## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Polyols
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Strain** & **Carbon Source 1** & **Product** & **Engineering 2** & **Strategy** & **Set-Up 3** & **Data from [Ref.]** \\ \hline Wratislavia K1 & CG & 170 g L\({}^{-1}\) erythroid & SE; ME & \begin{tabular}{c} Acetate negative \\ mutant; effect of pH \\ (2.5–6.5); pH 3.0 \\ \end{tabular} & B & [99] \\ \hline A UV’1; A-15; Wratislavia K1 & CG & \begin{tabular}{c} 80 g L\({}^{-1}\) erythroid \\ (Wratislavia K1); \\ 27.6 g L\({}^{-1}\) \\ Mannitol (A UV’1, \\ A-15) \\ \end{tabular} & SS; ME & \
\begin{tabular}{c} Addition of NaCl; \\ pH 3.0 \\ \end{tabular} & B & [100] \\ \hline LFMB 19 & CG & 7.1 g L\({}^{-1}\) mannitol & SS &
\begin{tabular}{c} Eukaryotic microbial \\ strains (yeasts and \\ Zygomycetes molds); \\ pH 5.0–6.0 \\ \end{tabular} & SF & [101] \\ \hline
2,021,417 & CG & 150 g L\({}^{-1}\) erythroid & SE & \begin{tabular}{c} Overexpression of \\ _GUT1, GUT2,_ and \\ _TKL1; Deletion of \\ _EYD1;_ pH 3.0 \\ \end{tabular} & B & [102] \\ \hline Wratislavia K1 & PG & 132 g L\({}^{-1}\) erythroid & ME & \begin{tabular}{c} Concentrations of \\ ammonium sulfate, \\ monopolousinsium \\ phosphate, and sodium \\ chloride; C/N ratio; \\ pH 3.0 \\ \end{tabular} & B & [76] \\ \hline A101 & PG & \begin{tabular}{c} 63.9 g L\({}^{-1}\) citric ; \\ 93 g L\({}^{-1}\) citric ; \\ 78 g L\({}^{-1}\) erythroid \\ \end{tabular} & SE & \begin{tabular}{c} Co-overexpression of \\ _GUT1_ and _GUT2_; effect \\ of pH (3.0 or 6.0) \\ \end{tabular} & B & [27] \\ \hline MK1 & PG & \begin{tabular}{c} 58 g L\({}^{-1}\) erythroid; \\ 62.5 g L\({}^{-1}\) \\ erythritol \\ \end{tabular} & SE & \begin{tabular}{c} Overexpression of \\ _TKL1, TAL1, ZWF1,_ and \\ _GNDI_; pH 3.0 \\ \end{tabular} & SF; B & [103] \\ \hline PoId & PG & \begin{tabular}{c} 80.6 g L\({}^{-1}\) \\ erythroid \\ \end{tabular} & SE & \begin{tabular}{c} Overexpression of \\ _GUT1_ and _TKL1;_ \\ disruption of _EYKI;_ \\ pH 3.0 \\ \end{tabular} & B & [104] \\ \hline Wratislavia K1 & PG & \
\begin{tabular}{c} 201.2 g L\({}^{-1}\) \\ erythroid \\ \end{tabular} & ME; PE &
\begin{tabular}{c} Addition of vitamins \\ and nitrogen sources; \\ batch and fed-batch; \\ pH 3.0 \\ \end{tabular} & B & [105] \\ \hline \hline \end{tabular}  
* CG, crude glycerol; PG, pure glycerol. 2 SE, strain engineering; ME, medium engineering; PE, process engineering; SS, strain screening. 3 SF, Shake flask; B, Bioreactor.  
\end{table} 
Table 3: _Cont._of _Y. lipolytica_. In addition, the authors pointed out the need to elevate NAD(P)H pool via engineering (overexpression of PPP genes), as ER requires NAD(P)H to reduce erythrose into erythnitol [106]. Using low-cost feedstocks such as raw beet molasses and crude glycerol, [PERSON] et al., engineered _Y. lipolytica_ and evaluated erythroid production. They overexpressed the _S. cerevisiae_ SUC2 gene and the native _GUT1_ gene in the Wratislavia K1 strain to allow for (i) sucrose assimilation by the mutant strain (wild type strains lack invertase activity being unable to hydrolyze sucrose), and (ii) efficient glycerol utilization due to the _GUT1_ overexpression. By applying a step-wise fed-batch culture with molasses (60 g L\({}^{-1}\)) added at the beginning of the process for yeast growth, and crude glycerol (150 g L\({}^{-1}\)) added after 24 h of culture for polyol production, they reached a maximum polyol production of 101 \(\pm\) 4 g L\({}^{-1}\), with productivity 1.1 \(\pm\) 0.9 g L\({}^{-1}\) h and yield 0.67 \(\pm\) 0.2 g g\({}^{-1}\)[97]. Similarly, [PERSON] et al., enhanced glycerol assimilation by co-overexpressing _GUT1_ and _GUT2_, resulting in rapid erythroid and citric acid synthesis. The engineered strains were able to utilize 150 g L\({}^{-1}\) glycerol within 44-48 h in a 5 L bioreactor. Erythnitol productivity for _GUT1_ overexpression and co-expression of _GUT1_ and _GUT2_ was 24 and 35%, respectively, over the control strain.