Metabolic engineering of Bacillus amyloliquefaciens LL 3 for enhanced poly‐γ‐glutamic acid synthesis

Summary 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 LL 3 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 LL 3. In this study, the γ‐ PGA synthesis by strain LL 3 was enhanced by chromosomally engineering its glutamate metabolism‐relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR , lysC , aspB , pckA , pro AB , rocG and gudB . The resulting strain NK ‐A6 synthesized 4.84 g l −1 γ‐ PGA , with a 31.5% increase compared with strain LL 3. 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 LL 3. The results demonstrated that the approaches described here efficiently enhanced γ‐ PGA production in B. amyloliquefaciens fermentation.