Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation.

丙二酸是一种平台化学品，已被用于合成许多有价值的化合物。在这里，酿酒酵母经过代谢工程改造，可通过丙二酰辅酶A途径产生丙二酸。为了构建将丙二酰辅酶A转化为丙二酸的关键步骤，将天然线粒体3-羟基异丁酰辅酶A水解酶基因EHD3突变为靶向细胞质并获得丙二酰辅酶A水解酶活性。Ehd3 的丙二酰辅酶A 水解酶活性是通过将丙二酰辅酶A 结合位点F121 突变为I121 并将活性位点E124 突变为七个氨基酸（S/T/H/K/R/N/Q）来实现的。我们发现具有 E124S 突变的菌株具有最高的丙二酸滴度，为 13.6 mg/L。突变EHD3和ACC1 的基因组整合**对 delta 序列位点的进一步探索，以增加它们的可靠表达。据此，构建了荧光检测、摇管发酵、摇瓶发酵工作流程的筛选方法，以高效筛选高拷贝delta序列。筛选出~1500个整合菌株后，丙二酸滴度提高到73.55 mg/L，比游离菌株提高了4.4倍。我们通过调节丙二酰辅酶A途径中关键酶的表达来进一步改造该菌株，以改善前体供应并抑制其竞争途径，最终工程菌株LMA-16在烧瓶中产生187.25 mg/L，是14倍与初始附加型表达菌株。最后，双方的共同努力将补料分批发酵中的丙二酸滴度提高到 1.62 g/L。