Zhu Zhiwen, Fu Bing, Lu Jiajie, Wang Peize, Yan Chuyang, Guan Fuyao, Huang Jianying, Yu Ping
College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, People's Republic of China.
College of Forestry Science and Technology, Lishui Vocational and Technical College, Lishui, People's Republic of China.
Prep Biochem Biotechnol. 2025 Apr;55(4):446-456. doi: 10.1080/10826068.2024.2423644. Epub 2024 Nov 5.
5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in BL21 to enhance ALA production. The ALA synthase genes , and were overexpressed in BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered in the future.
5-氨基乙酰丙酸（ALA）是一种非蛋白质氨基酸，已在医学和农业领域广泛应用。本研究旨在改造BL21中ALA生物合成的C5途径，以提高ALA产量。将ALA合酶基因、和在BL21中过表达，导致ALA产量增加。小RNA RyhB也过表达，以下调ALA脱水酶的表达，减少ALA向下游生物转化为胆色素原。接下来，通过CRISPR-Cas9技术敲除基因，打开三羧酸循环，促进菌株的呼吸代谢，以减少血红素对ALA的反馈抑制。通过响应面实验优化工程菌株的发酵条件。在1升摇瓶中进行了ALA产量的时间进程分析。通过这些努力，工程菌株在1升摇瓶中的ALA产量达到2953毫克/升。本研究有助于未来通过工程化的进行ALA的工业化生产。
