Metabolic engineering ofBacillus amyloliquefaciensLL3 for enhanced poly‐γ‐glutamic acid synthesis

Abstract

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 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 PC2up 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.

Bioconversion of poly-γ-glutamic acid (γ-PGA) from fulvic acid powder produced from the wastewater of yeast molasses fermentation

“…We determined whether GJ11 could produce γ-PGA by a glutamate-independent manner. GJ11 was cultured in LB medium overnight, then inoculated in 50 mL of basic medium or the basic medium with a concentration gradient of glutamate (10,20,30,40, 50, 60 and 70 g/L, respectively) in a 250 mL ask and incubated at 37 o C and 180 rpm for 36 h. After the incubation, the samples in different broths were collected for determining γ-PGA production.…”

“…Detecting compatibility of GJ11 to FA powder GJ11 was cultured in LB medium overnight, then inoculated in 50 mL of concentration -gradient FA powder medium (1,5,10,20,40, 60 and 100 g/L, respectively) in a 250 mL ask at a ratio of 3% (v/v), for further culture at 37 o C and 180 rpm for 24 h. After the incubation, the samples in different broths were collected for counting the colony-forming unit (cfu) of GJ11.…”

“…Analysis of hypersensitive reaction (HR) induced by γ-PGA and its hydrolyzates γ-PGA was recovered from the GJ11 broth [20]. 20 µL of γ-PGA in gradient concentration (1, 5, 10 and 15 g/L, respectively) was used for injecting the tobacco leaves with an 1 mL syringe without needles.…”

“…poly-γ-glutamic acid (γ-PGA)). γ-PGA is a natural anionic polymer of D/L-glutamic acids linked together via amide bonds between the α-amino group and the γ-carboxylic acid group, resulting in numerous properties, such as holding water, biodegradability and non-toxicity [20,21]. It has been widely used in food, medicine, cosmetic, and agriculture nowadays [22].…”

Microbial synthesis of poly- γ -glutamic acid ( γ -PGA) with fulvic acid powder, the waste from yeast molasses fermentation

“…Many Bacillus species, such as B. subtilis, B. licheniformis, B. amyloliquefaciens, and B. velezensis are generally used as hosts for producing fermentation-products including poly-γ-glutamic acid (γ-PGA). γ-PGA is a natural anionic polymer of D/Lglutamic acids linked together via amide bonds between the α-amino group and the γ-carboxylic acid group, resulting in numerous properties, such as holding water, biodegradability and non-toxicity [20,21].…”