Online measurement of the viscosity in shake flasks enables monitoring of γ-PGA production in depolymerase knockout mutants of Bacillus subtilis with the phosphate-starvation inducible promoter Ppst.

Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (Viscosity Monitoring Online System) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the Ppst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The Ppst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in gγ-PGA /gglucose of 190% could be achieved with the triple knockout mutant compared to the Ppst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production.

Poly-γ-glutamic acid production by Bacillus subtilis 168 using glucose as the sole carbon source: A metabolomic analysis.

The industrially relevant biopolymer poly-γ-glutamic acid (γ-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for γ-PGA synthesis were constructed. Efficient γ-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter Pveg or with the xylose-inducible promoter Pxyl. The carbon yield for γ-PGA increased by 129% to 0.131 C-mol C-mol-1 when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced γ-PGA demonstrated a time-dependent molecular weight of 1180-1850 kDa and a d-glutamic acid monomer content of 49-62%. To elucidate the consequences of γ-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in γ-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for γ-PGA synthesis from glucose.

Identification of Key Metabolites in Poly-γ-Glutamic Acid Production by Tuning γ-PGA Synthetase Expression.

Poly-γ-glutamic acid (γ-PGA) production is commonly achieved using glycerol, citrate, and L-glutamic acid as substrates. The constitutive expression of the γ-PGA synthetase enabled γ-PGA production with Bacillus subtilis from glucose only. The precursors for γ-PGA synthesis, D- and L-glutamate, are ubiquitous metabolites. Hence, the metabolic flux toward γ-PGA directly depends on the concentration and activity of the synthetase and thereby on its expression. To identify pathway bottlenecks and important metabolites that are highly correlated with γ-PGA production from glucose, we engineered B. subtilis strains with varying γ-PGA synthesis rates. To alter the rate of γ-PGA synthesis, the expression level was controlled by two approaches: (1) Using promoter variants from the constitutive promoter P veg and (2) Varying induction strength of the xylose inducible promoter P xyl . The variation in the metabolism caused by γ-PGA production was investigated using metabolome analysis. The xylose-induction strategy revealed that the γ-PGA production rate increased the total fluxes through metabolism indicating a driven by demand adaption of the metabolism. Metabolic bottlenecks during γ-PGA from glucose were identified by generation of a model that correlates γ-PGA production rate with intracellular metabolite levels. The generated model indicates the correlation of certain metabolites such as phosphoenolpyruvate with γ-PGA production. The identified metabolites are targets for strain improvement to achieve high level γ-PGA production from glucose.

Tailor-made poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), which is produced by several Bacillus species, is a chiral biopolymer composed of D- and L-glutamate monomers and has various industrial applications. However, synthesized γ-PGA exhibits great structural diversity, and the structure must be controlled to broaden its industrial use. The biochemical pathways for γ-PGA production suggest that the polymer properties molecular weight (MW) and stereochemical composition are influenced by (1) the affinity of γ-PGA synthetase for the two alternative glutamate enantiomers and (2) glutamate racemase activity; hence, the availability of the monomers. In this study, we report tailor-made γ-PGA synthesis with B. subtilis by combining PGA synthetase and glutamate racemase genes from several Bacillus strains. The production of structurally diverse γ-PGA was thereby achieved. Depending on the PGA synthetase and glutamate racemase origins, the synthesized γ-PGA contained 3-60% D-glutamate. The exchange of PGA synthetase changed the MW from 40 to 8500 kDa. The results demonstrate the production of low-, medium-, and high-MW γ-PGA with the same microbial chassis.

Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered Bacillus subtilis.

The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D- and L-glutamic acid monomers, currently relies on L-glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus, offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h-1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D-xylose/ D-glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D-xylose/ D-glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.

Biochemical and biocatalytic characterization of 17 novel halohydrin dehalogenases.

Halohydrin dehalogenases are rare but catalytically remarkable enzymes since they are able to form novel C-C, C-O, C-N, or C-S bonds. Very recently, a motif-based sequence database mining approach resulted in the identification of 37 novel halohydrin dehalogenase enzymes, many of them exhibiting only low sequence similarity to previously known halohydrin dehalogenases. In an attempt to explore the biocatalytic potential of these newly identified enzymes, 17 representatives from all six phylogenetic subtypes were heterologously produced in Escherichia coli, purified and characterized to determine their substrate scopes in the dehalogenation and epoxide ring-opening reaction. Several enzymes with broad substrate spectra were identified exhibiting high activities towards a selection of typical substrates. Moreover, four halohydrin dehalogenases were found to be significantly more thermostable than the previously known HheC from Agrobacterium radiobacter AD1. Investigation of the enzymes' stereoselectivity in the dehalogenation of racemic 2-chloro-1-phenylethanol revealed that their stereopreference correlates with the phylogenetic placing of the enzymes in subtypes A through G. Furthermore, the biocatalytic potential of these novel halohydrin dehalogenases was investigated in the preparation of ethyl 4-cyano-3-hydroxybutyrate, a statin side-chain precursor. Though none of the active enzymes selectively formed the required (R)-enantiomer, several halohydrin dehalogenases were identified with significantly higher activity in the conversion compared to HheC, making them promising candidates for this industrially relevant reaction.