Metabolic engineering of <italic>Saccharomyces cerevisiae</italic> chassis

Saccharomyces cerevisiae acts as a bio-foundry for producing natural products. S. cerevisiae ’s intrinsic pathways highlight microbial cell factories to produce pharmaceutics, food additive, and fine chemicals. For high-titer, yield, and conversion of target compounds, the critical point is that fine-tuning and optimizing intracellular metabolic flux in the yeast cell. Acetyl-CoA is the fundamental precursor for central metabolism and heterologous pathway. In comparison, acetyl-CoA compartmentalizes in mitochondria, nucleus, peroxisome subcellular, and cytosol. Most of the pathway enzymes are expressed in S. cerevisiae cytosol. So, we summarized the fine-tuning strategies of acetyl-CoA synthesis in cytosol matrix. The new acetyl-CoA pathway (phosphoketolase, phosphotransacetylase, and acetaldehyde dehydrogenase, PK/PTA-ADA) shows a high conversion ratio from the carbon source. The PK/PTA-ADA pathway presents improved redox balance, limited ATP requirement, and reduced carbon loss to CO2. Moreover, the heterologous ATP-dependent citrate lyase precisely converts mitochondria metabolism into acetyl-CoA. Knock-out acetyl-CoA depletion pathways retain high acetyl-CoA titer in the cytosol. The continuous metabolic engineering on PK/PTA-ADA pathway is expected for higher acetyl-CoA titer. The Mevalonate pathway has been engineered to produce terpenoids in S. cerevisiae . The exogenous HMG-CoA synthase and HMG-CoA reductase boost mevalonate pathway from acetyl-CoA. Further, the diversified terpenoids are generated from C5 unite Isopentenyl diphosphate and its’ isomer dimethylallyl diphosphate. Intrinsic farnesyl diphosphate synthase (Erg20) shows both dimethylallyltransferase (geranyl diphosphate, C10 product) and geranyltransferase (farnesyl diphosphate, C15 product) activity. The F96W/N127W mutations in Erg20 caused steric hindrance for geranyl diphosphate substrate. So, geranyl diphosphate is accumulated to produce C10 terpenoids or meroterpenoids. Moreover, geranylgeranyl diphosphate synthase (CrtE) convert farnesyl diphosphate (C15) into geranylgeranyl diphosphate (C20) for carotene, lycopene, astaxanthin, and taxol, etc. Interestingly, the novel isopentenol utilization pathway has been constructed in E. coli and Y. lipolytica to supply IPP and IMAPP precursors from isopentanol. This alternative pathway could relieve the acetyl-CoA supply burden in the mevalonate pathway. Fatty acid synthase (FAS) produces fatty acid with specific carbon length and derivatives from acetyl-CoA and malonyl-CoA. Acetyl-CoA carboxylase (ACC1) catalyzes acetyl-CoA to produce malonyl-CoA, which is utilized as the carbon unite in fatty acid pathway. Overexpression of ACC1 and mitochondria located isoenzyme Hfa1 significantly improves fatty acid production. FAS in S. cerevisiae is an α6β6 heterodimer protein and these catalytic domains are engineered to produce specific fatty acids. Directed mutations in ketoacyl synthase (KS), acetyltransferase (AT), and malonyl/palmitoyl transferase (MPT) domains engage FAS to generate high-yield short/medium fatty acid (C6 and C8). Furthermore, fatty acid reductase and acyl-ACP reductase convert fatty acid or fatty acyl-ACP to fatty aldehydes, respectively. Aldehyde deformylating oxygenase produces alkanes from fatty aldehydes substrate. Significantly, fatty acid-derived bio hydrocarbons showed closets properties to petroleum fuel. We reviewed the metabolic strategies of acetyl-CoA synthesis, the novel mevalonate pathway and fatty acid synthesis. These strategies may be valuable for producing high-yield terpene and fatty acid derivatives in Saccharomyces cerevisiae .