# Biosynthesis Progress of High-Energy-Density Liquid Fuels Derived from Terpenes
## 4 Strategies for Constructing Efficient Microbial Cell Factories for Terepenes
### Optimizing Metabolic Pathways
#### 4.2.2 Optimization of the Upstream and Downstream Pathways  
In microbial hosts, IPP and DMAPP are synthesized from glycerol-3-phosphate (G3P), pyruvate acid and acetyl-CoA, which are produced through the glycolytic pathway. Enhancing the supplement of these precursors is an effective means for improving the production of terpen in host cells (Figure 2B). For the MEP pathway, the imbalance supply of G3P and pyruvate acid is a major bottleneck. [PERSON] et al. found that downregulating _gapA_ in _E. coli_ was more effective than overexpressing _ppsA_ for balancing G3P and pyruvate acid, and downregulating _gapA_ effectively increased the production of lycopene (97% higher than the starting strain) [131]. Researchers found that in _E. coli_, the combination of the Entner-Doudoroff pathway (EDP) and the Hexose Monophosphate Pathway (HMP) was beneficial for precursor generation and energy supply, and activation of the EDP and PPP pathways increased the yield of isoprene by 1.9-fold [132; 133]. For the MVA pathway, it is essential to increase the accumulation of intracellular acetyl-CoA for terpene synthesis. Common methods, including strengthening the endogenous acetyl-CoA synthesis pathway or constructing an exogenous acetyl-CoA synthesis pathway, were applied [134]. For example, [PERSON] et al. knocked out the malate synthase encoding gene _MLS1_ and the citrate synthase encoding gene _CIT2_ in _S. cerevisiae_, leading to a 38% increase in the production of limonene, reaching 889.54 mg/L [135]. [PERSON] et al. increased the production of \(\beta\)-elemene to 707.18 mg/L (2.7-fold increase) in _E. coli_ by enhancing the accumulation of acetyl-CoA through knocking out the competing pathways in central carbon metabolism [136]. In _S. cerevisiae_, the expression of exogenous ACL can convert citric acid in the tricarboxylic acid cycle (TCA) into cytoplasmic acetyl-CoA. [PERSON] et al. found that ACL from _Aspergillus nidulans_ showed the best activity in yeast after screening five different sources of ACL [137]. Our research group significantly increased the production of \(\gamma\)-bisabolene in _S. cerevisiae_ by expressing ACL from _Yarrowia lipolytica_[56]. In addition to precursor supply, cofactors such as NADPH and ATP are also crucial for the synthesis of terpen. The consumption of cofactors for the MEP and MVA pathways differs. For the MEP pathway, the synthesis of one molecule of IPP/DMAPP requires the consumption of three molecules of NAD(P)H, one molecule of ATP and one molecule of CTP. For the MVA pathway, the synthesis of one molecule of IPP/DMAPP requires the consumption of two molecules of NADPH and three molecules of ATP. Common methods used to increase intracellular NADPH accumulation include reducing intracellular NADPH consumption or increasing NADPH synthesis. In _S. cerevisiae_, the production of cubebol was increased by 85% by knocking out the NADPH-dependent glutamate dehydrogenase gene _GDH1_, albeit impacting cell growth [138]. [PERSON] et al. found that overexpression the NADH-dependent glutamate dehydrogenase gene _GDH2_ could compensate for the effects on cell growth caused by the absence of _GDH1_[138]. Moreover, overexpressing the NADH kinase _POS5_ to increase intracellular NADPH accumulation led to a 1.8-fold increase in patchouli alcohol production in _S. cerevisiae_[139]. In _Rhodobacter capsulatus_, knocking out two NADPH-consuming enzymes, \(\beta\)-hydroxybutyrate synthase and glutamate synthase, led to a 2.15-fold increase in the production of bisabolene (390.3 mg/L) [140]. In _E. coli_, introducing the NADH kinase _POS5_ from yeast and knocking out the NADPH-dependent aldehyde reductase _YjgB_ led to a 1.1-fold increase in the production of protoilludene (512.7 mg/L) [141]. In addition to NADPH, an adequate ATP supply is also crucial for increasing the production of terpen. [PERSON] et al. overexpressed the endogenous adenine phosphoribosyltransferase (APRT), resulting in a 9.4% increase in intracellular ATP content, and also improved the yield of \(\alpha\)-farnesene by 10.3% [142].