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\begin{document}

\title{\emph{In vivo} interpretation of model predicted inhibition in acrylate
pathway engineered \emph{Lactococcus lactis}}



\author[1]{Sowmiya Balasubramanian}%
\author[1]{Priyadharshini  Chandrasekran}%
\author[1]{Vijayalaksmi  Kandasamy}%
\author[2]{Mark Eiteman}%
\author[1]{Ramalingam Subramanian}%
\affil[1]{Anna University Chennai}%
\affil[2]{University of Georgia}%


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  \date{March 13, 2025}


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\begin{abstract}
In order to maximize the productivity of engineered metabolic pathway,
\emph{in silico} model is an established means to provide features of
enzyme reaction dynamics. In our previous study, \emph{E.coli}
engineered with acrylate pathway yielded low propionic acid titre. To
understand the bottleneck behind this low productivity, a kinetic model
was developed that incorporates the enzymatic reactions of the acrylate
pathway. The resulting model was capable of simulating the fluxes
reported under \emph{in vitro} studies with good agreement, suggesting
repression of propionyl-CoA transferase by carboxylate metabolites as
the main limiting factor for propionate production. Furthermore, the
predicted flux control coefficients of the pathway enzymes under steady
state conditions revealed that the control of flux is shared between
propionyl-CoA transferase and lactoyl-CoA dehydratase. Increase in
lactate concentration showed gradual decrease in flux control coefficients
of propionyl-CoA transferase that in turn confirmed the control exerted
by the carboxylate substrate. To interpret these in silico predictions
under in vivo system, an organized study was conducted with a Lactic
Acid Bacteria (LAB) strain engineered with acrylate pathway. Analysis
reported a decreased product formation rate on attainment of inhibitory
titre by suspected metabolites and supported the model.%
\end{abstract}%



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