Xanthan: enzymatic degradation and novel perspectives of applications.

The extracellular heteropolysaccharide xanthan, synthesized by bacteria of the genus Xanthomonas, is widely used as a thickening and stabilizing agent across the food, cosmetic, and pharmaceutical sectors. Expanding the scope of its application, current efforts target the use of xanthan to develop innovative functional materials and products, such as edible films, eco-friendly oil surfactants, and biocompatible composites for tissue engineering. Xanthan-derived oligosaccharides are useful as nutritional supplements and plant defense elicitors. Development and processing of such new functional materials and products often necessitate tuning of xanthan properties through targeted structural modification. This task can be effectively carried out with the help of xanthan-specific enzymes. However, the complex molecular structure and intricate conformational behavior of xanthan create problems with its enzymatic hydrolysis or modification. This review summarizes and analyzes data concerning xanthan-degrading enzymes originating from microorganisms and microbial consortia, with a particular focus on the dependence of enzymatic activity on the structure and conformation of xanthan. Through a comparative study of xanthan-degrading pathways found within various bacterial classes, different microbial enzyme systems for xanthan utilization have been identified. The characterization of these new enzymes opens new perspectives for modifying xanthan structure and developing innovative xanthan-based applications. KEY POINTS: • The structure and conformation of xanthan affect enzymatic degradation. • Microorganisms use diverse multienzyme systems for xanthan degradation. • Xanthan-specific enzymes can be used to develop xanthan variants for novel applications.

Meltome atlas-thermal proteome stability across the tree of life.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.

Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii.

Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high cellulose-producing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC. KEY POINTS: • BcsAB1 induces formation of microcrystalline cellulose fibers. • Modifications by BcsAB2 and BcsAB3 alter diameter of cellulose fibers. • Complex regulatory network of DGCs on cellulose pellicle formation and motility.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol.

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g gCDW-1 h-1 in synthetic minimal medium when glycerol was added. By means of a 13C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two 'respiratory' carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.

The Roles of the Various Cellulose Biosynthesis Operons in Komagataeibacter hansenii ATCC 23769.

Cellulose is the most abundant biopolymer on earth and offers versatile applicability in biotechnology. Bacterial cellulose, especially, is an attractive material because it represents pure microcrystalline cellulose. The cellulose synthase complex of acetic acid bacteria serves as a model for general studies on (bacterial) cellulose synthesis. The genome of Komagataeibacter hansenii ATCC 23769 encodes three cellulose synthase (CS) operons of different sizes and gene compositions. This implies the question of which role each of the three CS-encoding operons, bcsAB1, bcsAB2, and bcsAB3, plays in overall cellulose synthesis. Therefore, we constructed markerless deletions in K. hansenii ATCC 23769, yielding mutant strains that expressed only one of the three CSs. Apparently, BcsAB1 is the only CS that produces fibers of crystalline cellulose. The markerless deletion of bcsAB1 resulted in a nonfiber phenotype in scanning electron microscopy analysis. Expression of the other CSs resulted in a different, nonfibrous extracellular polymeric substance (nfEPS) structure wrapping the cells, which is proposed to contain acetylated cellulose. Transcription analysis revealed that all CSs were expressed continuously and that bcsAB2 showed a higher transcription level than bcsAB1. Moreover, we were able to link the expression of diguanylate cyclase B (dgcB) to cellulose production. IMPORTANCE Acetic acid bacteria form a massive biofilm called "mother of vinegar," which is built of cellulose fibers. Bacterial cellulose is an appealing biomaterial with manifold applications in biomedicine and biotechnology. Because most cellulose-producing acetic acid bacteria express several cellulose synthase operons, a deeper understanding of their contribution to the synthesis of modified forms of cellulose fibers within a natural biofilm is of special interest. For the first time, we were able to identify the contribution of each of the three cellulose synthases to cellulose formation in Komagataeibacter hansenii ATCC 23769 after a chromosomal clean deletion. Moreover, we were able to depict their roles in spatial composition of the biofilm. These findings might be applicable in the future for naturally modified biomaterials with novel properties.

Anaeropeptidivorans aminofermentans gen. nov., sp. nov., a mesophilic proteolytic salt-tolerant bacterium isolated from a laboratory-scale biogas fermenter, and emended description of Clostridium colinum.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Unusual substrate specificity in GH family 12: structure-function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris.

In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear ß-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris, purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides and had transglycosylase activity on (1,3)-ß-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a ß-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. KEY POINTS: • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-ß-linkages in (1,3;1,4)-ß-D-glucooligosaccharides.

Variimorphobacter saccharofermentans gen. nov., sp. nov., a new member of the family Lachnospiraceae, isolated from a maize-fed biogas fermenter.

Strain MD1T is an anaerobic, Gram-stain-negative bacterium isolated from a lab-scale biogas fermenter fed with maize silage. It has a rod-shaped morphology with peritrichously arranged appendages and forms long chains of cells and coccoid structures. The colonies of MD1T were white, circular, slightly convex and had a smooth rim. The isolate is mesophilic, displaying growth between 25 and 45 °C with an optimum at 40 °C. It grew at pH values of pH 6.7-8.2 (optimum, pH 7.1) and tolerated the addition of up to 1.5% (w/v) NaCl to the medium. The main cellular fatty acids of MD1T are C14:0 DMA and C16:0. Strain MD1T fermented xylose, arabinose, glucose, galactose, cellobiose, maltose, maltodextrin10, lactose starch, and xylan, producing mainly 2-propanol and acetic acid. The genome of the organism has a total length of 4163427 bp with a G+C content of 38.5 mol%. The two closest relatives to MD1T are Mobilitalea sibirica P3M-3T and Anaerotaenia torta FH052T with 96.44 or 95.8 % 16S rRNA gene sequence similarity and POCP values of 46.58 and 50.58%, respectively. As MD1T showed saccharolytic and xylanolytic properties, it may play an important role in the biogas fermentation process. Closely related variants of MD1T were also abundant in microbial communities involved in methanogenic fermentation. Based on morphological, phylogenetic and genomic data, the isolated strain can be considered as representing a novel genus in the family Lachnospiraceae, for which the name Variimorphobacter saccharofermentans gen. nov., sp. nov. (type strain MD1T=DSM 110715T=JCM 39125T) is proposed.

Anaerosphaera multitolerans sp. nov., a salt-tolerant member of the family Peptoniphilaceae isolated from a mesophilically operated biogas fermenter fed with maize silage.

In this work, we succeeded in the isolation of a novel species out of a mesophilically operated biogas fermenter fed with maize silage. Strains GS7-6-2T, GS-7K2 and GS-0K3 were isolated from three individual enrichment cultures. 16S rRNA gene sequence comparisons indicated that the isolates had 100 % sequence identity and were most closely related to Anaerosphaera amininiphila WN036T, with which they shared a 16S rRNA gene sequence similarity of 93.1 %. As a representative, strain GS7-6-2T was further characterized. Strain GS7-6-2T was mesophilic with its growth optimum at 30 °C and a pH range from pH 5.5 to 9.5 (optimum, pH 6.0-8.5). Cells were spherical and sometimes arranged into short chains. Growth was possible with up to 3.6 % (w/v) NaCl, but best without additional NaCl. Strain GS7-6-2T produced butyric acid and acetic acid as main fermentation products while growing on GS2 medium. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C16 : 1ω9c. The Gram-stain result was negative. The DNA G+C content was 32.8 mol%. Strain GS7-6-2T was able to ferment 16 (comprising four carbohydrates, five amino acids, four organic acids and three nucleotides) out of the 95 tested substrates. Due to the ecological, genetic and phenotypic differences from the most closely affiliated and validly named organism, A. amininiphila WN036T, the isolates represent a novel species within the genus Anaerosphaera, family Peptoniphilaceae, for which the name Anaerosphaera multitolerans sp. nov. is proposed. The type strain is GS7-6-2T (=DSM 107952T=CECT 9705T).

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation.

Butanol is a platform chemical that is utilized in a wide range of industrial products and is considered a suitable replacement or additive to liquid fuels. So far, it is mainly produced through petrochemical routes. Alternative production routes, for example through biorefinery, are under investigation but are currently not at a market competitive level. Possible alternatives, such as acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia are not market-ready to this day either, because of their low butanol titer and the high costs of feedstocks. Here, we analyzed wheat middlings and wheat red dog, two wheat milling byproducts available in large quantities, as substrates for clostridial ABE fermentation. We could identify ten strains that exhibited good butanol yields on wheat red dog. Two of the best ABE producing strains, Clostridium beijerinckii NCIMB 8052 and Clostridium diolis DSM 15410, were used to optimize a laboratory-scale fermentation process. In addition, enzymatic pretreatment of both milling byproducts significantly enhanced ABE production rates of the strains C. beijerinckii NCIMB 8052 and C. diolis DSM 15410. Finally, a profitability analysis was performed for small- to mid-scale ABE fermentation plants that utilize enzymatically pretreated wheat red dog as substrate. The estimations show that such a plant could be commercially successful.Key points• Wheat milling byproducts are suitable substrates for clostridial ABE fermentation.• Enzymatic pretreatment of wheat red dog and middlings increases ABE yield.• ABE fermentation plants using wheat red dog as substrate are economically viable. Graphical abstract.

Hungateiclostridium mesophilum sp. nov., a mesophilic, cellulolytic and spore-forming bacterium isolated from a biogas fermenter fed with maize silage.

In this work, the isolation and characterization of a novel anaerobic, mesophilic and cellulolytic bacterium is described. Comparative analysis of the almost-complete sequence of the 16S rRNA gene showed that the closest relatives were Hungateiclostridium straminisolvens CSK1T (97.53  %) and Hungateiclostridium thermocellum DSM 2360T (95.42  %). Due to physiological and phylogenetic differences from its closest relatives, a new species is proposed. Cells of N2K1T were observed to be rod-shaped, non-motile, spore-forming, Gram-stain-positive and able to adhere directly to cellulose fibre. Cellulolytic activity and optimal growth were observed at 45 °C and neutral pH (optimum, pH 7.5). Of all tested substrates, only filter paper (cellulose) and cellobiose were used for growth. Arabinose, fructose, glucose, lactose, mannitol, mannose, ribose, starch, sucrose, trehalose, xylan and xylose did not support growth. The main fermentation products were acetic acid and isopropanol. The major cellular fatty acids (>5 %) were C16 : 0iso, C16 : 0 DMA and C16 : 0. The type strain, N2K1T, was isolated from a mesophilically operated, lab-scale biogas fermenter fed with maize silage in Freising, Germany in 2017. The genome assembly of strain N2K1T is 4.04 Mbp with a DNA G+C content of 38.36 mol%. The name Hungateiclostridiummesophilum sp. nov. is proposed for the novel organism. Strain N2K1T (=DSM 107956T; =CECT 9704T) represents the type strain of Hungateiclostridiummesophilum sp. nov.

Ruminiclostridium herbifermentans sp. nov., a mesophilic and moderately thermophilic cellulolytic and xylanolytic bacterium isolated from a lab-scale biogas fermenter fed with maize silage.

An anaerobic bacterial strain, designated MA18T, was isolated from a laboratory-scale biogas fermenter fed with maize silage. Cells stained Gram-negative and performed Gram-negative in the KOH test. The peptidoglycan type was found to be A1y-meso-Dpm direct. The major cellular fatty acids were C14 : 0 iso, C15 : 0 iso, anteiso and iso DMA as well as a C16 unidentified fatty acid. Oxidase and catalase activities were absent. Cells were slightly curved rods, motile, formed spores and measured approximately 0.35 µm in diameter and 3.0-5.0 µm in length. When cultivated on GS2 agar with cellobiose, round, arched, shiny and slightly yellow-pigmented colonies were formed. The isolate was mesophilic to moderately thermophilic with a growth optimum between 40 and 48 °C. Furthermore, neutral pH values were preferred and up to 1.2 % (w/v) NaCl supplemented to the GS2 medium was tolerated. Producing mainly acetate and ethanol, MA18T fermented arabinose, cellobiose, crystalline and amorphous cellulose, ribose, and xylan. The genome of MA18T consists of 4 817 678 bp with a G+C content of 33.16 mol%. In the annotated protein sequences, cellulosomal components were detected. Phylogenetically, MA18T is most closely related to Ruminiclostridium sufflavum DSM 19573T (76.88 % average nucleotide identity of the whole genome sequence; 97.23 % 16S rRNA gene sequence similarity) and can be clustered into one clade with other species of the genus Ruminiclostridium, family Oscillospiraceae, class Clostridia. Based on morphological, physiological and genetic characteristics, this strain represents a novel species in the genus Ruminiclostridium. Therefore, the name Ruminiclostridium herbifermentans sp. nov. is proposed. The type strain is MA18T (=DSM 109966T=JCM 39124T).

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans.

Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L-1 h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L-1 h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX-1 in the steady state. The product concentration (54 g L-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Novel endo-(1,4)-ß-glucanase Bgh12A and xyloglucanase Xgh12B from Aspergillus cervinus belong to GH12 subgroup I and II, respectively.

In spite of intensive exploitation of aspergilli for the industrial production of carbohydrases, little is known about hydrolytic enzymes of fungi from the section Cervini. Novel glycoside hydrolases Bgh12A and Xgh12B from Aspergillus cervinus represent examples of divergent activities within one enzyme family and belong to the GH12 phylogenetic subgroup I (endo-(1,4)-ß-glucanases) and II (endo-xyloglucanases), respectively. The bgh12A and xgh12B genes were identified in the unsequenced genome of A. cervinus using primers designed for conservative regions of the corresponding subgroups and a genome walking approach. The recombinant enzymes were heterologously produced in Pichia pastoris, purified, and characterized. Bgh12A was an endo-(1,4)-ß-glucanase (EC 3.2.1.4) hydrolyzing the unbranched soluble ß-(1,4)-glucans and mixed linkage ß-(1,3;1,4)-D-glucans. Bgh12A exhibited maximum activity on barley ß-glucan (BBG), which amounted to 614 ± 30 U/mg of protein. The final products of BBG and lichenan hydrolysis were glucose, cellobiose, cellotriose, 4-O-ß-laminaribiosyl-glucose, and a range of higher mixed-linkage gluco-oligosaccharides. In contrast, the activity of endo-xyloglucanase Xgh12B (EC 3.2.1.151) was restricted to xyloglucan, with 542 ± 39 U/mg protein. The enzyme cleaved the (1,4)-ß-glycosidic bonds of the xyloglucan backbone at the unsubstituted glucose residues finally generating cellotetraose-based hepta-, octa, and nona-oligosaccharides. Bgh12A and Xgh12B had maximal activity at 55 °C, pH 5.0. At these conditions, the half-time of Xgh12B inactivation was 158 min, whereas the half-life of Bgh12A was 5 min. Recombinant P. pastoris strains produced up to 106 U/L of the target enzymes with at least 75% of recombinant protein in the total extracellular proteins. The Bgh12A and Xgh12B sequences show 43% identity. Strict differences in substrate specificity of Bgh12A and Xgh12B were in congruence with the presence of subgroup-specific structural loops and substrate-binding aromatic residues in the catalytic cleft of the enzymes. Individual composition of aromatic residues in the catalytic cleft defined variability in substrate selectivity within GH12 subgroups I and II.

Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol.

BACKGROUND: The genus Bacillus includes a great variety of species with potential applications in biotechnology. While species such as B. subtilis or B. licheniformis are well-known and used to provide various products at industrial scale, other Bacillus species are less characterized and are not yet used in commercial processes. One reason for this is the fact that genetic manipulation of new isolates is usually complicated with conventional techniques which have to be adapted to each new strain. Even in well-established strains, the available transformation protocols often suffer from low efficiencies. RESULTS: In this paper, we provide a new broad host range E. coli/Bacillus shuttle vector, named pBACOV (Bacillus conjugation vector), that can be efficiently transferred to various Bacillus species using a single protocol. A variant of pBACOV carrying the sfGFP gene was successfully transferred to eight different species from the genus Bacillus and to one Paenibacillus species using triparental conjugation ("transmating"). This was achieved using a single protocol and worked for nine out of eleven tested acceptor species. The transmating procedure was used to test expression of the heterologous reporter gene sfGFP under control of the PaprE-promoter from B. subtilis in several Bacillus species in parallel. Expression of sfGFP was found in eight out of nine transmates. For several of the tested species, this is the first report of a method for genetic modification and heterologous gene expression. The expression level, analyzed by measuring the relative sfGFP-fluorescence normalized to the cell density of the cultures, was highest in B. mojavensis. CONCLUSIONS: The new shuttle vector pBACOV can be transferred to many different Bacillus and Paenibacillus species using a simple and efficient transmating protocol. It is a versatile tool facilitating the application of recombinant DNA technology in new as well as established strains, or selection of an ideal host for heterologous gene expression from a multitude of strains. This paves the way for the genetic modification and biotechnological exploitation of the broad diversity of species of Bacillus and related genera as well as different strains from these species.

Evaluation of promoter sequences for the secretory production of a Clostridium thermocellum cellulase in Paenibacillus polymyxa.

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

Handling gene and protein names in the age of bioinformatics: the special challenge of secreted multimodular bacterial enzymes such as the cbhA/cbh9A gene of Clostridium thermocellum.

An increasing number of researchers working in biology, biochemistry, biotechnology, bioengineering, bioinformatics and other related fields of science are using biological molecules. As the scientific background of the members of different scientific communities is more diverse than ever before, the number of scientists not familiar with the rules for non-ambiguous designation of genetic elements is increasing. However, with biological molecules gaining importance through biotechnology, their functional and unambiguous designation is vital. Unfortunately, naming genes and proteins is not an easy task. In addition, the traditional concepts of bioinformatics are challenged with the appearance of proteins comprising different modules with a respective function in each module. This article highlights basic rules and novel solutions in designation recently used within the community of bacterial geneticists, and we discuss the present-day handling of gene and protein designations. As an example we will utilize a recent mischaracterization of gene nomenclature. We make suggestions for better handling of names in future literature as well as in databases and annotation projects. Our methodology emphasizes the hydrolytic function of multi-modular genes and extracellular proteins from bacteria.

Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique.

Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

HPAEC-PAD for oligosaccharide analysis-novel insights into analyte sensitivity and response stability.

The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account. Graphical abstract HPAEC-PAD analysis of oligosaccharides and determination of PAD response drop leading to an improved data normalization.

Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression.

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.