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Summary  Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds.  Saccharomyces cerevisiae  is an attractive chassis for the native active MVA pathway. Here, the truncated form of  Enterococcus faecalis  MvaE with only 3‐Hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431‐fold and 9‐fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and β‐alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3‐4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl‐CoA supply and β‐alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3‐4‐CPS generated from expressing β‐caryophyllene synthase in SQ3‐4 produced 11.86 ± 0.09 mg l −1  β‐caryophyllene, while strain SQ3‐5 resulted from down‐regulation of  ERG1  in SQ3‐4 produced 408.88 ± 0.09 mg l −1  squalene in shake flask cultivations. Strain SQ3‐5 produced 4.94 g l −1  squalene in fed‐batch fermentation in cane molasses medium, indicating the promising potential for cost‐effective production of squalene.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β‐alanine metabolism