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

Poly-gamma-glutamic acid (gamma-PGA) is a biocompatible and biodegradable polypeptide with wide-ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce gamma-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 gamma-PGA production by LL3. In this study, the gamma-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) gamma-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 gamma-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 gamma-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 gamma-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 gamma-PGA production in B. amyloliquefaciens fermentation.