Ectoine　is　a　valuable　compatible　solute　with　extensive　applications　in　bioengineering,　cosmetics,　medicine,　and　the　food　industry.　While　certain　halophilic　bacteria　can　naturally　produce　ectoine,　as　a　model　organism　for　biomanufacturing,　Escherichia　coli　offers　significant　advantages　to　be　engineered　for　potentially　high-level　ectoine　production.　However,　complex　metabolic　flux　distributions　and　byproduct　formation　present　bottlenecks　that　limit　ectoine　production　in　E.　coli.　In　this　study,　we　aimed　to　enhance　ectoine　production　in　E.　coli　BL21(DE3)　through　systematic　metabolic　engineering　strategies.　We　investigated　the　effects　of　the　ectABC　gene　cluster　sequence,　plasmid　copy　number,　and　key　gene　copy　number　on　ectoine　synthesis.　Using　the　original　ectABC　sequence　with　the　high-copy-number　plasmid　pRSFDuet-1　resulted　in　the　highest　level　of　ectoine　production.　Knocking　out　genes　encoding　homoserine　dehydrogenase　and　diaminopimelate　decarboxylase　reduced　competing　pathways,　further　increasing　ectoine　yield.　Overexpression　of　aspartate　semialdehyde　dehydrogenase,　aspartate　kinase　I　(thrA*),　aspartate　aminotransferase,　and　aspartate　ammonia-lyase　(aspA)　was　performed,　and　optimal　gene　copy　numbers　were　determined.　When　the　copy　numbers　of　thrA*　and　aspA　were　both　three,　ectoine　synthesis　improved,　reaching　1.91　g/L.　Enhancing　the　oxaloacetate　pool　by　overexpressing　phosphoenolpyruvate　carboxylase　(ppc)　or　introducing　pyruvate　carboxylase　(pyc)　from　Corynebacterium　glutamicum　further　increased　ectoine　production　to　4.99　g/L.　Balancing　NADPH　and　ATP　levels　through　cofactor　engineering　contributed　to　additional　production　improvements.　Combining　these　strain　engineering　strategies,　we　ultimately　constructed　strain　C24,　which　produced　35.33　g/L　ectoine　in　a　5　L　fermenter　with　a　glucose　conversion　rate　of　0.21　g/g.　These　results　demonstrate　that　targeted　metabolic　engineering　can　significantly　enhance　ectoine　production　in　E.　coli,　providing　a　foundation　for　industrial-scale　production.