用于增强聚-γ-谷氨酸合成的解淀粉芽孢杆菌 LL3 的代谢工程。,Microbial Biotechnology

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用于增强聚-γ-谷氨酸合成的解淀粉芽孢杆菌 LL3 的代谢工程。
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2019-06-20 , DOI: 10.1111/1751-7915.13446
Weixia Gao _{1,

2} , Yulian He ₃ , Fang Zhang ₁ , Fengjie Zhao ₁ , Chao Huang ₁ , Yiting Zhang ₁ , Qiang Zhao ₂ , Shufang Wang ₂ , Chao Yang ₁

Affiliation

聚-γ-谷氨酸（γ-PGA）是一种生物相容性和可生物降解的多肽，在食品、化妆品、医药、农业和废水处理方面有着广泛的应用。解淀粉芽孢杆菌LL3 可以从蔗糖中产生 γ-PGA，而蔗糖可以从甘蔗和甜菜中轻松获得。在我们之前的工作中，发现细胞内谷氨酸浓度低是LL3产生γ-PGA的限制因素。在这项研究中，菌株 LL3 的 γ-PGA 合成通过染色体工程其谷氨酸代谢相关网络得到增强。首先，通过删除fadR、lysC、aspB、pckA、proAB、rocG和gudB来部分阻断下游代谢途径。所得菌株NK-A6合成了4.84 g l ^-1 γ-PGA，与菌株LL3相比增加了31.5%。其次，在icd基因上游插入强启动子PC _2up_，生成菌株NK-A7，进一步导致γ-PGA滴度提高33.5%，达到6.46 g l ^-1。通过调节pgi和gndA的表达来提高NADPH水平。第三，进行代谢进化以产生菌株 NK-A9E，其显示出与菌株 NK-A7 相当的 γ-PGA 滴度。最后，分别从原始菌株NK-A7和NK-A9E中删除srf和itu操纵子。所得菌株NK-A11表现出最高的γ-PGA滴度（7.53 g l ^-1），与LL3相比提高了2.05倍。结果表明，本文描述的方法有效提高了解淀粉芽孢杆菌发酵中 γ-PGA 的产量。

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Metabolic engineering of Bacillus amyloliquefaciens LL3 for enhanced poly-γ-glutamic acid synthesis.

Poly‐γ‐glutamic acid (γ‐PGA) is a biocompatible and biodegradable polypeptide with wide‐ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce γ‐PGA from sucrose that can be obtained easily from sugarcane and sugar beet. In our previous work, it was found that low intracellular glutamate concentration was the limiting factor for γ‐PGA production by LL3. In this study, the γ‐PGA synthesis by strain LL3 was enhanced by chromosomally engineering its glutamate metabolism‐relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR, lysC, aspB, pckA, proAB, rocG and gudB. The resulting strain NK‐A6 synthesized 4.84 g l⁻¹ γ‐PGA, with a 31.5% increase compared with strain LL3. Second, a strong promoter P_C_2up was inserted into the upstream of icd gene, to generate strain NK‐A7, which further led to a 33.5% improvement in the γ‐PGA titre, achieving 6.46 g l⁻¹. The NADPH level was improved by regulating the expression of pgi and gndA. Third, metabolic evolution was carried out to generate strain NK‐A9E, which showed a comparable γ‐PGA titre with strain NK‐A7. Finally, the srf and itu operons were deleted respectively, from the original strains NK‐A7 and NK‐A9E. The resulting strain NK‐A11 exhibited the highest γ‐PGA titre (7.53 g l⁻¹), with a 2.05‐fold improvement compared with LL3. The results demonstrated that the approaches described here efficiently enhanced γ‐PGA production in B. amyloliquefaciens fermentation.

更新日期：2019-06-20

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