized metal affinity \mbox{chromatography.} The 
\mbox{expressed} fusion proteins were
applied to a Ni$^{2+}$ chelating affinity column for purification. By
this procedure, 160~mg each of the purified enzymes TK$_{\mathrm{gst}}$
$(0.6~\mathrm{U}{\cdot}\mathrm{mg}^{-1})$ were effectively obtained
from the cells grown in 1~L culture 
($5~\mathrm{g}{\cdot}\mathrm{L}^{-1}$ of wet cells). We note that the
enzyme can be lyophilized, producing a powder easy to use for chemists
and enabling preservation over several months without loss of activity.
TK$_{\mathrm{gst}}$ also showed higher tolerance over time compared to
 other mesophilic TKs towards high-percentage cosolvents (up to
50\%) such as DMF and ionic liquids such as BMIMCl, which is a useful property
when substrates are not water  soluble~\cite{21}.

\subsection{Thermostability of TK$_{\mathrm{gst}}$}\label{sec2.2} 
As is characteristic of a microorganism, TK$_{\mathrm{gst}}$
has an optimum temperature range of  60--70~\textdegree C, leading to
an improvement of up to 10-fold when compared to the activity measured
at  20~\textdegree C. TK$_{\mathrm{gst}}$ retained 100\% activity for
1 week at  50~\textdegree C and 3 days at  70~\textdegree C, and its
activity decreased rapidly at  75~\textdegree C (half-life was about 
15~min)~\cite{16}. The thermostability of TK$_{\mathrm{gst}}$ was much
higher than that of other characterized TKs from microbial sources
commonly used in biocatalysis such as TK$_{\mathrm{sce}}$ having an
half-life of 35~min at  50~\textdegree C with immediate activity
loss at  70~\textdegree C or TK$_{\mathrm{eco}}$ more resistant (100\%
activity recovery at  50~\textdegree C for 90~min) but completely
vanishing during 5~min exposure\unskip\break 
at 70~\textdegree C. 

Given the TK$_{\mathrm{gst}}$ thermostability, an alternative
purification procedure by heat shock treatment at  65~\textdegree C for
45~min enabled an easy and rapid purification method, yielding 132~mg of
purified enzyme from the cells grown in 1~L of  culture~\cite{16}.

\subsection{Immobilization of transketolase from \textit{Geobacillus
stearothermophilus}}\label{sec2.3}
From an industrial point of view, the reusability of an enzyme is also a
crucial point. For that reason, we investigated different supports for
TK$_{\mathrm{gst}}$ immobilization. First in collaboration with
Forano et~al., we showed that layered double hydroxides  gave
efficient adsorption/entrapment of TK$_{\mathrm{gst}}$ with almost
total immobilization, recovery of initial activity, and reusability over
six cycles without loss of  activity~\cite{21,22}. The strategy is cheap,
rapid, and eco-friendly. More recently in collaboration with
Szymanska et~al., TK$_{\mathrm{gst}}$ was covalently
immobilized on silica monolithic pellets. This strategy allowed
performing the reaction in a basket-type  reactor~\cite{23}.

{\vspace*{-2pt}}

\subsection{Substrate scope of wild-type
TK$_{\mathrm{gst}}$}\label{sec2.4}

{\vspace*{-2pt}}

\subsubsection{Substrate scope of wild-type TK$_{\mathrm{gst}}$ towards
donor and acceptor substrates}\label{sec2.4.1}

{\vspace*{-2pt}}

As indicated before, the major advance for biocatalytic applications of
TKs including TK$_{\mathrm{gst}}$ was the use of HPA, rendering the
reaction almost irreversible due to the release of carbon dioxide 
(Figure~\ref{fig5}). We showed later that pyruvate can be accepted as a
donor substrate by TK$_{\mathrm{gst}}$ but with lower activity
compared to HPA.

\begin{figure*}
\includegraphics{fig05}
\vspace*{-2pt}
\caption{\label{fig5}Irreversible reaction catalyzed by
TK$_{\mathrm{gst}}$ in the presence of $\rmalpha$-ketoacid.}
\vspace*{-4pt}
\end{figure*}

As expected, knowing the high homology of the TK active sites, we
observed that the TK$_{\mathrm{gst}}$ acceptor substrate spectrum in
the presence of HPA as the  donor was very similar to those of 
TK$_{\mathrm{sce}}$ and TK$_{\mathrm{eco}}$ already described 
(Figure~\ref{fig6}). 

\begin{figure*}
\includegraphics{fig06}
\vspace*{-2pt}
\caption{\label{fig6}Specific activities of TK$_{\mathrm{gst}}$ towards
a panel of acceptor aldehydes (20--200~mM) were determined in the
presence of Li-HPA at 25~\textdegree C.}
\vspace*{-4pt}
\end{figure*}

TK$_{\mathrm{gst}}$ showed high activity towards short-chain 
$(2R)$\ $\rmalpha$-hydroxylated aldehydes (C2--C4). A significant
decrease in TK$_{\mathrm{gst}}$ activity was observed with aldehydes
displaying a long carbon chain length (C5--C6) or without a hydroxyl
group at the $\rmalpha$ or $\rmbeta$ position or hydrophobic and
aromatic aldehydes.

In contrast with other described microbial TKs, TK$_{\mathrm{gst}}$
showed significant activities towards $(2S)$-hydroxylated aldehydes
with three and four carbon  atoms~\cite{24} such as \textsc{l}-glyceraldehyde,
\textsc{l}-lactaldehyde, \textsc{l}-erythrose, and \textsc{d}-threose.
This is particularly interesting because these aldehydes can lead to the
production of rare, high-value ketoses. However, the
TK$_{\mathrm{gst}}$ activities were much lower than those determined
with their $(2R)$ epimers (approximately 20 and 30 times lower in the
case of lactaldehyde and glyceraldehyde, respectively).
TK$_{\mathrm{gst}}$ being thermostable, studies conducted at 
60~\textdegree C showed an improvement in activities by a factor 4 to 5
towards C3 (\textsc{l}-glyceraldehyde and \textsc{l}-lactaldehyde) and C4
aldoses (\textsc{l}-erythrose and \textsc{d}-threose) compared to
results obtained at  25~\textdegree C  (Figure~\ref{fig7}). These
analytical results were significant because the activities measured at
high temperatures were comparable to those obtained in the presence of
their $(2R)$ analogues at  25~\textdegree C.\looseness=-1

\begin{figure}
\includegraphics{fig07}
\caption{\label{fig7}Specific activity of TK$_{\mathrm{gst}}$ with
($2S$)-hydroxylated aldehydes and C3 (\textsc{l}-glyceraldehyde,
\textsc{l}-lactaldehyde) and C4 aldoses (\textsc{l}-erythrose,
\textsc{d}-threose) in the presence of Li-HPA at\ 
\protect\xinlinefig{fx01}~25~\textdegree C and\  
\protect\xinlinefig{fx02}~60~\textdegree C.}
\end{figure}

Considering all these results, the improvement and broadening of the
TK$_{\mathrm{gst}}$ substrate scope 
\mbox{particularly} towards other
$\rmalpha$-ketoacids as donors and long carbon chain aldoses and
hydrophobic and aromatic aldehydes as acceptors was investigated by
mutagenesis strategies.

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig08}
\caption{\label{fig8}Comparison of ThDP binding motifs of
TK$_{\mathrm{sce}}$ (green 1TRK.pdb), TK$_{\mathrm{eco}}$ (cyan
2R8O.pdb), and TK$_{\mathrm{ban}}$ (violet 3HYL.pdb).}
\end{figure*}

\subsection{Improvement and modification of TK$_{\mathrm{gst}}$ 
properties}\label{sec2.5}

\subsubsection{Strategies for generating TK$_{\mathrm{gst}}$ variant
libraries}\label{sec2.5.1} 
The strategies were based on the analysis of the active site to
determine the key positions that should be mutated to improve
TK$_{\mathrm{gst}}$ activity towards the targeted substrates. The 3D
structure of TK$_{\mathrm{gst}}$ with its cofactors was recently
determined showing as for the other TKs a dimeric enzyme with each monomer
containing an identical active  site~\cite{25}. However, as
this structure was not yet available for the studies presented here, a model of active
TK$_{\mathrm{gst}}$~\cite{26} was constructed by homology with
TK from \textit{Bacillus anthracis} (TK$_{\mathrm{ban}}$) using a template
already crystallized (PDB entry 3M49) belonging to the same
\mbox{microorganism} species and having 74\% identity. This model was 
validated by comparison with the 3D structure of TK$_{\mathrm{gst}}$.

It can be noted a high homology with the active sites of the other TK
sources used in biocatalysis such as TK$_{\mathrm{eco}}$ and 
TK$_{\mathrm{yeast}}$ and also with the active sites of all other
microbial TKs. The superimposition of TK$_{\mathrm{eco}}$, 
TK$_{\mathrm{sce}}$, and  TK$_{\mathrm{ban}}$ active sites shows the
same residues with a similar position to ThDP and divalent cation
Mg$^{2+}$  (Figure~\ref{fig8}). 

After the identification of the targeted residues, each targeted amino
acid of the wild-type sequence was replaced by the other 19  amino
acids by site saturation mutagenesis  (SSM)~\cite{27}. Different types
of degenerate codons can be used such as NNS and NDT. In NNS, ``N'' stands
for any nucleotide (A, T, G, or C) and ``S'' stands for G or C. NNS can
encode 32 possible codons $(4 \times 4 \times 2 = 32)$ including 20 codons for
all amino acids plus 12 additional codons that encode some amino acids
multiple times or encode stop codons. Alternative codons 
``NDT''~\cite{28} were designed to completely avoid stop codons while
encoding several representative amino acids in each category: anionic,
cationic, aliphatic, hydrophobic, hydrophilic, and aromatic. These ``NDT''
codons provide functional diversity without redundancy while reducing
the number of enzyme variants compared to NNS and consequently the
screening efforts. When more than two positions were investigated, NDT
strategy was preferred in the studies presented here to reduce the size
of the libraries.

\subsubsection{Screening assays}\label{sec2.5.2}
The principle of the generic assay developed in liquid or solid phase
was based on the TK-catalyzed decarboxylation of 
$\rmalpha$-ketoacid and the release of carbon dioxide, inducing the
variation in pH of the reaction medium monitored by a pH indicator. Indeed,
during the reaction catalyzed by TK in the presence of 
$\rmalpha$-ketoacid as the donor, a proton is consumed in each cycle, which
releases an equivalent of  $\mathrm{HCO}_3^{-}$ bicarbonate ions,
leading to a pH increase in the reaction medium  (Figure~\ref{fig9}).

\begin{figure*}
\includegraphics{fig09}
\vspace*{-2pt}
\caption{\label{fig9}Catalytic cycle of TK in the presence of Li-HPA.}
\vspace*{-2pt}
\end{figure*}

The bicarbonate ion is the dissociated form of carbonic acid 
(H$_{2}$CO$_{3}$) and is involved in a dynamic equilibrium of
dissociation in water. This leads to a basification of the solution
due to the partial 
\mbox{formation} of hydroxide ion raising the pH of the medium,
which is approximately 7.5. Based on this pH variation in reaction
medium, we first developed in collaboration with Fessner et~al.\ a
colorimetric screening assay in the liquid phase with purified 
variants~\cite{29}. More recently, the same principle was applied
to a semi-solid phase, allowing the direct detection of clones expressing
active TK$_{\mathrm{gst}}$  variants~\cite{30}.

For the qualitative liquid screening  assay~\cite{29}, upon release of
carbon dioxide from  $\rmalpha$-ketoacid, the pH increase in the
reaction mixture can be determined photometrically by the color change
of phenol red. This pH indicator was selected for its 
$\mathrm{p}K_{\mathrm{a}}$ of 
7.4 and its turning zone (6.8 ${&lt;}$ pH ${&lt;}$ 8.2) being compatible with
the optimal pH of the reaction catalyzed by TK, which is approximately
7.5~\cite{50}. Phenol red changes from bright yellow at acidic pH to
bright pink at basic pH, allowing easy visualization of the progress of
the reaction  (Figure~\ref{fig10}). In addition, phenol red in its
deprotonated form has a high molar absorption coefficient:
$\rmvarepsilon= \text{56,000}~\mathrm{M}^{-1}{\cdot}\mathrm{cm}^{-1}$
at $\rmlambda_{\mathrm{max}} = 557$~nm, detected by spectrophotometry with high
sensitivity. At low buffer concentration (2~mM of
triethanolamine, pH 7.5), this generic, cheap, rapid method allowed
continuous monitoring for quantitative determination of  kinetic 
parameters~\cite{29}.

\begin{figure*}
\includegraphics{fig10}
\caption{\label{fig10}TK pH-metric activity test based on the use of
phenol red as the colored indicator.}
\end{figure*}

The qualitative solid phase assay procedure~\cite{30} was based on the
strategies already developed with other enzymes by 
Turner et~al.~\cite{31} and Bornscheuer et~al.~\cite{32}, allowing one to detect
the color 
\mbox{variation} directly in clones expressing the targeted variant.
In our case, the pH indicator used to detect the 
\mbox{variation} in the
reaction medium was bromothymol blue offering higher contrast than
phenol red described previously in the liquid phase. The libraries of
clones expressing TK$_{\mathrm{gst}}$ variants were first cultured on
Petri dishes and then transferred on a nitrocellulose membrane, which
was placed on a semi-solid medium containing the substrates, cofactors,
and a pH indicator bromothymol blue. The colonies became blue if TK
variants were active towards the substrates  
\mbox{(Figure~\ref{fig11}).} 

\begin{figure*}
\includegraphics{fig11}
\caption{\label{fig11}pH-metric activity test based on semi-solid
medium containing bromothymol blue as the  colored indicator.}
\end{figure*}

\begin{figure*}
\includegraphics{fig12}
\caption{\label{fig12}Model of wild-type TK$_{\mathrm{gst}}$ based on
the X-ray crystal structure of TK$_{\mathrm{ban}}$ (PDB entry 3M49)
with the physiological acceptor substrate
\textsc{d}-erythrose-4-phosphate (E4P).} 
\end{figure*}

\subsubsection{Extension of TK$_{\mathrm{gst}}$ substrate
spectra}\label{sec2.5.3} 

\paragraph*{Enhancement of TK$_{\mathrm{gst}}$ activity towards (2$S$ or
2$R$)  $\rmalpha$-hydroxyaldehydes (C3--C6) as acceptors with Li-HPA as
donor}\label{sec2.5.3.1} 
Due to their scarcity in nature and complex synthesis, natural and
unnatural ketoses particularly with long carbon chains have not been
widely assayed for their potential properties on different biological
targets, but recent studies have  started considering these forgotten
compounds. For these reasons, the improvement of TK$_{\mathrm{gst}}$
towards the appropriate substrates for obtaining such ketoses and
analogues with different types of stereochemistry is of great interest.

Our strategy was based on the analysis of the wild-type
TK$_{\mathrm{gst}}$ active site with the physiological acceptor
substrate \textsc{d}-erythrose-4-phosphate  (Figure~\ref{fig12}). We
applied SSM to key positions in direct interaction 
of this substrate depending on the structure of the novel
targeted aldehyde as the substrate.

Some examples of ketoses obtained with specific variants selected from
TK$_{\mathrm{gst}}$ libraries are presented in  Table~\ref{tab1}. 

\begin{table*}
\caption{\label{tab1}Synthesis of ketoses (C5--C8) from aldoses (C3--C6)
in the presence of the most efficient
TK$_{\mathrm{gst}}$ variants}
\tabcolsep=2.5pt
\begin{tabular}{ccccccc}
\thead
\parbox[t]{2cm}{\centering C$_{n}$ aldose acceptors} &amp; 
\parbox[t]{2cm}{\centering C$_{n+2}$ ketose product} &amp; 
\parbox[t]{1.5cm}{\centering TK$_{\mathrm{gst}}$ variant} &amp; 
\parbox[t]{2cm}{\centering Reaction progress (\%)} &amp; 
\parbox[t]{1.5cm}{\centering Reaction time} &amp;
\parbox[t]{1.5cm}{\centering Isolated yield (\%)} &amp; 
\parbox[t]{1cm}{\centering de (\%)}
\vspace*{2pt}\\
\endthead 
\raisebox{4em}{}
\xinlinefig{fx03} &amp; \xinlinefig{fx04} &amp;
\morerows{1}{L382D/D470S} &amp;
\morerows{1}{98} &amp;
\morerows{1}{24~h} &amp;
\morerows{1}{63} &amp;
\morerows{1}{${&gt;}$95} \\
\parbox[t]{3cm}{\centering \textsc{l}-glyceraldehyde\unskip\break C3} &amp;
\parbox[t]{3cm}{\centering \textsc{l}-ribulose\unskip\break C5} &amp; &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

\xinlinefig{fx05} &amp; \xinlinefig{fx06} &amp;
\morerows{1}{L382D/D470S} &amp;
\morerows{1}{98} &amp;
\morerows{1}{24~h} &amp;
\morerows{1}{55} &amp;
\morerows{1}{${&gt;}$95} \\
\parbox[t]{3cm}{\centering \textsc{l}-lactaldehyde\unskip\break C3} &amp;
\parbox[t]{3cm}{\centering 5-deoxy-\textsc{l}-ribulose\unskip\break C5} &amp;  &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

\xinlinefig{fx07} &amp; \xinlinefig{fx08} &amp;
\morerows{1}{L382F/F435Y} &amp;
\morerows{1}{${&gt;}$95} &amp;
\morerows{1}{48~h} &amp;
\morerows{1}{62} &amp;
\morerows{1}{${&gt;}$95}\\
\parbox[t]{3cm}{\centering \textsc{d}-threose\unskip\break C$_{4}$} &amp;
\parbox[t]{3cm}{\centering \textsc{d}-tagatose\unskip\break C6} &amp; &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

\xinlinefig{fx09} &amp; \xinlinefig{fx10} &amp;
\morerows{1}{L382F/F435Y} &amp;
\morerows{1}{${&gt;}$95} &amp;
\morerows{1}{72~h} &amp;
\morerows{1}{84} &amp;
\morerows{1}{${&gt;}$95}\\
\parbox[t]{3cm}{\centering \textsc{l}-erythrose\unskip\break C4} &amp;
\parbox[t]{3cm}{\centering \textsc{l}-psicose\unskip\break C6} &amp; &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

\xinlinefig{fx11} &amp;
\xinlinefig{fx12} &amp;
\morerows{1}{H462N/R521Y} &amp;
\morerows{1}{${&gt;}$95} &amp;
\morerows{1}{32~h} &amp;
\morerows{1}{67} &amp;
\morerows{1}{${&gt;}$95}\\
\parbox[t]{3cm}{\centering \textsc{d}-ribose\unskip\break C$_5$} &amp;
\parbox[t]{3cm}{\centering \textsc{d}-\textit{altro}-heptulose\unskip\break C7} &amp;  &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

\xinlinefig{fx13} &amp;
\xinlinefig{fx14} &amp;
\morerows{1}{H462N/R521Y} &amp;
\morerows{1}{${&gt;}$95} &amp;
\morerows{1}{96~h} &amp;
\morerows{1}{59} &amp;
\morerows{1}{${&gt;}$95}\\
\parbox[t]{3cm}{\centering \textsc{d}-xylose\unskip\break C$_5$} &amp;
\parbox[t]{3cm}{\centering \textsc{d}-\textit{ido}-heptulose\unskip\break C7} &amp;  &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

\xinlinefig{fx15} &amp;
\xinlinefig{fx16} &amp;
\morerows{1}{\parbox[t]{2cm}{\centering S385D/H462S/\unskip\break R521V}} &amp;
\morerows{1}{93} &amp;
\morerows{1}{7 days} &amp;
\morerows{1}{70} &amp;
\morerows{1}{${&gt;}$95}\\
\parbox[t]{3cm}{\centering \textsc{d}-allose\unskip\break C6} &amp; 
\parbox[t]{3cm}{\centering \textsc{d}-\textit{glycero}-\textsc{d}-\ubreak\textit{altro}-octulose\unskip\break C8} &amp;  &amp;  &amp;  &amp;  &amp; 
\vspace*{3pt}
\botline
\end{tabular}
\end{table*}

As we reported that wild-type TK$_{\mathrm{gst}}$ tolerates at 
60~\textdegree C $(2S)$-hydroxylated aldehyde as an acceptor substrate
contrary to the other  TKs~\cite{25}, we first investigated  extending
these activities towards short carbon chain aldehydes (C3) such as
(2$S$)-lactaldehyde and (2$S$)-glyceraldehyde through 
SSM at two key positions: D470, which interacts with the
C2($R$) hydroxyl group of the physiological erythrose-4-phosphate
(DE4P) in the wild-type enzyme, and L382, located at the opposite side
of the C2 aldehyde  (Figure~\ref{fig12}). The best variant,
L382D/D470S, increased activity by factors 4 and 5 towards
(2$S$)-lactaldehyde and (2$S$)-glyceraldehyde, respectively, forming
the corresponding products 5-deoxy-\textsc{l}-ribulose and
\textsc{l}-ribulose  with 63\% and 55\% isolated yields, respectively.

Based on these first results, a second generation of
TK$_{\mathrm{gst}}$ variants was investigated to extend 
TK$_{\mathrm{gst}}$ activity towards longer carbon chain aldoses
(C3--C6) by targeting four other positions S385, F435, H462, and R521
near, or by direct interaction with, the phosphate group of the
physiological acceptor substrate \textsc{d}-E4P 
(Figure~\ref{fig12})~\cite{33,34}. Three efficient TK$_{\mathrm{gst}}$
variants were identified following the screening of 
TK$_{\mathrm{gst}}$ variant libraries. The L382F/F435Y variant showed
an improvement by factors 3.7 and 4 compared to the wild-type
TK$_{\mathrm{gst}}$ towards the (2S)-tetroses \textsc{d}-threose and
\textsc{l}-erythrose, respectively, producing the corresponding Cn${+}$2
ketoses \textsc{d}-tagatose and \textsc{l}-psicose, respectively. The double variant H462N/R521Y
demonstrated an increase in conversion by factors 3.5 and 4 towards
the pentoses \textsc{d}-ribose and \textsc{d}-xylose compared to the
wild-type enzyme, forming \textsc{d}-\textit{altro}- and
\textsc{d}-\textit{ido}-heptulose,  respectively, with 67\% and 59\%
yields. The triple variant S385D/H462S/R521V allowed the synthesis of
\textsc{d}-\textit{glycero}-\textsc{d}-\textit{altro}-octulose from
\textsc{d}-allose while this was impossible with wild-type 
TK$_{\mathrm{gst}}$.

In conclusion, the aldoses (C3--C6) were almost totally converted into
the expected ketose products with good to excellent isolated yields 
(62\%--80\%) and in reasonable reaction time (24~h--72~h) 
except the hexose
\textsc{d}-allose requiring 7 days of reaction time with the double
variant H462S/R521V. Globally, the mutations of variants on targeted
positions near the phosphate group of the physiological substrate gave
higher or de novo conversions compared to the wild-type enzyme. In
addition, all compounds were obtained with excellent
diastereoselectivities, one stereoisomer being obtained by in situ 
nuclear magnetic resonance (NMR)
analysis of the reaction mixture. In all cases, the ($S$)-configuration
of the C3 ketose product was confirmed by NMR spectrum comparison with the 
already described product.

Among the ketoses listed in Table~\ref{tab1}, some of them have been
already described for their valuable biological properties. Hexoses include
\textsc{d}-tagatose, which is a sweetener and antidiabetic 
compound~\cite{6a,6b}, and \textsc{l}-psicose, which is used for the synthesis of
\textsc{l}-fructose, a precursor of an inhibitor of 
glucosidases~\cite{35,36}. The heptulose
\textsc{d}-\textit{altro}-heptulose is a marker of sugar metabolism
disorders such as cystinosis~\cite{37,38}.
The \textsc{d}-\textit{ido}-heptulose has been reported to be a precursor
of valiolamine and $N$-substituted derivatives, glucosidase
inhibitors useful for the treatment of hyperglycemic 
symptoms~\cite{39}. Among octuloses,
\textsc{d}-\textit{glycero}-\textsc{d}-\textit{altro}-octulose has been
identified in the plant \textit{Spinacia oleracea}, and its
(5$S$)-epimer
\textsc{d}-\textit{glycero}-\textsc{d}-\textit{ido}-octulose, which is
very abundant in \textit{Craterostigma plantagineum}, has been
described as a plant antioxidant involved in desiccation tolerance and
could be a potential reactive oxygen species  scavenger with
applications in nutrition and  healthcare~\cite{40,41,41a}. 
Recent studies have also shown the essential role of octuloses in the
metabolism of parasites such as \textit{Trypanosoma} and various
\textit{Leishmania} species, opening the way for potential octulose
analogue-based inhibitors to treat the diseases caused by these 
parasites~\cite{42}. 

\paragraph*{Enhancement of TK$_{\mathrm{gst}}$ activity towards aromatic
aldehydes with Li-HPA as donor}\label{sec2.5.3.2} 
In collaboration with the Fessner group, we investigated novel
electrophiles ortho-, meta-, and para-substituted nitrosoarenes, which
had not been previously investigated with  TKs~\cite{43a,43b}. These
substrates led to the formation of corresponding aromatic
hydroxamic acids, which have a wide range of medical applications
(antifungal, antibacterial, antioxidant, anti-inflammatory) due to
their chelating properties 
\mbox{(Table~\ref{tab2})}~\cite{44a,44b}.

\begin{table}
\caption{\label{tab2}Panel of nitroarenes (ortho-, 
\mbox{meta-,} and
para-substituted) used as acceptor substrates for  TK$_{\mathrm{gst}}$
variant in the presence of Li-HPA to obtain the corresponding
hydroxamic acids}
\begin{tabular}{ccc}
\thead
Aromatic group~(X)$^{\mathrm{a},\mathrm{b}}$ &amp; Acyl group (R) &amp; 
Yield (\%)\\
\endthead 
H &amp; CH$_{2}$OH &amp; 41/50 \\ 
4-Br &amp; CH$_{2}$OH &amp; 28/54 \\ 
4-Cl &amp; CH$_{2}$OH &amp; 41 \\ 
3-Cl &amp; CH$_{2}$OH &amp; 20 \\ 
3,4-Cl$_{2}$ &amp; CH$_{2}$OH &amp; 10 \\ 
4-CH$_{3}$ &amp; CH$_{2}$OH &amp; 49 \\ 
3-CH$_{3}$ &amp; CH$_{2}$OH &amp; 29 \\ 
4-Cl, 3-CH$_{3}$ &amp; CH$_{2}$OH &amp; 37 \\ 
3-Cl, 4-CH$_{3}$ &amp; CH$_{2}$OH &amp; 32 \\ 
4-CF$_{3}$ &amp; CH$_{2}$OH &amp; 9 \\ 
3-CF$_{3}$ &amp; CH$_{2}$OH &amp; 17 \\ 
4-N(Me)$_{2}$ &amp; CH$_{2}$OH &amp; 0 \\ 
H &amp; CH$_{3}$ &amp; 5
\botline
\end{tabular}
\tabnote{$^{\mathrm{a}}$~Substituent position relative to the nitroso
group.}
\tabnote{$^{\mathrm{b}}$~DMSO as cosolvent for X${=}$H and 4-Br and acetone as
cosolvent for the other aromatic group.} 
\end{table}

A key aspect of these reactions was the 
formation of a carbon--nitrogen
bond instead of the typical carbon--carbon bond formed by TKs with
other aldehydes as electrophiles. The TK$_{\mathrm{gst}}$ variant
L382N/D470S 
(positions previously identified for improved activity
towards hydrophobic aldehydes) 
\mbox{exhibited} the highest activity towards
these substrates, with a sevenfold increase in activity compared to
wild-type TK$_{\mathrm{gst}}$. The replacement of aspartate at position
470 with serine, a non-charged but polar amino acid, seems to enhance
interaction during the carboligation step. This mutation had been
described in a previous study with non-hydroxylated acceptor 
substrates~\cite{45}. Additionally, this variant has a melting
temperature $(T_{\mathrm{m}})$ of  73.9~\textdegree C (compared to  75.5~\textdegree
C for wild-type TK$_{\mathrm{gst}}$) and remains highly stable in the
presence of cosolvents, retaining 80\% to 100\% residual activity in
DMF, acetone, and acetonitrile.

\paragraph*{Enhancement of TK$_{\mathrm{gst}}$ activity towards novel
hydrophobic $\rmalpha$-ketoacids as donors and different aldehydes as
acceptors}\label{sec2.5.3.3} 
In 2017, a challenging work developed jointly with Fessner et~al.\ 
was conducted to expand the donor substrate spectrum of
TK$_{\mathrm{gst}}$ to $\rmalpha$-ketoacids with hydrophobic side
chains, which had not previously been considered for  TKs~\cite{45}.
The selection of  key positions in the active site was guided by a
structural study based on the model of TK$_{\mathrm{gst}}$ with HPA as
the donor substrate, constructed from the crystal structure of
TK$_{\mathrm{ban}}$  (Figure~\ref{fig13}). 

\begin{figure*}
\includegraphics{fig13}
\caption{\label{fig13}Model of wild-type TK$_{\mathrm{gst}}$ based on
the X-ray crystal structure of TK$_{\mathrm{ban}}$ (PDB entry 3M49)
with hydroxypyruvate (HPA) as donor. The model was built using Modeller
9.14 and Chimera.}
\vspace*{1.5pc}
\end{figure*}

The active site residues interacting with the hydroxyl group of HPA,
namely H68, H102, G116, and H474, were targeted. After screening
libraries created by the SSM technique at positions H102 and H474, the
variant H102L/H474S showed relative rate increases by factors of 
4.4--32 with pyruvate,  2-oxobutyrate, and 3-methyl-2-oxobutyrate 
\mbox{(Figure~\ref{fig14}).}

\begin{figure*}
\includegraphics{fig14}
\caption{\label{fig14}Activity of TK$_{\mathrm{gst}}$ variants
(k$_{\mathrm{cat}}$) with glycolaldehyde as acceptor against
non-natural nucleophile substrates relative to
wt-1-deoxy-\textsc{d}-xylulose-5-phosphate-synthase (DXS$_{\mathrm{eco}}$),
wt-TK$_{\mathrm{gst}}$, and wt-TK$_{\mathrm{eco}}$ 
(\protect\xinlinefig{fx17}~pyruvate\ 
\protect\xinlinefig{fx18}~2-oxobutanoate\ 
\protect\xinlinefig{fx19}~3-methyl-2-oxobutanoate).}
\end{figure*}

In position His474, it seems to be essential to preserve a hydrogen
bond with the carbonyl group, which was attained by the replacement by
serine, providing a slightly reduced size. The replacement of His102 by
a non-polar leucine residue not only provided extra space but also
improved the binding of the hydrophobic alkyl chain of the substrate in
the otherwise initially highly polar active site of wild-type
TK$_{\mathrm{gst}}$. The best variants using glycolaldehyde as the 
acceptor were H102L with pyruvate giving a 6-fold increase,
H102L/474S with 2-oxobutyrate giving a 30-fold increase, and H102T with
3-methyl-2-oxobutyrate giving a 11.4-fold increase while wild-type 
TK$_{\mathrm{gst}}$ produced any activity with 2-oxobutyrate and
3-methyl-2-oxobutyrate and slight activity with pyruvate 
(Figure~\ref{fig16}). We note that the H102T activity towards pyruvate
reached 80\% of that obtained~with
1-deoxy-\textsc{d}-xylulose-5-phosphate-synthase (DXS$_{\mathrm{eco}}$) when using
pyruvate as the physiological substrate.\looseness=1

These results offered interesting prospects for the combination of
these new donors with hydroxylated or 
non-hydroxylated  aldehydes as acceptors. 

\paragraph*{Enhancement of TK$_{\mathrm{gst}}$ activity towards
hydrophobic $\rmalpha$-ketoacid as donors and hydrophobic aldehyde as
acceptors}\label{sec2.5.3.4}
The hydrophobic donors particularly 2-oxobutyrate were investigated
with hydrophobic aldehyde as acceptors to produce the corresponding
unsymmetrical $\rmalpha$-hydroxyketones (acyloins), which are valuable
as flavors or non-ionic surfactants when compounds display long carbon
chains  (Figure~\ref{fig15})~\cite{5,7}.

\begin{figure*}
\includegraphics{fig15}
\caption{\label{fig15}Synthesis of monohydroxylated
$\rmalpha$-hydroxyketones from hydrophobic $\rmalpha$-ketoacid as
donors and hydrophobic aldehyde as acceptors.}
\end{figure*}

\begin{figure*}
\includegraphics{fig16}
\caption{\label{fig16}Synthesis of 1- and 1,2-deoxy-ketoses from
aliphatic $\rmalpha$-ketoacids combined with aldoses 
\mbox{(C4--C5).}}
\end{figure*}

New TK$_{\mathrm{gst}}$ variants were created by combining the most
beneficial mutations identified previously with hydrophobic donors
(H102L/H474S) with those identified earlier for hydrophobic aldehydes 
(F435I)~\cite{46}. The triple variant H102L/H474S/F435I was able to
transfer the acyl group from {2-oxobutyrate} to aliphatic aldehydes,
enabling the synthesis of the corresponding monohydroxylated
$\rmalpha$-hydroxyketones (C3--C7) while except with propanal, any
activity of the wild-type TK$_{\mathrm{gst}}$ was observed with the
longer carbon chain aldehydes. 
Even if the yields and enantiomeric excess (ee) were globally
lower than those obtained with HPA, the products obtained with
oxobutyrate as the donor are not easy to obtain with other enzymatic or
chemical routes and were isolated and purified with accepted yields
except for  C8 (Table~\ref{tab3})~\cite{47}.

\begin{table*}
\caption{\label{tab3}Combination of hydrophobic donor ketoacids and
aldehydes for the synthesis of monohydroxylated
$\rmalpha$-hydroxyketones in the presence of the triple variant
H102L/H474S/F435I}
\begin{tabular}{ccccccc}
\thead
\parbox[t]{1.5cm}{\centering Ketoacid donor} &amp; 
\parbox[t]{1.5cm}{\centering Aldehyde acceptor} &amp; 
\parbox[t]{1.5cm}{\centering TK$_{\mathrm{gst}}$ variant} &amp; 
\parbox[t]{2.4cm}{\centering Fold increase for TK$_{\mathrm{gst}}$ wt} &amp;
\parbox[t]{1.5cm}{\centering Reaction time (h)}&amp; 
\parbox[t]{2cm}{\centering Isolated yield (\%)} &amp; 
\parbox[t]{.5cm}{\centering ee (\%)} 
\vspace*{2pt}\\
\endthead 
\morerows{5}{\parbox[t]{2.5cm}{\centering HPA\Lbreak
$\mathrm{R}_1=\ndash\mathrm{CH}_{2}\mathrm{OH}$}} &amp; 
$\mathrm{R}_2=\mathrm{Me}$ &amp; L382F &amp; 4 &amp; 24 &amp; 56 &amp; 94 
\vspace*{2pt}\\
 &amp; $\mathrm{R}_{2}= \mathrm{Et}$ &amp;
 L382F &amp; 6 &amp; 24 &amp; 43 &amp; 99 \\
 &amp; $\mathrm{R}_{2}= \mathrm{Pr}$ &amp; 
 L382F &amp; 0.5 &amp; 24 &amp; 44 &amp; 99 \\
 &amp; $\mathrm{R}_{2}= \mathrm{But}$ &amp;
 L382F &amp; 2 &amp; 24 &amp; 41 &amp; 98 \\
 &amp; $\mathrm{R}_{2}= \mathrm{Pent}$ &amp;
 L382F &amp; ${\infty}$ &amp; 24 &amp; 21 &amp; 87 \\
 &amp; $\mathrm{R}_{2}= \mathrm{Hex}$ &amp;
 L382F &amp; 6 &amp; 24 &amp; \07 &amp; 91\vspace*{6pt}\\
 
\morerows{4}{\parbox[t]{2cm}{\centering Oxobutyrate\Lbreak
$\mathrm{R}_{1}=-\mathrm{Et}$}} &amp;
$\mathrm{R}_{2}=\mathrm{Me}$ &amp; 
H102L/H474S/F435I &amp; 20 &amp; 24 &amp; 25 &amp; 12 
\vspace*{2pt}\\
 &amp; $\mathrm{R}_{2}= \mathrm{Et}$ &amp; 
 H102L/H474S/F435I &amp; ${\infty}$ &amp; 24 &amp; 47 &amp; \06 \\
 &amp; $\mathrm{R}_{2}= \mathrm{But}$ &amp; 
 H102L/H474S/F435I &amp; ${\infty}$ &amp; 24 &amp; 20 &amp; 31 \\
 &amp; $\mathrm{R}_{2}= \mathrm{Pent}$ &amp; 
 H102L/H474S/F435I &amp; ${\infty}$ &amp; 24 &amp; 25 &amp; 33 \\
 &amp; $\mathrm{R}_{2}= \mathrm{Hex}$ &amp; 
 H102L/H474S/F435I &amp; ${\infty}$ &amp; 24 &amp; ${&lt;}$5 &amp; nd 
\botline 
\end{tabular}
\tabnote{nd $=$ not determined.}
\end{table*}

\paragraph*{Enhancement of TK$_{\mathrm{gst}}$ activity towards
hydrophobic $\rmalpha$-ketoacid as donors and hydroxylated aldehyde as
acceptors}\label{sec2.5.3.5} 
The objective was to produce 1- or 1,2-deoxy-ketoses when using
pyruvate or 2-oxobutyrate, respectively, as donors  appropriately
polyhydroxylated with increased carbon chain length aldoses such as two
tetroses (\textsc{d}-erythrose (C4) and \textsc{l}-threose (C4)) and two
pentoses (\textsc{d}-ribose (C5) and \textsc{d}-xylose (C5)) 
(Figure~\ref{fig16}, Table~\ref{tab4})~\cite{48a,48b}.

\begin{table*}
\caption{\label{tab4}Synthesis of 1- and 1,2-deoxy-ketoses at
50~\textdegree C using TK$_{\mathrm{gst}}$ variants in the presence of
2-oxobutyrate and pyruvate as donors and aldoses as acceptors
(\textsc{d}-erythrose, \textsc{l}-threose, \textsc{d}-ribose,
\textsc{d}-xylose)}
\tabcolsep=2pt
\begin{tabular}{cccccccc} 
\thead
Donor &amp; 
\parbox[t]{.5cm}{\centering 
Acceptor} &amp; 
\parbox[t]{.8cm}{\centering 
Product} &amp; 
\parbox[t]{.5cm}{\centering 
Time (h)} &amp; 
\parbox[t]{1.5cm}{\centering 
TK$_{\mathrm{gst}}$ variant} &amp;
\parbox[t]{1.2cm}{\centering 
TK$_{\mathrm{gst}}$\unskip\break
(mg/mL)} &amp; 
\parbox[t]{1.2cm}{\centering 
In situ yield$^{\mathrm{a}}$ (\%)} &amp; 
\parbox[t]{1.2cm}{\centering 
Isolated yield (\%)$^{\mathrm{a},\mathrm{b}}$}
\vspace*{2pt}\\
\endthead
\raisebox{4em}{}
\morerows{7}{\parbox[t]{2cm}{\centering 
R$_1$\ ${=}$ Et\Lbreak
Oxobutyrate}}  &amp; 
\xinlinefig{fx20} &amp;
\xinlinefig{fx21} &amp; 
\morerows{1}{24} &amp;
\morerows{1}{H102L/L118I} &amp;
\morerows{1}{0.5} &amp;
\morerows{1}{85} &amp;
\morerows{1}{71} 
\vspace*{2pt}\\
 &amp; 
\textsc{d}-erythrose &amp;
\parbox[t]{4cm}{\centering 
1,2-deoxy-\textsc{d}-\textit{arabino}-{\ubreak}hept-3-ulose} &amp;
 &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt}\\

 &amp; \xinlinefig{fx22} &amp;
 \xinlinefig{fx23} &amp;
\morerows{1}{\06} &amp;
\morerows{1}{\parbox[t]{1.5cm}{\centering 
H102L/L118/{\ubreak}H474S}} &amp;
\morerows{1}{0.5} &amp;
\morerows{1}{89} &amp;
\morerows{1}{86}\\
 &amp;
 \textsc{l}-threose &amp; 
\parbox[t]{3.5cm}{\centering  
 1,2-deoxy-\textsc{d}-\textit{ido}-hept-{\ubreak}3-ulose} &amp; 
  &amp;  &amp;  &amp;  &amp;  
\vspace*{10pt}\\

 &amp; \xinlinefig{fx24} &amp; \xinlinefig{fx25} &amp;
\morerows{1}{72} &amp; 
\morerows{1}{H102L/L118I} &amp;
\morerows{1}{3} &amp;
\morerows{1}{68} &amp; 
\morerows{1}{68}\\ 
 &amp; \textsc{d}-ribose &amp;
\parbox[t]{3cm}{\centering   
1,2-deoxy-\textsc{d}-\textit{altro}-{\ubreak}oct-3-ulose} &amp; 
 &amp;  &amp;  &amp;  &amp;  
\vspace*{10pt}\\

 &amp; \xinlinefig{fx26} &amp; \xinlinefig{fx27} &amp;
\morerows{1}{48} &amp;
\morerows{1}{H102L/L118I} &amp;
\morerows{1}{3} &amp;
\morerows{1}{82} &amp;
\morerows{1}{61}\\
 &amp; \textsc{d}-xylose &amp;
\parbox[t]{3.5cm}{\centering  
1,2-deoxy-\textsc{d}-\textit{ido}-oct-{\ubreak}3-ulose} &amp;
 &amp;  &amp;  &amp;  &amp; 
\vspace*{16pt}\\

\morerows{3}{\parbox[t]{2cm}{\centering R$_1$\ ${=}$ Me\Lbreak
Pyruvate}} &amp;
\xinlinefig{fx28} &amp;
\xinlinefig{fx29} &amp;
\morerows{1}{24} &amp;
\morerows{1}{\parbox[t]{1.5cm}{\centering 
H102L/L118/{\ubreak}H474S}} &amp;
\morerows{1}{1} &amp;
\morerows{1}{74} &amp;
\morerows{1}{68}
\vspace*{2pt}\\
  &amp; \textsc{d}-erythrose &amp; 
1-deoxy-\textsc{d}-fructose &amp; 
 &amp;  &amp;  &amp;  &amp; 
\vspace*{10pt} \\

 &amp; \xinlinefig{fx30} &amp;
\xinlinefig{fx31} &amp;
\morerows{1}{24} &amp;
\morerows{1}{\parbox[t]{1.5cm}{\centering 
H102L/L118/{\ubreak}H474S}} &amp;
\morerows{1}{1} &amp;
\morerows{1}{81} &amp;
\morerows{1}{60}
\vspace*{2pt}\\

&amp;
\textsc{l}-threose  &amp; 
1-deoxy-\textsc{l}-sorbose &amp; 
 &amp;  &amp;  &amp;  &amp;
\vspace*{3pt} 
\botline
\end{tabular}
\tabnote{$^{\mathrm{a}}$~Reactions were carried out with
TK$_{\mathrm{gst}}$ (0.5--3~mg), ThDP (0.1~mM), MgCl$_{2}$ (1~mM),
donors (50--60~mM), and $\rmalpha$-hydroxyaldehydes (50~mM) at pH 7 and
50~\textdegree C.}
\tabnote{$^{\mathrm{b}}$~Determined by in situ $\mbox{}^{1}$H NMR 
analysis.\vspace*{-2pt}}
\end{table*}

Among the previous TK$_{\mathrm{gst}}$ variants, we combined the best
mutations identified earlier, H102L and H474S, for hydrophobic
$\rmalpha$-ketoacids (pyruvate and oxobutyrate) with a new mutation on
L118I, which is involved in the stabilization of the thiazolium cycle of
ThDP and which could also stabilize the aliphatic donor substrates when
histidine replaced isoleucine by interacting with an
appropriate longer carbon chain residue. 

Even if the use of higher enzyme quantities was required when using
pentoses as acceptors, both donors 2-oxobutyrate and pyruvate formed the
expected 1,2-deoxy-hexoses or 1,2-heptuloses and 1-deoxyhexoses,
respectively, in pure form (one stereoisomer was identified in the
reaction mixture by NMR) with good to excellent isolated yields. Some
of them have been already described as \mbox{displaying} \mbox{interesting}
properties; for example,  1-deoxy-\textsc{d}-fructose is a potential metabolic
inhibitor and antimetabolite.

\subsection{Multienzymatic synthesis strategies involving
TKs}\label{sec2.6}
We applied the strategy of cascade reaction for in situ generation of
TK$_{\mathrm{gst}}$ donor and acceptor substrates, which were unstable
or costly or not commercially available. To compete with the
productivity of traditional methods, the use of two or even more
enzymes in cascade can considerably improve the efficiency of a
multistage synthesis by obviating the isolation of intermediates, thus
saving time, resources, reagents, and energy while reducing 
waste~\cite{49a,49b}. Cascade reactions can be performed along a
simultaneous one-pot strategy when all the enzyme requirements are
compatible. To meet limitations, such as substrate/product/reagent
inhibition or incompatibility of reaction conditions (pH, temperature),
a telescoped, sequential one-pot procedure can be used. 

Knowing that Li-HPA was obtained from toxic bromopyruvate with modest 
yields~\cite{8} and its instability at higher temperatures than 
25~\textdegree C~\cite{11,24}, a different strategy for HPA in situ
generation was investigated. We first reported a procedure with 
\textsc{l}-$\rmalpha$-transaminase from \textit{Thermosinus
carboxydivorans}  (TA$_{\mathrm{tca}}$) able to produce HPA from
\textsc{l}-serine, developed in collaboration with de Berardinis 
et~al.\ 
(Figure~\ref{fig17})~\cite{50}.

\begin{figure*}
\includegraphics{fig17}
\vspace*{-2pt}
\caption{\label{fig17}In situ generation of HPA catalyzed by
TA$_{\mathrm{tca}}$ coupled with TK$_{\mathrm{gst}}$ for the synthesis
of (3$S$, 4$S$) ketoses.}
\end{figure*}

This is the first example of TA--TK coupling at high temperature cited
in the literature. The  TA$_{\mathrm{tca}}$ being thermostable, it
could be coupled in a ``one-pot'' at  60~\textdegree C with the
thermostable TK$_{\mathrm{gst}}$. This approach was applied to the
synthesis of rare \textsc{l}-\textit{erythro}-ketoses (3$S$,
4$S$) in the presence of (2$S$)-aldehydes for which TK$_{\mathrm{gst}}$
has low activity at  20~\textdegree C. The reaction products were
obtained in good to excellent yields (51\%--98\%) with no accumulation
of HPA in the medium and complete conversion of \textsc{l}-serine. 

Ward et~al.\ has since discovered more efficient thermostable TAs
within metagenomic  libraries~\cite{15a} from \textit{Thermobifida
fusca} (TA$_{\mathrm{tfu}}$) and \textit{Geobacillus
stearothermophilus}  (TA$_{\mathrm{gst}}$), which showed opposite
stereoselectivities. The two thermostable TAs retain approximately 70\%
of their activity after 24~h of incubation at 60~\textdegree C. 
They were selected to generate HPA from a racemic mixture
of serine. 

Based on our bi-enzymatic cascade reaction, an additional third
enzymatic step was investigated to generate (2$S$)-aldehydes
such as \textsc{l}-glyceraldehyde, \textsc{d}-threose, and
\textsc{l}-erythrose in collaboration with Clapes et~al.~\cite{51}
The precursors of $\rmalpha$-hydroxylated aldehydes (2$S$) are
glycolaldehyde and formaldehyde, which are inexpensive and achiral in the presence
\textsc{d}-fructose-6-phosphate aldolase from \textit{E.~coli} 
(FSA$_{\mathrm{eco}}$)  (Figure~\ref{fig18}). 

\begin{figure*}
\includegraphics{fig18}
\vspace*{-2pt}
\caption{\label{fig18}In situ generation of aldehydes ($2S$) with
FSA$_{\mathrm{eco}}$ and HPA with TA$_{\mathrm{tca}}$ coupled with
TK$_{\mathrm{gst}}$ in a one-pot sequential cascade.}
\vspace*{-2pt}
\end{figure*}

The simultaneous coupling of FSA$_{\mathrm{eco}}$ and
TK$_{\mathrm{gst}}$ in a one-pot was not possible since glycolaldehyde
and formaldehyde would react directly with HPA in the presence of
TK$_{\mathrm{gst}}$ to form \textsc{l}-erythrulose and DHA,
respectively. Therefore, a sequential process was developed in which
the $\rmalpha$-hydroxylated (2$S$) aldehyde is produced in the
first step catalyzed by  FSA$_{\mathrm{eco}}$ and then used in
solution without intermediate purification as the acceptor substrate of
TK$_{\mathrm{gst}}$. The synthesis of the corresponding
\textsc{l}-\textit{erythro}-ketoses (3$S$, 4$S$) was carried out from
HPA also in situ generated from \textsc{l}-serine in the presence of
the thermostable TA$_{\mathrm{tca}}$ described previously. The
simultaneous coupling of  TA$_{\mathrm{tca}}$ and TK$_{\mathrm{gst}}$
makes it possible to carry out the synthesis at  60~\textdegree C and
thus to increase the low affinity of TK$_{\mathrm{gst}}$ for
(2$S$)-hydroxylated aldehydes.

This process made  possible the synthesis of three rare ketoses (3$S$,
4$S$)---\textsc{l}-ribulose from \textsc{l}-glyceraldehyde, \textsc{d}-tagatose
from \textsc{d}-threose,  and \textsc{l}-psicose from
\textsc{l}-erythrose---which are compounds with high added value, with
diastereomeric 
excess (de) greater than 95\% and product isolated yields of 53, 55,
and 49\%, respectively.

The main disadvantage of these TA strategies for HPA generation is the
release of alanine as the 
\mbox{by-product,} which is not in favor of atom economy.
Another enzymatic strategy avoiding the use of cosubstrate has been
considered via serine, allowing the oxidation of the amine function by
a microbial amino acid oxidase (AAO, EC 1.4.3.3) in the presence of 
O$_{2}$ and catalase (EC 1.11.1.6) to dismutate the 
\mbox{hydrogen} peroxide
formed during the oxidation stage. 
We turned to \textsc{d}AAO$_{\mathrm{rg}}$~\cite{52}, 
already reported by  Pollegioni et~al.~\cite{53a,53b}, to accept the
polar amino  acid \textsc{d}-serine
with higher specific activity compared to \textsc{d}AAO from pig kidney.
But \textsc{d}AAO$_{\mathrm{rg}}$ has never been investigated for producing
$\rmalpha$-ketoacid HPA from \mbox{\textsc{d}-serine,} especially at
the \mbox{preparative} scale. In a 
\mbox{sequential} reaction, we also investigated 
producing \textsc{d}-serine in collaboration with Prot\'eus by Seqens from achiral
glycine and formaldehyde as precursors with \textit{E.~coli} cells
expressing threonine aldolase~\cite{54}. After total
conversion of the substrate, the crude extract containing \textsc{d}-serine was used
as the precursor of HPA  (Figure~\ref{fig19}).

\begin{figure*}
\includegraphics{fig19}
\caption{\label{fig19}In situ generation of $\rmalpha$-ketoacids from
\textsc{d}AAO (\textsc{d}AAO$_{\mathrm{rg}}$ for HPA and \textsc{d}AAO4536 for pyruvate and
oxobutyrate) coupled with TK$_{\mathrm{gst}}$ for the synthesis of
ketoses and analogues.}
\end{figure*}

In parallel, we discovered in collaboration with Prozomix a
thermostable \textsc{d}AAO4536 from metagenomic library screening not applicable
for producing HPA but pyruvate and oxobutyrate from \textsc{d}-alanine and
\textsc{d}-homoalanine, respectively  (Figure~\ref{fig21})~\cite{47}. 

Finally, \textsc{d}AAO$_{\mathrm{rg}}$ (or \textsc{d}AAO4536) was coupled with the 
TK$_{\mathrm{gst}}$ variant in a one-pot, two-step simultaneous or
sequential cascade sequence with different aldehydes (hydrophobic or
polyhydroxylated with increased carbon chain length) as
TK$_{\mathrm{gst}}$ acceptor substrates introduced into the reaction
mixture at the same concentration as \textsc{d}-amino acids. \textsc{A}
complete conversion of all substrates was observed, and targeted
compounds were recovered with high enantio- and
diastereoselectivities. These approaches 
\mbox{appeared} particularly
appropriate for expensive, unstable, and commercially unavailable
\textit{a}-ketoacid synthesis.

\subsection{Promiscuous cross-acyloin condensation reaction catalyzed
by TK$_{\mathrm{gst}}$}\label{sec2.7} 
Previous studies conducted with propanal showed that the variant
H102L/L118I/H474S was capable of catalyzing the self-acyloin
condensation of propanal to yield propioin 
(Figure~\ref{fig20})~\cite{55}.

\begin{figure*}
\includegraphics{fig20}
\caption{\label{fig20}Self-condensation of two propanal molecules
giving (${\pm}$)-4-hydroxyhexan-3-one (propioin) catalyzed by
TK$_{\mathrm{gst}}$ variant H102L/L118I/H474S.}
\end{figure*}

\begin{figure*}
\includegraphics{fig21}
\caption{\label{fig21}Proposed mechanism for acyloin condensation
according to pathway B compared to pathway A giving the common
$\rmalpha$-hydroxy-$\rmbeta$-(polyhydroxy)alkylthiamine diphosphate
(acylThDP) carbanion \textbf{II} generated from aliphatic
$\rmalpha$-ketoacids (pathway A) or from corresponding aliphatic
aldehydes 
\mbox{(pathway~B).}}
\end{figure*}

\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig22}
\vspace*{-2pt}
\caption{\label{fig22}Synthesis of $\rmalpha$-hydroxyketones catalyzed
by TK$_{\mathrm{gst}}$ variants from two possible nucleophiles,
aliphatic $\rmalpha$-ketoacids (pyruvate, 2-oxobutyrate,
3-methyl-2-oxobutyrate; pathway A) and the corresponding aliphatic
aldehydes (propanal, butanal, iso-butanal; pathway B) in
the presence of hydroxylated aldehydes as electrophiles.}
\vspace*{-2pt}
\end{figure*}

According to these results, a possible mechanism for the acyloin
condensation reaction was investigated in the presence of two aldehydes
as TK$_{\mathrm{gst}}$ substrates by in silico studies using
H102L/L118I/H474G(S)  TK$_{\mathrm{gst}}$ variant active site 
models~\cite{56}. The presence of leucine in place of histidine in the
102 position (H102L) may contribute to the stabilization of the first
aldehyde molecule in the active site. Then, an attack of ThDP carbanion
\textbf{I} on the carbonyl of the first molecule of aldehyde acting as
a nucleophile followed by a proton transfer of \textbf{I}--\textbf{B}
via a water molecule to His263 led to acylThDP carbanion
\textbf{II}  (Figure~\ref{fig21}). In the last step, acylThDP carbanion
\textbf{II} attacked the carbonyl group of the second aldehyde molecule
acting as the electrophile to form the acyloin product. In this mechanism,
the \mbox{aldehyde} acting as the nucleophile can replace the corresponding acyl
group of corresponding $\rmalpha$-ketoacids but avoids 
decarboxylation and the release of carbon dioxide. We proved that both
pathways led to the same acylThDP carbanion \textbf{II} and 
products~\cite{56}. 

To extend this reaction to the more challenging cross-acyloin
condensation, we used three aldehydes' (ethanal, propanal, and
\textit{iso}-butanal) analogues of  $\rmalpha$-ketoacids (pyruvate,
2-oxobutyrate, and 3-methylbutyrate used previously) combined with
hydroxylated aldehydes (C2--C4; glycolaldehyde, \textsc{d}-glyceraldehyde, and
\textsc{d}-erythrose) in the presence of  TK$_{\mathrm{gst}}$ variants
H102L/L118I/H474S(G) identified earlier  (Figure~\ref{fig22})~\cite{56}.

For each synthesis with stoichiometric amounts of both different
aldehydes  (Table~\ref{tab5}), the in situ analysis of the reaction
medium by NMR showed the formation of only one
$\rmalpha$-hydroxyketone, highlighting the selectivity of the variants.
Indeed, the uncontrolled cross-acyloin condensation can lead to four
products. All $\rmalpha$-hydroxyketones were obtained with good
isolated yields comparable to those obtained by pathway A from
the carboxylation of $\rmalpha$-ketoacids  
\mbox{(Table~\ref{tab5})}~\cite{56}. 

\begin{table*}
\caption{\label{tab5}Synthesis of $\rmalpha$-hydroxyketones using
TK$_{\mathrm{gst}}$ variants in the presence of ethanal, propanal, and
iso-butanal as nucleophiles (50~mM) and hydroxylated aldehydes
(glycolaldehyde, \textsc{d}-glyceraldehyde, \textsc{d}-erythrose) as
electrophiles (50~mM). 
Reactions were performed at 37~\textdegree C in
phosphate buffer (50~mM) at pH 7.0\vspace*{-1pt}}
\tabcolsep=3pt
\begin{tabular}{cccccccc} 
\thead
\parbox[t]{.8cm}{\centering 
Donor} &amp;
\parbox[t]{2cm}{\centering 
Acceptor} &amp;
\parbox[t]{.5cm}{\centering 
Product} &amp; 
\parbox[t]{.5cm}{\centering 
Time (h)} &amp;
\parbox[t]{3cm}{\centering 
TK$_{\mathrm{gst}}$ variant} &amp; 
\parbox[t]{1.5cm}{\centering 
In situ yield (\%)$^{\mathrm{a}}$} &amp; 
\parbox[t]{1.5cm}{\centering 
Isolated yield (\%)} &amp;
\parbox[t]{1cm}{\centering 
de$^{\mathrm{b}}$ (\%)}\\
\endthead
\morerows{5}{\parbox[t]{1cm}{\centering 
\xinlinefig{fx32}\Lbreak
propanal}} &amp;
\xinlinefig{fx33} &amp; 
\xinlinefig{fx34} &amp;
\morerows{1}{72} &amp;
\morerows{1}{\parbox[t]{1cm}{\centering
H102L/L118I/{\ubreak}H474G}} &amp;
\morerows{1}{80} &amp;
\morerows{1}{50} &amp;
\morerows{1}{69 $(S)$}
\vspace*{2pt}\\
&amp; 
glycolaldehyde &amp; &amp; &amp; &amp; &amp; &amp;
\vspace*{10pt}\\

 &amp; \xinlinefig{fx35} &amp; \xinlinefig{fx36} &amp;
\morerows{1}{72} &amp;
\morerows{1}{\parbox[t]{1.5cm}{\centering
H102L/L118I/{\ubreak}H474S}} &amp;
\morerows{1}{48} &amp;
\morerows{1}{31} &amp;
\morerows{1}{${&gt;}$95}\\
 &amp; \textsc{d}-glyceraldehyde &amp; &amp; &amp; &amp; &amp; &amp; 
\vspace*{10pt}\\

 &amp; \xinlinefig{fx37} &amp;
\xinlinefig{fx38} &amp;
\morerows{1}{48} &amp;
\morerows{1}{\parbox[t]{1.5cm}{\centering
H102L/L118I/{\ubreak}H474S}} &amp;
\morerows{1}{75} &amp;
\morerows{1}{50} &amp;
\morerows{1}{${&gt;}$95}
\vspace*{2pt}\\
&amp;
\textsc{d}-erythrose &amp; &amp; &amp; &amp; &amp; &amp;  
\vspace*{18pt}\\

\morerows{3}{\parbox[t]{1.2cm}{\centering 
\xinlinefig{fx39}\Lbreak
\textit{Iso}-butanal}}  &amp; 
\xinlinefig{fx40} &amp;
\xinlinefig{fx41} &amp; 
\morerows{1}{72} &amp;  
\morerows{1}{\parbox[t]{1cm}{\centering
H102L/L118I/{\ubreak}H474G}} &amp; 
\morerows{1}{82} &amp;  
\morerows{1}{60} &amp;
\morerows{1}{34 $(S)$}\\
 &amp; glycolaldehyde &amp; &amp; &amp; &amp; &amp; &amp;
\vspace*{10pt}\\

 &amp; \xinlinefig{fx42} &amp;
\xinlinefig{fx43} &amp;
\morerows{1}{48} &amp;
\morerows{1}{\parbox[t]{1cm}{\centering 
H102L/L118I/{\ubreak}H474S}} &amp; 
\morerows{1}{39} &amp;
\morerows{1}{31} &amp;
\morerows{1}{${&gt;}$95}\\
&amp; \textsc{d}-glyceraldehyde &amp; &amp; &amp; &amp; &amp; &amp; 
\vspace*{3pt}
\botline
\end{tabular}
\tabnote{$^{\mathrm{a}}$~Determined by in situ  $\mbox{}^{1}$H  NMR
using TSP-d$_{4}$ as an internal standard and calculated based on in
situ product formation.} 
\tabnote{$^{\mathrm{b}}$~Determined by chiral GC-MS analysis after
derivatization.}
\vspace*{-1pt}
\end{table*}

The formation of the targeted $\rmalpha$-hydroxyketones by
cross-acyloin condensation offers the great advantage of ensuring atom
economy by avoiding the release of carbon dioxide generated by the
decarboxylation of $\rmalpha$-ketoacids. This novel enzymatic acyloin
condensation reaction catalyzed by TK should broaden the synthetic
toolkit for creating unsymmetrical $\rmalpha$-hydroxyketones and
enhance the efficiency of previous enzymatic and chemical approaches in
terms of mass metrics.

\section{Conclusion}\label{sec3}
Wild-type TKs in cells exclusively transfer a ketol unit from a
ketose phosphate to an aldose phosphate Cn leading to a Cn${+}$2 ketose
phosphate by a reversible reaction. The results reported in this
paper showed that TK$_{\mathrm{gst}}$ engineering allowed the synthesis
of a wide range of non-phosphorylated $\rmalpha$-hydroxyketones
(aliphatic, hydroxylated, or aromatic) in one step with excellent yields
and stereoselectivities. The noteworthy evolvability of thermostable TK$_{\mathrm{gst}}$
was mostly based on its robust 3D structure
against destabilizing mutagenesis factors. The best variants selected
from libraries \mbox{created} by rational design based on active site analysis
\mbox{enhanced} the wild-type TK$_{\mathrm{gst}}$ towards targeted
nucleophiles, analogues of pyruvate, coupled with electrophiles such as
aliphatic,  increased carbon chain length polyhydroxylated, and
aromatic aldehydes. An essential tool to select the best variants was
the development of a generic rapid and cheap pH-shift-based screening
assay applied in the liquid or solid phase. The latter enabled visual
detection of clones expressing active TK$_{\mathrm{gst}}$ by their
specific 
coloration. 

The best efficient TK$_{\mathrm{gst}}$ variants were first used to
catalyze the usual TK reaction from the $\rmalpha$-ketoacid and aldehyde as the
nucleophile and electrophile, 
\mbox{respectively.} As the main hurdle for
biocatalytic application is the instability/cost of 
$\rmalpha$-ketoacids, one-pot strategies were investigated for in situ
generation of $\rmalpha$-ketoacids from corresponding \textsc{l}- or
\textsc{d}-amino acids with \textsc{l}-transaminase or 
\textsc{d}-amino acid oxidase. The novel
promising approach was based on a promiscuous  TK$_{\mathrm{gst}}$
reaction, allowing selective cross-acyloin condensation of two
aldehydes, one playing the role of the nucleophile in place of 
$\rmalpha$-ketoacid and the other aldehyde acting as the electrophile. This
original TK$_{\mathrm{gst}}$ catalyzed reaction provides atom economy
while avoiding carbon dioxide release and achieving similar efficiency
compared to the commonly used pathway. 

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could \mbox{benefit} from this article, and have
declared no affiliations other than their research organizations.

\section*{Funding}
This work was funded by the Fonds R\'{e}gional Innovation
Laboratoire (grant DOS00494484/00 to LH), Pack Ambition Recherche ID 
1701105201-61617, the Agence Nationale de la Recherche (grants
ANR-09-BLAN-0424-CSD3 and ANR-22-CE07-0038-01), ERA CoBioTech
TRALAMINOL---ID: 64 (grant to LH), and MSCA-ITN-ETN-2020 CC-TOP---ID: 956931.

\section*{Acknowledgments}
The authors thank Lionel Nauton and Vincent Thery for molecular modeling
studies, Muriel Joly for  TK$_{\mathrm{gst}}$ variant production,
Martin Leremboure for GC-MS analysis, PhD students (Juliane
Abdoul-Zabar, Marion Lorilli\`{e}re, Nazim Ocal, Giuseppe Arbia, and
Camille Gadona), and postdocs (Ghina Ali, Thomas Moreau, Sandrine
Sorel, Romain Dumoulin, M\'{e}lanie L'Enfant, Hubert Casajus, and
Aur\'{e}lie Lagarde). 

\CDRGrant[FRIL]{DOS00494484/00}
\CDRGrant[PAR]{ID 1701105201-61617}
\CDRGrant[ANR]{ANR-09-BLAN-0424-CSD3}
\CDRGrant[ANR]{ANR-22-CE07-0038-01}
\CDRGrant[ERA CoBioTech TRALAMINOL]{64}
\CDRGrant[MSCA-ITN-ETN-2020 CC-TOP-ID]{956931}

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