Genome Shuffling Of Bacillus Amyloliquefaciens ES-2-4for Improving Lipopeptide Yield And Differential Proteomics Analysis In Its Mutant Strain

Bacillus strains produce many kinds of bioactive lipopeptides synthesized nonribosomally by a large multifunctional enzyme complex. Of these, the lipopeptide surfactin is well characterized at the genetic level. Surfactin is biosynthesized by three NRPSs, SrfA-C; the thioesterase/acyltransferase enzyme SrfD stimulates the initiation of this process. Surfactin is an extraordinarily powerful biosurfactant that is known to decrease the surface tension of water; it exerts a detergent-like action on biological membranes, and is distinguished by its exceptional emulsifying, foaming, antiviral and anti-mycoplasma activities. Surfactin has a great number of potential applications in plant disease biocontrol and biomedicine. Moreover, lipopeptide can be widely used in the food, cosmetic industries and for enhanced oil recovery and for the bioremediation of oil-contaminated sites. However, the production of antimicrobial peptides in Bacillus is generally lower than1.0g/L, some even below0.1g/L, thereby it is particularly important to improve the antimicrobial peptide production. There have been many attempts to increase lipopeptide production, but almost all of them have focused on traditional mutagenesis, fermentation optimization, isolation and purification, or on the regulation of lipopeptide synthesis using genetic engineering methods. Although rational methods and global techniques have been successfully applied to strain improvement, the need to engineer more complex phenotypes requires a more combinatorial approach.Bacillus amyloliquefaciens ES-2was an endophytic bacterium isolated from the Chinese medicinal plant Scutellaria baicalensis Georgi by Sun Lijun in our laboratory, which strongly inhibited plant pathogens, foodborne pathogens, spoilage bacteria and fungi. B. amyloliquefaciens ES-2-4was a high-yield mutant strain obtained with N+ion beam implantation. The concentration of the lipopeptides in fermentation broth increased by 15.2%compared to ES-2. In this study, the strain ES-2-4was determined for the initial strain. The technology of genome shuffling has been used as a novel whole-genome engineering approach for the rapid improvement of antimicrobial lipopeptide yield. Comparative RT-PCR and proteomic analysis were conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. The detailed works were described as following:1. Make the diversity of phenotype in parental library by UV, NTG and N+ion implantation mutagenesis. To start the genome shuffling process, parent strain library should be constructed in the first place. The initial strain was firstly engineered to generate more genotypes, and then the strains of interest were collected to form the parental library for the next step of recursive protoplast fusion. In order to make the diversity of phenotype in parental library, ES-2-4cells were mutagenized with either nitrosoguanidine, ultraviolet irradiation, or ion implantation (implantation sources were produced by an ion-beam bioengineering instrument devised by Chinese Academy of Sciences, Institute of plasma physics) and then spread on PDA agar plates. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. The mutation conditions were as follows, UV treatment:UV for20W,15cm,60s; NTG treatment:0.4mg/ml NTG,37℃for30min; N+ion implantation treatment:10kev of energy,10s of time. The six mutant strains with high yield, namely UV89、UV101、N114、N32、Ni49、Ni5, were obtained by UV、NTG and N+ion implantation mutagenesis, respectively. These six mutants showed small increases, from40.5to45.8mg/L in the production of surfactin. In addition, their high producing capacity was stable and was maintained after at least10transfers in shake-flasks. Consequently, these six mutants were used as the starting population for genome shuffling.2. The conditions of Bacillus amyloliquefaciens ES-2-4protoplast preparation, regeneration, inactivation and fusion were studied. The optimized conditions for protoplast preparation and regeneration were as follows:cell culture for4h, lysozyme concentration of0.2mg/mL, incubation at37℃for15min to allow cell wall lysis; The PDA as a basis for the regeneration medium,0.6mol/L NaCl as medium osmotic stabilizer, and the regeneration rate of up to22.7%. An equal number of protoplasts from different populations were mixed and then divided equally into two fractions. One fraction was inactivated with UV for60min, and the other was heat treated at100℃for30min. Both inactivated protoplasts were fused by PEG6000at concentration of40%, pH9.0and37℃for12min. The fusion rate was up to1.04×10"5.3. A surfactin high-yield recombinant was obtained by genome shuffling. This study was undertaken to enhance the yield of surfactin produced by Bacillus amyloliquefaciens ES-2-4using genome shuffling. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant F2-38strain that exhibited3.5-and10.3-fold increases in surfactin production in1L shake flask and19L fermenter respectively, was obtained.4. The surfactin synthetase gene srfA expression in the recombinant was quantitated by FQ RT-PCR. We focused on the relative quantification of target gene transcript in comparision to a reference gene transcript.16S rDNA was selected as the housekeeping gene, the surfactin synthetase gene srfA gene is the gene of interest. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. The normalized expression was calculated by averaging three CT values for the reference gene (16S rDNA) and for the gene of interest (srfA). Delta CT (threshold cycle) relative quantitation analysis revealed that surfactin synthetase gene (srfA) expression at the transcriptional level in the F2-38strain was15.7-fold greater than in the ES-2-4. It is consistent with the increase in production of antibacterial lipopetide.5. The2-DE protocol was optimized.2-DE is one of the key techniques used in proteomic studies and, production of a high quality2-DE image is crucial for analysis of the whole-cell proteins. In this experiment, an optimized protocol was developed paying special attention to the whole-cell protein analyses of Bacillus amyloliquefaciens based on2-DE. The optimization included sample preparation, sample loading, gel staining, IPG strips of pH scope, electrophoresis conditions, protein spots resolution and reproducibility. The optimized conditions are as follows:stained by silver staining; strips pH value of4-7;200μg sample loading of24cm strip. The results indicated that, use of rehydration solution containing7M urea and2M thiourea, in combination with low-voltage desalination in isoelectric focus (IEF) gave rise to satisfactory2-DE images. The maximal detectable protein spots number were estimated to be more than1000per gel under silver staining. This protocol may lay the foundation for2-DE studies in our laboratory.6. The differentially expressed proteins in the recombinant were studied. The proteome was separated by2-DE and analysed by MS. In the shuffled strain FMB38,51differentially expressed protein spots with higher than2-fold spot density were detected in gel image comparison.46protein spots were detectable by silver staining and sampled for further MS analysis. The results showed that46proteins were successfully identified by MALDI-TOF-MS, among which3proteins were transcription regulation proteins concerned with lipopeptide synthesis previously reported, the abundance change of ComA and DegU in our results are consistent with others work, but that of CodY is in contrast. According to COG function category and related references, these proteins may approximately be classified into several functional categories such as, proteins involved in metabolism, energy production and conversion, cell division and chromosome partitioning, translation, ribosomal structure and biogenesis, DNA replication, cell motility and secretion, posttranslational modification, general function prediction, etc. The analysis also elucidated that84.8%of proteins detected are located in cytoplasm.