Cost-competitive γ-PGA production from low costs feedstock using an engineered Bacillus subtilis lab strain

Poly-γ-glutamic acid (γ-PGA) represents one of the most promising biomaterial naturally secreted by microorganisms mainly belonging to the Bacillales order. This uncommon homo-polyamide is formed by D-/L- glutamic acid units polymerized by γ-amide linkages. The polymer, resistant to common proteases, is endowed with characteristics that are suitable for many biotechnological applications, e.g. as metal flocculant, drug carrier, food additive and many more. However it is crucial to improve the economic viability of its production for γ-PGA industrial exploitation. Nowadays valorization of agro-food wastes is also a crucial issue. This work aims at improving the cellulolytic capabilities of a B. subtilis strain to obtain γ-PGA exploiting the abundant and low-cost organic fraction present in rice straw as feedstock. Unfortunately rice straw is rich in silica which inhibits bacterial growth. However, an alkali treatment was established in which hemicelluloses are collected in the liquid fraction and silica can be extracted from the solid lignocellulosic part, concurrently enhancing cellulose bioavailability. The liquid hemicellulose-rich fraction and the remaining solid cellulose-rich part can then be used for bacterial fermentation. Preliminary results demonstrate that B. subtilis can grow on such a substrate as sole carbon source. Conveniently, the strain carrying the degU32(Hy) mutation, necessary for hyper-production of γ-PGA, shows more efficient hydrolysis of cellobiose and xylan than the wild type. To further improve the strain cellulolytic potential genome and transcriptome data analyses were performed to select endogenous genes coding for enzymes important for saccharification of lignocellulose matrixes that could be modified. Genes bglC and xynA (encoding an endo-1,3,(4)-beta-glucanase and an endo-1,4-beta-xylanase, respectively) were chosen as targets, since their expression is poor and limited to the transition phase. An allelic-exchange approach was set up to increase xynA and bglC expression levels by optimizing the endogenous promoters and translation signals as well as by inserting a promoter UP element. The cellulolytic properties and γ-PGA yield of the engineered strains will be presented and discussed

Spore-forming bacteria in soil cultivated with GM white poplars: isolation and characterization

The impact of transgenic white poplars (Populus alba L. cv. ‘Villafranca’) was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 106–2.5 × 108 CFU/g dry soil and of 104–5 × 108 CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 103–5 × 104 CFU/g dry soil and 102–2 × 105 CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide- resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil leve

Optimization of γ-PGA biosynthesis supported by synthetic biology and metabolic engineering strategies

Poly-γ-glutamate (γ-PGA) is a natural polymer composed by glutamic acid residues, synthesized by the pgs operon of Bacillus subtilis. γ-PGA has a wide range of applications as food, cosmetic and pharmaceutical additive. However, to increase its industrial attractiveness, it is necessary to cut production costs utilizing cost-competitive feedstocks for fermentation. A low-cost by-product that can be used as feedstock is raw glycerol, that accounts for 10% (w/w) of the total biodiesel production. To achieve cost-competitive γ-PGA production from glycerol a multifaceted approach has been set up that includes: 1) improvement of pgs expression; 2) accumulation of γ-PGA precursors by metabolic engineering; 3) enhancement of glycerol metabolism. 1) The strength of the pgs operon regulatory elements has been analysed both by a synthetic biology approach, exploiting the well-characterized expression operating unit (EOU) inserted in amyE, and by a classical in-locus transcriptional fusion. Results from the two settings will be compared. These data will be then used to finely tune pgs expression and optimize γ-PGA yield. To this end, an inducible pgs operon has been constructed. 2) A genome-scale metabolic model was used to identify suitable targets for enhancing central carbon pathway flux toward γ-PGA synthesis. The first two B. subtilis strains, engineered following this analysis, showed enhanced polymer production. Other target genes are currently under investigation. 3) B. subtilis tolerance to raw glycerol obtained from a biodiesel plant (from both vegetable and animal origin) was verified. Further investigations are underway to improve glycerol uptake and consumption

Integration of enzymatic data in <i>Bacillus subtilis</i> genome-scale metabolic model improves phenotype predictions and enables in silico design of poly-γ-glutamic acid production strains

Abstract Background Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. Results Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis, which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis. We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. Conclusions This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis

The molecular function of SwrA: an auxiliary factor modulating DegU transcriptional activity

We recently demonstrated that the main fla/che promoter PA(fla/che) can be bound by the phosphorylated form of DegU (DegU~P) with two opposite outcomes: complete repression if DegU~P alone is bound to PA(fla/che) DNA; transcriptional stimulation if DNA is bound by DegU~P complexed with SwrA. Thus SwrA, which is necessary for swarming motility, constitutes an auxiliary factor that modulates the transcriptional activity of the response regulator DegU turning it from a repressor into an activator of PA(fla/che). Evidences indicate that SwrA might modulate other DegU~P-regulated promoters. Also, we demonstrated that DegU32(Hy) is a mutant protein unable to functionally interact with SwrA at the fla/che promoter; the phenotype of degU32(Hy) strains differs from that of the wild type DegU~P and we suggest the use of degS200(Hy) mutant strains for studies aimed at analyzing the effect of the level of DegU phosphorylation. SwrA is coded by a gene containing a slippery poly-adenine tract that allows phase variations between a functional and a non-functional allelic state. In swrA+ cells (typically in undomesticated strains) fla/che transcription oscillates from the basal/medium level to the activated state that is required for swarming. When swrA is in the non-functional form (e.g. in the 168 laboratory strain) fla/che transcription can oscillate between a repressed state in which no flagella are made and a basal/medium level of transcription sufficient for a limited swimming motility. While in both swrA- and swrA+ strains oscillations depend on phosphorylation of DegU mediated by environmental stimuli, in swrA- cells the secondary fla/che promoter PD3(fla/che) plays an important role that might constitute the bistable switch acting on motility

Poly-gamma-glutamate production in Bacillus subtilis

Poly-gamma-glutamic acid (gamma-PGA) is an anionic polymer of increasing industrial interest, composed of thousands of glutamic acid residues linked by gamma-glutamyl bonds. Secretion of the polymer into the medium confers a mucoid colony morphology to the Bacillus producer strains grown on LB agar plates. Although Bacillus subtilis possesses the functional biosynthetic pgs operon, containing four genes, laboratory strains do not have the ability to produce the polymer because pgs transcription is not active. We dissected the genetic elements involved in the conversion of laboratory non-producer strains into gamma-PGA producers and established that the synergic action of two gene products is required. The co-presence of the wild-type swrAA allele, a gene involved in swarming motility, and the hyperphosphorylated form of the transcriptional factor degU, belonging to the two-component system degS/degU, is sufficient to drive pgs transcription and gamma-PGA production

Transcriptional autoregulation of swrAA, a gene involved in Bacillus subtilis motility

The swrAA gene, required for swarming migration in B. subtilis, is involved in transcriptional regulation of the fla/che operon which contains the genes necessary for flagellum biosynthesis and chemotaxis and the gene coding for the alternative sigma factor SigD. Nevertheless SwrAA does not bear resemblance to DNA binding proteins nor does it show any particular feature by in silico analysis. In order to gain insight into its biological role we studied its expression profile in relationship with swarming and swimming motility. We demonstrate that transcription of swrAA is driven by two promoters: a sigD-dependent promoter, active during growth in liquid LB medium, and a putative sigA-dependent promoter, triggered by the phosphorylated form of the two-component response regulator DegU and active during swarming migration. Our data indicate that DegU is necessary not only for swarming (2, 3), but also for swimming and this requirement is independent from swrAA transcription, since SwrAA over-expression does not restore motility in strains carrying a degU deletion, suggesting that DegU concurs with SwrAA to achieve complete motility in B. subtilis

Does SwrA affect DegU-mediated transcriptional activation of aprE?

Bacillus subtilis subtilisin E (AprE) is one of main industrial proteases. Expression of aprE is regulated by a complex network of activators and repressors that comprises AbrB, SinR, ScoC, CodY and DegU. The latter is a global regulator activated via phosphorylation mediated by its cognate sensor kinase DegS. It has been shown that DegU~P can physically interact with SwrA, a small protein essential for swarming motility. The cooperative effect of DegU~P and SwrA has been demonstrated on two promoters: PA(fla/che), which codes for the major structural components of the flagellum; and Ppgs, that encodes the genes for γ-PGA biosynthesis. On those promoters, SwrA appears to act as a modulator of DegU activity, promoting transcriptional activation. To date, no studies addressed the role of SwrA on the regulation exerted by DegU~P on PaprE. The aim of this work is to investigate whether SwrA is involved in modifying DegU activity also at the subtilisin promoter. To this end, the construct containing a GFP reporter gene under the control of PaprE, kindly provided by Jan-Willem Veening, was used to carry out quantitative and qualitative analyses of aprE expression. The construct was inserted in isogenic Bacillus strains, differing for the status of both the swrA allele (swrA+, swrA- and ΔswrA) and the degUS TCS (degUSwt, degU32Hy, degS200Hy). The PaprE transcriptional profile will be presented together with fluorescent microscopy data

Autoregulation of swrAA and Motility in Bacillus subtilis▿

We demonstrate that transcription of the gene swrAA, required for swarming migration in Bacillus subtilis, is driven by two promoters: a sigD-dependent promoter and a putative sigA-dependent promoter, which is inactive during growth in liquid Luria-Bertani medium and becomes active in the presence of the phosphorylated form of the response regulator DegU or on semisolid surfaces. Since sigD transcription is enhanced by SwrAA, this finding reveals that swrA expression is controlled by a positive feedback loop. We also demonstrate that the positive action of SwrAA in swimming and swarming motility is prevented in strains carrying a deletion of the two-component system degS-degU and that this effect is independent of swrAA transcription. Therefore, both DegU and SwrAA must be present to achieve full motility in B. subtilis