Isoprene,　as　a　versatile　bulk　chemical,　has　wide　industrial　applications.　Here,　we　attempted　to　improve　isoprene　biosynthesis　in　Saccharomyces　cerevisiae　by　simultaneous　strengthening　of　precursor　supply　and　conversion　via　a　combination　of　pathway　compartmentation　and　protein　engineering.　At　first,　a　superior　isoprene　synthase　mutant　ISPSLN　was　created　by　saturation　mutagenesis,　leading　to　almost　4-fold　improvement　in　isoprene　production.　Subsequent　introduction　of　ISPSLN　to　strains　with　strengthened　precursor　supply　in　either　cytoplasm　or　mitochondria　implied　an　imperfect　match　between　the　synthesis　and　conversion　of　the　isopentenyl　pyrophosphate　(IPP)/dimethylallyl　diphosphate　(DMAPP)　pool.　To　reconstruct　metabolic　balance　between　the　upstream　and　downstream　flux,　additional　copies　of　diphosphomevalonate　decarboxylase　gene　(MVD1)　and　isopentenyl-diphosphate　delta-isomerase　gene　(IDI1)　were　introduced　into　the　cytoplasmic　and　mitochondrial　engineered　strains.　Finally,　the　diploid　strain　created　by　mating　the　above　haploid　strains　produced　11.9　g/L　of　isoprene,　the　highest　ever　reported　in　eukaryotic　cells.　©　2018　American　Chemical　Society.