Abstract:

Gentamicin is a kind of aminoglycoside antibiotic, widely used to treat severe Gram-negative bacterial infections. As an important secondary metabolite produced by Micromonospora echinospora, its biosynthetic pathway has been studied for years and scientists have a clear understanding for the biosynthetic gene cluster. In order to increase the titer of gentamicin, this study used the industrial strain M. echinospora J1-020 to determine the gentamicin synthetic gene cluster and established a stable genetic manipulation method. On this basis, three promoters with strong (kasOp*), medium (rpsLp-cf ), and weak (ermE*) strengths were used to evaluate the optimal overexpression level of phosphotransferase GenP, and the corresponding attB/attP site integration mutant strains YC002, YC003, YC001 was then constructed. After shaking flask fermentation, the results showed that the titer of gentamicin C component of YC001, YC002 and YC003 strains were increased by 16.9 % [(1178±39)mg/L], 30.8 % [(1319±29)mg/L] and 18.8%[(1198±46)mg/L] respectively, compared with the original strain [(1008±57)mg/L]. At the same time, combined with the impurity content, it was determined that the medium-strength promoter has the best effect on controlling the overexpression of gene genP in the above three strains, so that the corresponding overexpression strain YC004 of the genP stably integrated in the genome was constructed through homologous recombination. After shaking flask fermentation, the results showed that the titer of gentamicin C component increased by 34.5 %[(1427±37)mg/L]. Then, using M. echinospora J1-020 as the chassis, the genQ knockout strain YC005 was constructed to produce the G418 as the single component. The results showed that the titer of G418 was 460 mg/L. Finally, the gene genP overexpression strain YC004 as the starting strain, in which genB4 and genK were knocked out, was used to construct a double knockout mutant YC007 in order to produce sisomicin as a single component. After shaking flask fermentation, the titer of sisomicin was 1046 mg/L. It is expected that overproduction strains of various aminoglycoside antibiotics can be readily constructed by rational metabolic engineering strategies in the industrial chassis.

Key words: gentamicin, G418, sisomicin, aminoglycoside antibiotics, Micromonospora echinospora

摘要：

庆大霉素是一种氨基糖苷类抗生素，在临床上广泛应用于治疗由革兰氏阴性菌引起的严重感染。它可由棘孢小单孢菌Micromonospora echinospora产生，生物合成途径清晰。为了提高庆大霉素的产量，本文以工业菌株M. echinospora J1-020为基础，确定庆大霉素合成基因簇信息，建立了稳定的遗传操作方法。在此基础上，使用强（kasOp*）、中（rpsLp-cf）、弱（ermE*）三种强度的启动子评估磷酸转移酶GenP的最适过表达水平，构建对应attB/attP位点整合突变株YC002、YC003、YC001。摇瓶发酵结果显示，YC001、YC002、YC003菌株的庆大霉素C组分的产量较原始菌株［（1008±57） mg/L］分别提高了16.9%［（1178±39） mg/L］、30.8 %［（1319±29） mg/L］和18.8 %［（1198±46） mg/L］；同时，结合杂质含量，在以上三株菌株中确定了中强度启动子控制genP过表达的效果最佳，以此构建对应的稳定整合在基因组上的genP过表达菌株YC004。使得庆大霉素C组分摇瓶发酵产量提高了34.5 %［（1427±37） mg/L］。此外，以工业菌株M. echinospora J1-020为底盘，构建genQ敲除菌株，获得了只产生G418单一组分的菌株YC005，其摇瓶发酵产量为460 mg/L。以YC004为出发菌株，依次敲除genB4、genK，获得了只产生西索米星单一组分的菌株YC007，其摇瓶发酵产量达1046 mg/L。综上，以该工业菌株M. echinospora J1-020为底盘，借助合理的代谢工程策略有望快速获得多种氨基糖苷类抗生素的高产菌株。

关键词: 庆大霉素, G418, 西索米星, 氨基糖苷类抗生素, 棘孢小单孢菌