Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis.

N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC-MS and NMR. KEY POINTS: • A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified. • The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum. • Synthesis of acyl amino acids was achieved in good yields.

Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans.

Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236T . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC3.5.1.14), designated SgAA, and an ε-lysine acylase (EC3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl amino acids at the Nα -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of Nε -acetyl-l-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine.

Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822T.

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822T (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H2 O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2 O2 , suggest it has potential for biotechnological applications.

New robust subtilisins from halotolerant and halophilic Bacillaceae.

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. KEY POINTS: • Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins. • Four new subtilisins were biochemically characterised in detail. • The four proteases show potential for future biotechnological applications.

Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis.

BACKGROUND: Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. RESULTS: We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl ß-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. CONCLUSION: Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.

Phylogenetic survey of the subtilase family and a data-mining-based search for new subtilisins from Bacillaceae.

The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.

A convenient ninhydrin assay in 96-well format for amino acid-releasing enzymes using an air-stable reagent.

An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates.

Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T.

Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T . The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0-12.0 and temperature 20-80 °C, optimally at pH 9.0-9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2 O2 for 1 h while stimulated at 1% (v/v) H2 O2 . Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.

Synthesis of α-hydroxy ketones and vicinal diols with the Bacillus licheniformis DSM 13T butane-2,3-diol dehydrogenase.

The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled.

Synthesis of α-hydroxy ketones and vicinal (R,R)-diols by Bacillus clausii DSM 8716T butanediol dehydrogenase.

α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716T (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-diketones to the corresponding HK and, in some cases, the reduction of the same to the corresponding 1,2-diols. Aliphatic diketones, like 2,3-pentanedione, 2,3-hexanedione, 5-methyl-2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione are well transformed. In addition, surprisingly alkyl phenyl dicarbonyls, like 2-hydroxy-1-phenylpropan-1-one and phenylglyoxal are accepted, whereas their derivatives with two phenyl groups are not substrates. Supplementation of Mn2+ (1 mM) increases BcBDH's activity in biotransformations. Furthermore, the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated.

Elucidation of auxotrophic deficiencies of Bacillus pumilus DSM 18097 to develop a defined minimal medium.

BACKGROUND: Culture media containing complex compounds like yeast extract or peptone show numerous disadvantages. The chemical composition of the complex compounds is prone to significant variations from batch to batch and quality control is difficult. Therefore, the use of chemically defined media receives more and more attention in commercial fermentations. This concept results in better reproducibility, it simplifies downstream processing of secreted products and enable rapid scale-up. Culturing bacteria with unknown auxotrophies in chemically defined media is challenging and often not possible without an extensive trial-and-error approach. In this study, a respiration activity monitoring system for shake flasks and its recent version for microtiter plates were used to clarify unknown auxotrophic deficiencies in the model organism Bacillus pumilus DSM 18097. RESULTS: Bacillus pumilus DSM 18097 was unable to grow in a mineral medium without the addition of complex compounds. Therefore, a rich chemically defined minimal medium was tested containing basically all vitamins, amino acids and nucleobases, which are essential ingredients of complex components. The strain was successfully cultivated in this medium. By monitoring of the respiration activity, nutrients were supplemented to and omitted from the rich chemically defined medium in a rational way, thus enabling a systematic and fast determination of the auxotrophic deficiencies. Experiments have shown that the investigated strain requires amino acids, especially cysteine or histidine and the vitamin biotin for growth. CONCLUSIONS: The introduced method allows an efficient and rapid identification of unknown auxotrophic deficiencies and can be used to develop a simple chemically defined tailor-made medium. B. pumilus DSM 18097 was chosen as a model organism to demonstrate the method. However, the method is generally suitable for a wide range of microorganisms. By combining a systematic combinatorial approach based on monitoring the respiration activity with cultivation in microtiter plates, high throughput experiments with high information content can be conducted. This approach facilitates media development, strain characterization and cultivation of fastidious microorganisms in chemically defined minimal media while simultaneously reducing the experimental effort.

Development and characterization of a field-effect biosensor for the detection of acetoin.

A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.

(R,R)-Butane-2,3-diol dehydrogenase from Bacillus clausii DSM 8716T: Cloning and expression of the bdhA-gene, and initial characterization of enzyme.

The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33-43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12U/mg and 23U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.

High-resolution proteome maps of Bacillus licheniformis cells growing in minimal medium.

Bacillus licheniformis is an important host for the industrial production of enzymes mainly because of its ability to secrete large amounts of protein. We analyzed the proteome of B. licheniformis cells growing in a minimal medium. Beside the cytosolic proteome, the membrane and the extracellular proteome were studied. We could identify 1470 proteins; 1168 proteins were classified as cytosolic proteins, 195 proteins with membrane-spanning domains were classified as membrane proteins, and 107 proteins, with either putative signals peptides or flagellin-like sequences, were classified as secreted proteins. The identified proteins were grouped into functional categories and used to reconstruct cellular functions and metabolic pathways of growing B. licheniformis cells. The largest group was proteins with functions in basic metabolic pathways such as carbon metabolism, amino acid and nucleotide synthesis and synthesis of fatty acids and cofactors. Many proteins detected were involved in DNA replication, transcription, and translation. Furthermore, a high number of proteins employed in the transport of a wide variety of compounds were found to be expressed in the cells. All MS data have been deposited in the ProteomeXchange with identifier PXD000791 (http://proteomecentral.proteomexchange.org/dataset/PXD000791).

Cell physiology of the biotechnological relevant bacterium Bacillus pumilus-an omics-based approach.

Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.

Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer.

BACKGROUND: Since volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis. RESULTS: To develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel's industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer. CONCLUSIONS: In this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the basis for a next generation production host, since the strain has still a large potential for further genetic engineering. The final amylase titer of 65% in reference to B. licheniformis protease titer suggests that the developed B. pumilus expression platform is also suitable for an efficient production of non-proteolytic enzymes reaching a final titer of several grams per liter without complex process modifications.

Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress.

Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.

Fermentation stage-dependent adaptations of Bacillus licheniformis during enzyme production.

BACKGROUND: Industrial fermentations can generally be described as dynamic biotransformation processes in which microorganisms convert energy rich substrates into a desired product. The knowledge of active physiological pathways, reflected by corresponding gene activities, allows the identification of beneficial or disadvantageous performances of the microbial host. Whole transcriptome RNA-Seq is a powerful tool to accomplish in-depth quantification of these gene activities, since the low background noise and the absence of an upper limit of quantification allow the detection of transcripts with high dynamic ranges. Such data enable the identification of potential bottlenecks and futile energetic cycles, which in turn can lead to targets for rational approaches to productivity improvement. Here we present an overview of the dynamics of gene activity during an industrial-oriented fermentation process with Bacillus licheniformis, an important industrial enzyme producer. Thereby, valuable insights which help to understand the complex interactions during such processes are provided. RESULTS: Whole transcriptome RNA-Seq has been performed to study the gene expression at five selected growth stages of an industrial-oriented protease production process employing a germination deficient derivative of B. licheniformis DSM13. Since a significant amount of genes in Bacillus strains are regulated posttranscriptionally, the generated data have been confirmed by 2D gel-based proteomics. Regulatory events affecting the coordinated activity of hundreds of genes have been analyzed. The data enabled the identification of genes involved in the adaptations to changing environmental conditions during the fermentation process. A special focus of the analyses was on genes contributing to central carbon metabolism, amino acid transport and metabolism, starvation and stress responses and protein secretion. Genes contributing to lantibiotics production and Tat-dependent protein secretion have been pointed out as potential optimization targets. CONCLUSIONS: The presented data give unprecedented insights into the complex adaptations of bacterial production strains to the changing physiological demands during an industrial-oriented fermentation. These are, to our knowledge, the first publicly available data that document quantifiable transcriptional responses of the commonly employed production strain B. licheniformis to changing conditions over the course of a typical fermentation process in such extensive depth.

Stress responses of the industrial workhorse Bacillus licheniformis to osmotic challenges.

The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.

RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation.

BACKGROUND: The production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. Regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. This complex network of genes is orchestrated by proteins as well as regulatory RNA elements. Here we present an RNA-Seq based study considering selected phases of an industry-oriented fermentation of Bacillus licheniformis. RESULTS: A detailed analysis of 20 strand-specific RNA-Seq datasets revealed a multitude of transcriptionally active genomic regions. 3314 RNA features encoded by such active loci have been identified and sorted into ten functional classes. The identified sequences include the expected RNA features like housekeeping sRNAs, metabolic riboswitches and RNA switches well known from studies on Bacillus subtilis as well as a multitude of completely new candidates for regulatory RNAs. An unexpectedly high number of 855 RNA features are encoded antisense to annotated protein and RNA genes, in addition to 461 independently transcribed small RNAs. These antisense transcripts contain molecules with a remarkable size range variation from 38 to 6348 base pairs in length. The genome of the type strain B. licheniformis DSM13 was completely reannotated using data obtained from RNA-Seq analyses and from public databases. CONCLUSION: The hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. The identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of Bacillus licheniformis strains.