## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Lipids
#### 3.1.3 Engineering Redox and Energy Equivalents  
Engineering redox and energy equivalents is also a strategy to increase lipogenesis in _Y. lipolytica_. NADPH is mainly generated via the PPP [25; 40; 95]. In addition, the MAE catalyzed decarboxylation of malate to pyruvate is proposed as another important NADPH generator. [PERSON] et al., however, did not observe any significant improvement in NADPH regeneration in _Y. lipolytica_ overexpressing the _MAE_ gene, suggesting the presence of alternative routes [46]. Given that NADPH can limit FA synthesis, it was suggested that capturing most of the electrons generated from substrate catabolism could possibly increase the FA conversion yield, and therefore increase substrate-to-product yields [85]. To achieve this goal, [PERSON] et al., employed the conversion of NADH to NADPH by engineering 13 strains of _Y. lipolytica_ with synthetic pathways that convert glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA. The three synthetic pathways tested were:  
1. The NADP\({}^{+}\)-dependent GPD (cofactor switch by using NADP\({}^{+}\)-dependent GPDs i.e., GapC from _Clostridium acetobutylicum_ and GPD1 from _Kluyveromyces lactis_);
2. The pyruvate/oxaloacetate/malate which that converts 1 mol NADH to 1 mol NADPH at a cost of 1 mol ATP (by using the NADP\({}^{+}\)-dependent malic enzyme MCE2 from _Mucor circinelloides_);
3. The non-oxidative glycolytic pathway which yields 3 mol acetyl-CoA from 1 mol glucose, bypassing the Embden-Meyerhof-Parnas (EMP) pathway (by co-expressing phosphoketolase from _Leuconostoc mesenteroides_ and phosphate acetyltransferase from _Clostridium kluyveri_). Their established quantitative model pinpointed the yield of the lipid pathway as a crucial factor that determines the overall process yield. The best-engineered strain achieved a productivity of 1.2 g L\({}^{-1}\) h\({}^{-1}\) and a process yield of 0.27 g fatty acid methyl esters/g glucose that is 25% improvement compared to previously engineered strains. Due to the decreased NADH oxidation by aerobic respiration, the oxygen requirements of the highest producer were reduced [85]. The work of [PERSON] et al., evaluated three potential NADPH sources that could contribute to fatty acid synthesis, i.e., the oxidative PPP, a cytosolic specific NADP\({}^{+}\) isocitrate dehydrogenase, and a NADP\({}^{+}\) specific glutamate dehydrogenase. As mentioned above, the overexpression of the only native malic enzyme isoform, MAE1, was shown to have no effect on the lipid synthesis in _Y. lipolytica_. It is suggested that in _Y. lipolytica_ the MAE1 may be specific mainly for NAD\({}^{+}\) and not NADP\({}^{+}\). _SOL3_ (6-hosphogluconolactone), catalyzing the intermediate step of the oxPPP, was the only gene of this group that significantly enhanced lipid synthesis when overexpressed. Specifically, _SOL3_ overexpression increased the lipid titer, content, and yield on glucose by 23%, 18%, and 27%, respectively, over the control strain. It should be noted, that in the oxPPP, SOL3 does not generate NADPH, but the reactions before and after generate, catalyzed by _ZWF1_ and _GND1_, respectively. When using acetate as carbon source, _SOL3_ overexpression significantly increased the lipid content (97% over control), but dramatically reduced growth rate; thus, yielding a lower overall titer. It seems that on acetate, _SOL3_ overexpression directs carbon flux into lipids synthesis and not into non-lipid biomass formation. Interestingly, the overexpression of oxPPP NADPH producing genes did not significantly increase lipid production [47]. The work of [PERSON] et al., studied how synergistic co-feeding of carbon sources which generate ATP and NADPH can advance bio-product synthesis, overcome undesirable substrate preferences and improve carbon reduction. Their study's concept was that the direct access of substrate mixtures to multiple pathways might optimize the biosynthetic requirements balancing carbon, ATP, and reducing agents.