National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
Metab Eng. 2022 Sep;73:1-10. doi: 10.1016/j.ymben.2022.05.007. Epub 2022 May 25.
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.
丙二酸盐是一种平台化学品，已被用于合成许多有价值的化学化合物。在这里，通过丙二酰辅酶 A 途径对酿酒酵母进行了代谢工程改造，以生产丙二酸盐。为了构建将丙二酰辅酶 A 转化为丙二酸盐的关键步骤，将天然的线粒体 3-羟异丁酰辅酶 A 水解酶基因 EHD3 突变，使其靶向细胞质并获得丙二酰辅酶 A 水解酶活性。通过将丙二酰辅酶 A 结合位点 F121 突变为 I121 和活性位点 E124 突变为七个氨基酸（S/T/H/K/R/N/Q），实现了 Ehd3 的丙二酰辅酶 A 水解酶活性。我们发现，E124S 突变株的丙二酸盐产量最高，为 13.6mg/L。进一步探索了突变体 EHD3 和 ACC1**整合到 delta 序列位点的基因组整合，以增加其可靠表达。因此，构建了一种具有荧光检测、摇瓶发酵和摇瓶发酵工作流程的筛选方法，以有效地筛选高拷贝数的 delta 序列。通过筛选约 1500 个整合株，丙二酸盐的产量提高到 73.55mg/L，比质粒菌株提高了 4.4 倍。我们通过调节丙二酰辅酶 A 途径中的关键酶的表达来进一步工程化该菌株，以增加前体供应并抑制其竞争途径，最终工程菌株 LMA-16 在摇瓶中产生 187.25mg/L，比初始质粒表达菌株提高了 14 倍。最后，通过联合努力，在分批补料发酵中使丙二酸盐的产量提高到 1.62g/L。
