Browsing by Subject "Molecular biology"

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Evaluation and method development for the biosynthesis of microbial lipopeptides by bacillus species
(2023) Vahidinasab, Maliheh; Hausmann, Rudolf
Microbial lipopeptides are secondary metabolites produced by bacteria and single-celled microorganisms. They consist of a cyclic or linear peptide chain linked to a lipid residue. Due to their high-foaming biosurfactant properties, they have various industrial applications such as in detergents, food emulsifiers, bioremediation, and enhanced oil recovery. Additionally, they possess other functional properties such as antifungal activity, making them an environmentally friendly alternative to synthetic fertilizers and fungicides. Bacillus species produce cyclic lipopeptides known for their potent antifungal activity, which makes them a potential source of bio-fungicides in agriculture. However, the production titer of wild-type Bacillus species does not meet industrial needs. Thereby, genetic modification of producer strains and bioprocess engineering can help increase the production of lipopeptides. Nevertheless, the regulation and basis of biosynthesis for Bacillus lipopeptides are still not completely understood, and ongoing research aims to enhance their production. In general, three main lipopeptide families, including surfactins, iturins, and fengycins are produced by different Bacillus species. Among these, surfactin as the strong biosurfactant is the most extensively studied lipopeptide produced by Bacillus species. The focus of this doctoral thesis was mainly to evaluate the biosynthesis of iturin and fengycin families, which are strong antimicrobial lipopeptides produced by Bacillus subtilis and Bacillus velezensis. This involved developing strains through genetic engineering and enhancing the lipopeptide titer by evaluating the cultivation medium. Initially, the entire genome of the bacteria used in this thesis was examined in terms of lipopeptide biosynthesis, and the structure and yield of the different produced lipopeptides were analyzed. Regarding the lipopeptide producer derivatives of the domesticated laboratory model strain B. subtilis 168 and B. subtilis 3NA, a spore deficient strain appropriate for bioreactor cultivation, surfactin is the lipopeptide with the highest yield, while plipastatin which is a member of fengycin family, is produced in lower quantities. In the present thesis, the biosynthesis of plipastatin by B. subtilis BMV9 as the lipopeptide producer derivative of strain 3NA was evaluated. The study aimed to convert BMV9 to a constitutive plipastatin mono-producer strain. In this sense, overexpressing plipastatin biosynthesis operon using the stronger constitutive Pveg promoter led to a five-fold increase in plipastatin production. Interestingly, it was observed that deletion of srfAA-AD operon in BMV9 and the constructed constitutive plipastatin producer strain has not improved plipastatin production. Therefore, it can be stated that presumably the biosynthesis of plipastatin may be positively influenced in a post-transcriptional manner by the surfactin synthetase or some of its subunits. However, the regulatory mechanism behind this effect remained unknown and requires further research. Another attempt to enhance the plipastatin biosynthesis in strain BMV9 was repairing the degQ expression. One main genome characterization of strains with B. subtilis 168 and 3NA background is that the pleiotropic degQ gene expression, which is known to have a positive effect on plipastatin biosynthesis, is silenced due to a mutation in the promoter area. However, while repair of degQ expression in BMV9 increased the plipastatin production, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) has not significantly increased the plipastatin yield. To further evaluate the impact of degQ expression on surfactin and plipastatin biosynthesis, two strains of B. subtilis were selected: JABs24, a lipopeptide producer derived from the 168 strain, and DSM10T, the wild-type strain expressing native degQ. The findings demonstrated that surfactin biosynthesis is negatively affected by DegQ-associated DegU regulation, while increased plipastatin biosynthesis is achieved in the presence of native degQ expression. In addition to production of lipopeptides, the DegU regulatory system also plays a role in the formation of secretory proteases. A comparison of extracellular protease activities between JABs24 and DSM10T showed that degQ expression led to DSM10T having five times higher protease activity than JABs24. Interestingly, production of extracellular proteases has not affected the stability of both plipastatin and surfactin during cultivation, suggesting that lipopeptides are less targeted by extracellular proteases. The identification of proficient wild-type strains is critical to the advancement of bio-fungicide in agriculture. Therefore, the subsequent approach of this thesis centered on the production of microbial lipopeptide by wild-type B. velezensis strains. Here, the lipopeptide productivity and antifungal ability of B. velezensis UTB96 was higher than B. velezensis FZB42, as a well-established strain for biocontrol of plant pathogens in agriculture. Furthermore, addition of certain amino acids stimulated lipopeptide production, and using a bioreactor system resulted in enhancement of lipopeptide production, especially iturin A by UTB96. Overall, the doctoral thesis evaluates the biosynthesis of antimicrobial lipopeptides produced by B. subtilis and B. velezensis. The study involves genetic engineering such as promoter exchange, deletion of genes involved in competing biosynthetic pathways and cultivation medium development with amino acid supplementation to enhance the lipopeptide titer. The thesis also identifies B. velezensis UTB96 as a promising candidate for further research to be used as a wild-type antifungal agent in agriculture.