## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Polyols
Gene co-expression enhanced erythroid productivity from glycerol up to 1.08 g L\({}^{-1}\) h\({}^{-1}\) and also the production of citric acid from 4.4 to 64 g L\({}^{-1}\) (0.69 g L\({}^{-1}\) h\({}^{-1}\)) [27]. Using a mutant that overexpressed _GUT1_ and _TKL1_, and in which _EYKI_ encoding erythnulose kinase (erythnitol catabolism) was disrupted, [PERSON] et al., increased erythroid productivity 75% compared to the wild type. Furthermore,  
Figure 2: Biosynthetic pathway of erythroid in _Y. lipolytica_ using glycerol. The enzymes encoded by corresponding genes are listed as follows: G3 PP, G3P phosphatase; GK, glycerol kinase (_GUT1_); GPDH, G3P-dehydrogenase (_GUT2_); TIM, triose-phosphate isomerase (TP1); TK, transketolase (_TKL1_); G6 PD, glucose 6-phosphate dehydrogenase (_ZWT1_); 6 PGL, 6-phosphogluconolactone (_SOL_); 6 PGD, 6-phosphogluconate dehydrogenase (_GNDI_); RKI, ribose 5-phosphate isomerase (_RKII_); TAL, transaldolase (_TAL1_); E4 PP, erythrose 4-phosphate phosphatase; ER, erythrose reductase; EYD, erythroid dehydrogenase; EYK, erythnulose kinase. Abbreviations of metabolites are: F-1,6-P, fructose-1,6-bisphosphate; F-6-P, fructose-6-phosphate; 6-PGL, 6-phosphogluconolactone; 6-TGA, 6-phosphogluconate; Ribu-5-P, ribulose-5-phosphate; X-5-P, xylulose-5-phosphate, R-5-P, ribose-5-phosphate; GA-3-P, Glyceraldehyde 3-phosphate S-7-P, sedoheptulose-7-phosphate; E-4-P, erythrose-4-phosphate. The red arrows indicate deleted reactions while the green arrows gene overexpression as discussed in this study. the culturing time needed to achieve maximum concentration reduced by 40%. Due to _EYKI_ deletion, the strain was unable to consume the synthesized erythroid, further increasing the process efficiency [104]. On the other hand, [PERSON] et al., proposed a multiple gene integration strategy to efficiently improve the production of erythroid in _Y. lipolytica_ from glycerol. For that, seven genes which are involved in the erythroid synthesis pathway were individually evaluated, with the _TKL1_ and _TAL1_ genes playing important roles in enhancing erythroid production. The co-expression of four genes (_GUTTI_, _TPII_, _TKL1_, _TAL1_), together with the disruption of the _EYD1_ gene (encoding erythroid dehydrogenase), resulted in approximately 40 g L\({}^{-1}\) erythroid from glycerol. Further enhanced erythroid synthesis was achieved by overexpressing the _RKI1_ gene (encoding ribose 5-phosphate isomerase) and the _AMPD_ gene (encoding AMP deaminase), suggesting for the first time that these two genes also determine erythroid production in _Y. lipolytica_[107]. Osmophilic microorganisms in response to osmotic stress also produce erythroid [108]. Osmotic stress combined with heterologous expression of sugar alcohol phosphatase (_PYP_, YNL010W) from _S. cerevisiae_ also increased sugar alcohol production in _Y. lipolytica_. It was found that osmotic stress increased the expression of genes involved in glycerol catabolism and the PPP, while intracellular metabolites such as amino acids, sugar alcohols, and polyamines produced at higher levels. Overexpression of the heterologous _PYP_ gene increased both erythroid production and glycerol consumption, suggesting that this new enzyme creates an orthogonal route for increasing production. The expression of genes encoding the native glycerol kinase and transketolase further increased erythroid production. This strain was able to produce 27.5 \(\pm\) 0.7 g L\({}^{-1}\) and 58.8 \(\pm\) 1.68 g L\({}^{-1}\) erythroid from glycerol during batch and fed-batch growth in shake-flask experiments, respectively. Additionally, the glycerol utilization was increased by 2.5-fold. The engineered strain was also able to efficiently produce erythroid from crude glycerol [109].