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.