Li Xujie, Zhang Meng, Lu Yu, Wu Ningyang, Chen Jian'gang, Ji Zhixia, Zhan Yangyang, Ma Xin, Chen Junyong, Cai Dongbo, Chen Shouwen
State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, 430062, China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China.
Synth Syst Biotechnol. 2023 Jun 7;8(3):378-385. doi: 10.1016/j.synbio.2023.05.002. eCollection 2023 Sep.
Owing to the feature of strong α-glucosidase inhibitory activity, 1-deoxynojirimycin (1-DNJ) has broad application prospects in areas of functional food, biomedicine, etc., and this research wants to construct an efficient strain for 1-DNJ production, basing on HZ-12. Firstly, using the temperature-sensitive shuttle plasmid T2 (2)-Ori, gene in phosphotransferase system (PTS) was weakened by homologous recombination, and non-PTS pathway was strengthened by deleting its repressor gene , and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold, reached 110.72 mg/L. Then, to increase precursor fructose-6-phosphate (F-6-P) supply, phosphofructokinase was weaken, fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement, moreover, regulator gene was deleted, 1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold. Subsequently, promoter of 1-DNJ synthetase cluster was optimized, as well as 5'-UTRs of downstream genes in synthetase cluster, and 1-DNJ produced by the final strain reached 478.62 mg/L. Last but not the least, 1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter, which was the highest yield of 1-DNJ reported to date. Taken together, our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis, this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.
由于具有较强的α-葡萄糖苷酶抑制活性，1-脱氧野尻霉素（1-DNJ）在功能食品、生物医药等领域具有广阔的应用前景，本研究欲以HZ-12为基础构建一株高效生产1-DNJ的菌株。首先，利用温度敏感型穿梭质粒T2(2)-Ori，通过同源重组弱化磷酸转移酶系统（PTS）中的基因，并通过缺失其阻遏基因来强化非PTS途径，所得菌株HZ-S2的1-DNJ产量提高了4.27倍，达到110.72mg/L。然后，为增加前体果糖-6-磷酸（F-6-P）的供应，弱化磷酸果糖激酶，通过启动子替换强化果糖磷酸酶GlpX和6-磷酸葡萄糖异构酶Pgi，此外，缺失调控基因，1-DNJ产量进一步提高2.41倍，达到267.37mg/L。随后，对1-DNJ合成酶簇的启动子以及合成酶簇下游基因的5'-UTR进行优化，最终菌株产生的1-DNJ达到478.62mg/L。最后，在3L发酵罐中获得了1632.50mg/L的1-DNJ产量，这是迄今为止报道的1-DNJ的最高产量。综上所述，我们的结果表明代谢工程是1-DNJ合成的有效策略，本研究为基于1-DNJ的功能食品和药物的工业化奠定了基础。
