## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### TCA Cycle Intermediates
Recently, [PERSON] et al., characterized the _Y. lipolytica_ strains _Asdh5_ (PGC01003) and _Asdh5 Anch1_ (PGC202, additional deletion of acetyl-CoA hydrolase 1 gene to avoid acetate synthesis as a co-product) and compared them to the wild type strain W29 in defined medium. By performing fed-batch cultivation with glycerol and under nitrogen deficiency conditions, they investigated how carbon flux can be rerouted toward SA synthesis. The _Asdh5_ produced 19 g L\({}^{-1}\) SA with an overall yield of 0.23 g g\({}^{-1}\) and an overall productivity of 0.23 g L\({}^{-1}\) h\({}^{-1}\), while the _Asdh5 Anch1_ produced 33 g L\({}^{-1}\) with an overall yield of 0.12 g g\({}^{-1}\) and a productivity of 0.57 g L\({}^{-1}\) h\({}^{-1}\)[127]. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Strain** & \begin{tabular}{c} **Carbon** \\ **Source 1** \\ \end{tabular} & **Product** & **Engineering 2** & **Strategy** & **Set-Up 3** & \begin{tabular}{c} **Data from** \\ **[Ref.]** \\ \end{tabular} \\ \hline \multirow{4}{*}{\begin{tabular}{c} VKM \\ Y-2378 \\ \end{tabular} } & \multirow{4}{*}{CG} & 41 g L\({}^{-1}\) pyruvate, _a_-ketoglutate & \multirow{4}{*}{SE; ME; PE} & 33 taxonomically different & \multirow{4}{*}{B} & \multirow{4}{*}{[78]} \\  & & & yeast strains; thiamine and & & & \\  & & & biotin concentration; & & & \\  & & & & nitrogen source; & & & \\  & & & & temperature, aeration, and & & & \\  & & & & glycerol concentration; & & & \\  & & & pH 4.5 & & & & \\ \hline \multirow{2}{*}{Po1f} & \multirow{2}{*}{CG} & \multirow{2}{*}{160 g L\({}^{-1}\) succinic acid} & \multirow{2}{*}{SE; PE} & \begin{tabular}{c} Inactivation of \(SDH5\); \\ effect of pH, oxygen \\ supply; pH 6.0 \\ \end{tabular} & \multirow{2}{*}{[124]} \\  & & & 81.11 g L\({}^{-1}\) citric acid; 24.90 g L\({}^{-1}\) & & ME & \
\begin{tabular}{c} Optimization of the carbon \\ and nitrogen source; pH \\ 4.0 \\ \end{tabular} & \multirow{2}{*}{B} & \multirow{2}{*}{[72]} \\  & & & & Deletion of _Ylach_; & & \\  & & & & overexpression of & \\  & & & heterologous _ScPCK_ from & & \\  & & & acid & _S. cerevisiae_ and native & \multirow{2}{*}{B} & \multirow{2}{*}{[126]} \\  & & & & _YlSCS2_; fed-batch & & \\  & & & fermentation; pH not & & \\  & & & regulated & & \\ \hline \multirow{2}{*}{Po1f} & \multirow{2}{*}{PG} & \multirow{2}{*}{45.5 g L\({}^{-1}\) succinic acid} & \multirow{2}{*}{SE; ME} & Ts-mutants with blocked & \multirow{2}{*}{SF} & \multirow{2}{*}{[123]} \\  & & & & SDH activity; medium & & \\  & & & buffering; CaCO\({}_{3}\); pH \(<\) 3.5 & & & \\ \hline \multirow{2}{*}{WSH-Z06} & \multirow{2}{*}{PG} & 66.2 g L\({}^{-1}\) \(a\)-ketoglutate acid & ME; PE & Two-stage pH control & \multirow{2}{*}{B} & \multirow{2}{*}{[128]} \\  & & & & strategy; constant feeding; & & \\  & & & pH 3.0 & & & \\ \hline \multirow{2}{*}{374/4} & \multirow{2}{*}{PG} & 8.2 g L\({}^{-1}\) pyruvate acid & \multirow{2}{*}{SS; ME} &
\begin{tabular}{c} Thiamine-auxotrophic \\ strain; thiamine \\ concentration; periodical \\ addition of glycerol; pH 4.5 \\ \end{tabular} & \multirow{2}{*}{B} & \multirow{2}{*}{[129]} \\  & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Engineering strategies (strain, medium, process) and cultivation systems using pure glycerol as carbon source for the production of organic acids by _Y. lipolytica_. Glycerol was also exploited for _a_-KG production, a valuable industrial compound with various medical, pharmaceutical, food and feed, and chemical synthesis applications. The biotechnological synthesis of _a_-KG is particularly attractive not only due to its value (15-20 $/kg in comparison to citric acid 0.6 $/kg), but also due to tedious production and downstream processes [130].