## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### Terpenoids
Specifically, two genes encoding nervyl diphosphate synthase 1 (NDPS1) (_Solanum lyopersicum_) and LS (_Agastache rugosa_) were codon-optimized and heterologously expressed in _Y. lipolytica_. To enhance limonene production, genes of the MVA pathway were overexpressed, either in different copies of the same gene or in combination, with the overexpression of _ERG12_ (mevalonate kinase) and _HMG1_ (3-hydroxy-3-methylglutaryl-coenzyme A reductase 1) improving limonene production (Figure 3). Finally, with an optimized pyruvate acid and dodecane concentration in their flask culture, a maximum limonene titer and content of 23.56 mg L\({}^{-1}\) and 1.36 mg g\({}^{-1}\) CDW were achieved in the final engineered strain Po1f-LN-051, showing an approximately 226-fold increase compared with the initial yield of 0.006 mg g\({}^{-1}\) CDW [118]. Using the engineered strain Po1f-LN-051, the introduction of an additional _LS_ gene copy, further increased limonene production. This engineered strain was used to further optimize production. Various carbon sources  
Figure 3: Terpenoids biosynthesis in _Y. lipolytica_. Enzyme abbreviations are: ERG10, acetyl-CoA C-acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMG1, HMG-CoA reductase; ERG12, mevalonate kinase; GPPS, geranyl diphosphate synthase; NDPS1, nervyl diphosphate synthase 1; LS, limonene synthase; ERG20, farnesyl diphosphate synthase; FS, farresene synthase; ERG9, squalene synthase; ERG1, squalene monooxygenase; LUP1, lupeol synthase; CYP16A12, cytochrome P450 monooxygenase; CPR, cytochrome P450 reductase. Abbreviations of metabolites are: HMG-CoA, 3-Hydroxy-3-methylglutaryl-coenzyme A; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl diphosphate; NPP, nervyl diphosphate; FPP, farnesyl diphosphate. Green arrows indicate gene overexpression as discussed in this study. were tested, with glycerol leading to the highest limonene production and citrate selected as an auxiliary carbon source. In fed-batch fermentation with an optimized medium, the engineered strain produced 165.3 mg L\({}^{-1}\) limonene, which corresponds to the highest yield to date for the production of limonene in _Y. lipolytica_[119]. Glycerol was also used to produce betulinic acid by _Y. lipolytica_, a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug. In this case, the heterologous biosynthesis of betulinic acid was achieved by co-expressing lupeol synthase _opAtLIP1_, NADPH-cytochrome P450 monooxygenase _opCYP16A12_, and NADPH-cytochrome P450 reductase _opAtCPR1_ (Figure 3). The engineered _Y. lipolytica_ produced 0.32 mg L\({}^{-1}\) betulinic acid. P450 enzyme fusion and overexpression of key genes from the upstream MVA pathway further increased production to 9.41 mg L\({}^{-1}\). Using glycerol further enhanced the production to 16.98 mg L\({}^{-1}\), 1.8-fold higher than that obtained with glucose. 26.53 mg L\({}^{-1}\) betulinic acid was obtained with 40 g L\({}^{-1}\) glycerol as the sole carbon source, which was comparable to the titer obtained from _S. cerevisiae_, with 50 g L\({}^{-1}\) glucose in shake flask cultivation. The authors stated that glycerol improved betulinic acid titer by increasing the expression of key genes in the MVA pathway and by increasing the supply of acetylCoA [120].