Competence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.

Screening soil samples collected from a diverse range of slightly alkaline soil types, we have isolated 22 competent phosphate solubilizing bacteria (PSB). Three isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 hydrolyzed inorganic and organic phosphate compounds effectively. Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. In general, a close association was evident between phosphate solubilizing ability and growth rate which is an indicator of active metabolism. All three PSB were able to withstand temperature as high as 42°C, high concentration of NaCl upto 5% and a wide range of initial pH from 5 to 11 while hydrolyzing phosphate compounds actively. Such criteria make these isolates superior candidates for biofertilizers that are capable of utilizing both organic and mineral phosphate substrates to release absorbable phosphate ion for plants.

Strain engineering is often a method of choice towards increasing the yields of the biosurfactant surfactin which is naturally synthesized by many Bacillus spp., most notably Bacillus subtilis . In the current study, a genome reduced B. subtilis 168 strain lacking 10% of the genome was established and tested for its suitability to synthesize surfactin under aerobic and anaerobic conditions at 25 °C, 30 °C, 37 °C and 40 °C. This genome reduced strain was named IIG-Bs20-5-1 and lacks, amongst others, genes synthesizing the lipopeptide plipastatin, the antibiotic bacilysin, toxins and prophages, as well as genes involved in sporulation. Amongst all temperatures tested, 37 °C was overall superior. In comparison to the reference strain JABs24, a surfactin synthesizing variant of B. subtilis 168, strain IIG-Bs20-5-1 was both aerobically and anaerobically superior with respect to specific growth rates µ and yields Y X/S . However, in terms of surfactin production, strain JABs24 reached higher absolute concentrations with up to 1147.03 mg/L and 296.37 mg/L under aerobic and anaerobic conditions, respectively. Concomitant, strain JABs24 reached higher Y P/S and Y P/X . Here, an outstanding Y P/X of 1.541 g/g was obtained under anaerobic conditions at 37 °C. The current study indicates that the employed genome reduced strain IIG-Bs20-5-1 has several advantages over the strain JABs24 such as better conversion from glucose into biomass and higher growth rates. However, regarding surfactin synthesis and yields, the strain was overall inferior at the investigated temperatures and oxygen conditions. Further studies addressing process development and strain engineering should be performed combining the current observed advantages of the genome reduced strain to increase the surfactin yields and to construct a tailor-made genome reduced strain to realize the theoretically expected advantages of such genome reduced strains. Electronic supplementary material The online version of this article (10.1186/s13568-019-0806-5) contains supplementary material, which is available to authorized users.

Bacillus subtilis 3NA is a strain capable of reaching high cell densities. A surfactin producing sfp+ variant of this strain, named JABs32, was utilized in fed-batch cultivation processes. Both a glucose and an ammonia solution were fed to set a steady growth rate μ of 0.1 h-1. In this process, a cell dry weight of up to 88 g L-1 was reached after 38 h of cultivation, and surfactin titers of up to 26.5 g L-1 were detected in this high cell density fermentation process, achieving a YP/X value of 0.23 g g-1 as well as a qP/X of 0.007 g g-1 h-1. In sum, a 21-fold increase in surfactin titer was obtained compared with cultivations in shake flasks. In contrast to fed-batch operations using Bacillus subtilis JABs24, an sfp+ variant derived from B. subtilis 168, JABs32, reached an up to fourfold increase in surfactin titers using the same fed-batch protocol. Additionally, a two-stage feed process was established utilizing strain JABs32. Using an optimized mineral salt medium in this high cell density fermentation approach, after 31 h of cultivation, surfactin titers of 23.7 g L-1 were reached with a biomass concentration of 41.3 g L-1, thus achieving an enhanced YP/X value of 0.57 g g-1 as well as a qP/X of 0.018 g g-1 h-1. The mutation of spo0A locus and an elongation of AbrB in the strain utilized in combination with a high cell density fed-batch process represents a promising new route for future enhancements on surfactin production. Key points • Utilization of a sporulation deficient strain for fed-batch operations • High cell density process with Bacillus subtilis for lipopeptide production was established • High titer surfactin production capabilities confirm highly promising future platform strain

P lant biomass is the main carbon supply in the soil. The plant cell wall is a lignocellulosic complex consisting of hydrophilic polymers, i.e., cellulose, hemicellulose, and pectin, along with the hydrophobic aromatic heteropolymer lignin (1, 2). The integrity of the plant cell wall depends on its lignin content, which is crosslinked to polysaccharides by hydroxycinnamic acids bridges, mainly ferulate (3-5). These feruloylated polysaccharides, especially the ferulate-arabinoxylan complex, serve as initiation sites for lignification in the cell walls (6, 7). Lignin is degraded by lignolytic enzymes, including lignin peroxidase, manganese peroxidase, or laccase, into ␤-aryl ether, di-aryl ether, and biphenyl, which are further catabolized to other aromatic compounds, such as vanillin and vanillate. Likewise, ferulate bound through ester linkages to hemicellulose is released by esterases and degrades to vanillin and vanillate (for a review, see references 4, 5, 8, 9, and 10).Phenolic acids released by degradation of lignin, e.g., ferulate or p-coumarate, are toxic for many Gram-positive bacteria, such as Bacillus subtilis, at low pH. Thus, there is a system for phenolic acid stress response which detoxifies phenolic acid by decarboxylation and generation of vinyl phenol derivatives (11). In contrast to B. subtilis, Corynebacterium glutamicum utilizes ferulate, vanillin, and vanillate derived from lignin degradation as a carbon source. Generally, aromatic compounds are channeled via gentisate, catechol, protocatechuate, 1,2,4-trihydroxybenzene, or phenylacetyl coenzyme A (phenylacetyl-CoA) intermediates into the central carbon metabolism of C. glutamicum (12). Ferulate is catabolized via vanillin and vanillate as the intermediate products to protocatechuate (3,4-dihydroxybenzoate) (see Fig. S1 in the supplemental material) (13). Protocatechuate is further metabolized in the aerobic ␤-ketoadipate pathway and finally flows into the carbon and energy cycle (14). So far, only the genes for degradation of vanillate are identified in C. glutamicum. Conversion of vanillate to protocatechuate is carried out by vanillate O-demethylase (see Fig. S1) (13). The vanillate O-demethylase enzyme has two subunits, which are encoded by vanA (NCgl2300) and vanB (NCgl2301) (13,15). In addition to vanillate O-demethylase, the vanillate utilization system consists of a vanillate transporter encoded by vanK (NCgl2302), which forms the vanABK operon along with vanA and vanB (16). Transcription of the vanABK operon is regulated by VanR (NCgl2299), which forms a divergon with the vanABK operon (12).VanR belongs to the PadR-like transcriptional regulator family (17). The PadR-like protein family (Pfam accession no. PF03551) contains 26 reported structures deposited in the Protein Data Bank (http://www.rcsb.org/). Generally, PadR-like regulators play an important role in their bacterial host, especially concerning virulence and stress. Structurally, PadR-type regulators contain a highly conserved N-terminal winged helix-turn-helix (wHTH)