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Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16‐5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time‐consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9‐based genome editing system with high efficiency for single‐gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D‐lactic acid. Compared with the wild‐type Bacillus sp. N16‐5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D‐lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9‐based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis. [ABSTRACT FROM AUTHOR]

Steroids have been widely used in birth‐control, prevention, and treatment of various diseases, representing the largest sector after antibiotics in the global pharmaceutical market. The steroidal active pharmaceutical ingredients (APIs) have been produced via partial synthetic processes first mainly from sapogenins, which was converted into 16‐dehydropregnenolone by the famous "Marker Degradation". Traditional mutation and screening, and process engineering have resulted in the industrial production of 4‐androstene‐3,17‐dione (AD), androst‐1,4‐diene‐3,17‐dione (ADD), 9α‐hydroxy‐androsta‐4‐ene‐3,17‐dione (9α‐OH‐AD), and so on, which serve as the key intermediates for the synthesis of steroidal APIs. Recently, genetic and metabolic engineering have generated highly efficient microbial strains for the production of these precursors, leading to the replacement of sapogenins with phytosterols as the starting materials. Further advances in synthetic biology hold promise in the design and construction of microbial cell factories for the industrial production of steroidal intermediates and/or APIs from simple carbon sources such as glucose. Integration of biotransformation into the synthesis of steroidal APIs can greatly reduce the number of reaction steps, achieve lower waste discharge and higher production efficiency, thus enabling a greener steroidal pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Summary: Nα‐acetyl‐α‐lysine was found as a new type of compatible solutes that acted as an organic cytoprotectant in the strain of Salinicoccus halodurans H3B36. A novel lysine Nα‐acetyltransferase gene (shkat), encoding an enzyme that catalysed the acetylation of lysine exclusively at α position, was identified from this moderate halophilic strain and expressed in Escherichia coli. Sequence analysis indicated ShKAT contained a highly conserved pyrophosphate‐binding loop (Arg‐Gly‐Asn‐Gly‐Asn‐Gly), which was a signature of the GNAT superfamily. ShKAT exclusively recognized free amino acids as substrate, including lysine and other basic amino acids. The enzyme showed a wide range of optimal pH value and was tolerant to high‐alkali and high‐salinity conditions. As a new member of the GNAT superfamily, the ShKAT was the first enzyme recognized free lysine as substrate. We believe this work gives an expanded perspective of the GNAT superfamily, and reveals great potential of the shkat gene to be applied in genetic engineering for resisting extreme conditions. [ABSTRACT FROM AUTHOR]

The chiral N‐substituted 1,2‐amino alcohol motif is found in many natural and synthetic bioactive compounds. In this study, enzymatic asymmetric reductive amination of α‐hydroxymethyl ketones with enantiocomplementary imine reductases (IREDs) enabled the synthesis of chiral N‐substituted 1,2‐amino alcohols with excellent ee values (91–99 %) in moderate to high yields (41–84 %). Furthermore, a one‐pot, two‐step enzymatic process involving benzaldehyde lyase‐catalyzed hydroxymethylation of aldehydes and subsequent asymmetric reductive amination was developed, offering an environmentally friendly and economical way to produce N‐substituted 1,2‐amino alcohols from readily available simple aldehydes and amines. This methodology was then applied to rapidly access a key synthetic intermediate of anti‐malaria and cytotoxic tetrahydroquinoline alkaloids. [ABSTRACT FROM AUTHOR]

Summary: Propionic acid (PA) has been widely used as a food preservative and chemical intermediate in the agricultural and pharmaceutical industries. Environmental and friendly biotechnological production of PA from biomass has been considered as an alternative to the traditional petrochemical route. However, because PA is a strong inhibitor of cell growth, the biotechnological host should be not only able to produce the compound but the host should be robust. In this study, we identified key PA tolerance factors in Pseudomonas putida KT2440 strain by comparative transcriptional analysis in the presence or absence of PA stress. The identified major facilitator superfamily (MFS) transporter gene cluster of PP_1271, PP_1272 and PP_1273 was experimentally verified to be involved in PA tolerance in P. putida strains. Overexpression of this cluster improved tolerance to PA in a PA producing strain, what is useful to further engineer this robust platform not only for PA synthesis but for the production of other weak acids. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9‐guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double‐stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome‐targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome‐targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9‐fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5‐bp editing window to 7‐bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user‐friendly online tool named gBIG was provided for designing guide RNAs for base editing‐mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG). [ABSTRACT FROM AUTHOR]

5‐Aminolevulinic acid (ALA) is a value‐added compound with potential applications in the fields of agriculture and medicine. Although massive efforts have recently been devoted to building microbial producers of ALA through metabolic engineering, few studies focused on the cellular response and tolerance to ALA. In this study, we demonstrated that ALA caused severe cell damage and morphology change of Escherichia coli via generating reactive oxygen species (ROS), which were further determined to be mainly hydrogen peroxide and superoxide anion radical. ALA treatment activated the native antioxidant defense system by upregulating catalase (CAT) and superoxide dismutase (SOD) expression to combat ROS. Further overexpressing CAT (encoded by katG and katE) and SOD (encoded by sodA, sodB, and sodC) not only improved ALA tolerance but also its production level. Notably, coexpression of katE and sodB in an ALA synthase expressing strain enhanced the biomass and final ALA titer by 81% and 117% (11.5 g/L) in a 5 L bioreactor, respectively. This study demonstrates the importance of tolerance engineering in strain development. Reinforcing the antioxidant defense system holds promise to improve the bioproduction of chemicals that cause oxidative stress. [ABSTRACT FROM AUTHOR]

Ursodeoxycholic acid (UDCA), a pharmaceutical ingredient widely used in clinics, can be prepared from chenodeoxycholic acid (CDCA) by the epimerization of the 7α‐OH group. In this study, a nicotinamide adenine dinucleotide (NAD+) regeneration system was developed by using flavin oxidoreductase (FR) and flavin mononucleotide (FMN). Only catalytic amount of FMN is required for the effective NAD+ recycling. FR/FMN system was then applied in the oxidation of CDCA to 7‐ketolithocholic acid (7‐keto‐LCA) by NAD+‐dependent 7α‐hydroxysteroid dehydrogenase (Bs‐7α‐HSDH) from Brevundimonas sp. which showed extremely high enzyme activity toward CDCA (kcat/Km=8050 s−1 ⋅ mM−1). When Escherichia coli whole cells coexpressing Bs‐7α‐HSDH and FR genes were used as biocatalyst, CDCA (50 mM) was completely converted to 7‐keto‐LCA with the turnover number of FMN being 227 and 58.8 g ⋅ L−1 ⋅ d−1 space‐time yield of 7‐keto‐LCA. For the reduction of 7‐keto‐LCA, nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent 7‐β‐hydroxysteroid dehydrogenase (Cm‐7β‐HSDH) from Clostridium sp. Marseille was employed with alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) and iso‐propanol as co‐factor regeneration system. When E. coli whole cells coexpressing Cm‐7β‐HSDH and TbADH genes were used as biocatalyst, 40 mM 7‐keto‐LCA was reduced to UDCA with 26.8 g ⋅ L−1 ⋅ d−1 space‐time yield. The oxidation and reduction were then carried in a one‐pot concurrent mode, 12.5 mM CDCA was completely converted to UDCA. The epimerization of CDCA to UDCA proceeded to completion at the substrate concentration of 30 mM in the one‐pot sequential process. Therefore, the complete conversion of CDCA to UDCA in one‐pot has been realized by employing 7α‐HSDH and 7β‐HSDH of different co‐factor specificities with independent co‐factor recycling systems. The cholic acids, especially UDCA, exert inhibitive effect on the activities of these enzymes, preventing the complete epimerization of 7α‐OH at higher substrate loading. This inhibition issue should be solvable by engineering the involved enzymes, that is currently pursued in our laboratory. [ABSTRACT FROM AUTHOR]

D‐Allulose 3‐epimerase (DAE) has been applied to produce D‐allulose, a low‐calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food‐grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harboring three DAE genes in an expression vector exhibited the highest enzyme activity with 22.7 U/mg. Whole‐cell transformation of DAE16 produced 225 g/L D‐allulose with a volumetric productivity of 353 g·g −1·hr −1. The catalytic efficiency of strain C‐DAE9 integrating total 11 DAE genes in chromosome was 16.4‐fold higher than strains carrying one DAE. Fed‐batch culture of C‐DAE9 gave enzyme activity of 44,700 U/L. We also expressed a thermostable invertase in C. glutamicum and obtained enzyme activity of 29 U/mg. Immobilized cells expressing DAE or invertase exhibited 80% of retained activity after 30 cycles of catalytic reactions. Those immobilized cells were coupled to produce 61.2 g/L D‐allulose from cane molasses in a two‐step reaction process. This study provided an efficient approach for enzyme preparation and allowed access to produce D‐allulose from other abundant and low‐cost feedstock enriched with sucrose. A tandem isoenzyme gene expression strategy has been applied to express multiple D‐allulose 3‐epimerases (DAEs) in one cell and facilitates constructing food‐grade expression systems based on Corynebacterium glutamicum. Abundant raw materials cane molasses have been converted to D‐allulose by one‐pot, two‐step reaction process. [ABSTRACT FROM AUTHOR]

We report the first X-ray structure of the unique 'head-to-middle' monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase. [ABSTRACT FROM AUTHOR]

A salt-tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p-nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three-dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat-resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890-899, 2015 [ABSTRACT FROM AUTHOR]

A xylanase gene ( xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module ( CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry. [ABSTRACT FROM AUTHOR]

ABSTRACT Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C40 long-chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans-prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo-form as well as in complexes with IPP and a FPP thio-analog, FsPP, at resolutions of 2.2-2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active-site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand-bound crystal structures of a long-chain trans-prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37-45. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars. Biotechnol. Bioeng. 2015;112: 168-180. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Pullulanase is a debranching enzyme that specifically hydrolyzes the α-1,6 glycosidic linkage of α-glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18-11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg−1 and 16.4 μmol L−1, respectively. PulA has a half-life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75-2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β-strand and the third α-helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate-binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685-1693. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild-type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo-form wild-type TsXylA and the xylobiose-, xylotriose-, and xylotetraose-bound E146A and E251A mutants were solved by X-ray diffraction to high resolution (1.32-1.66 Å). The protein forms a classic (β/α)8 folding of typical GH10 xylanases. The ligands in substrate-binding groove as well as the interactions between sugars and active-site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering. Proteins 2013; 81:1256-1265. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Flux balance analysis (FBA) has been widely used in calculating steady-state flux distributions that provide important information for metabolic engineering. Several thermodynamics-based methods, for example, quantitative assignment of reaction directionality and energy balance analysis have been developed to improve the prediction accuracy of FBA. However, these methods can only generate a thermodynamically feasible range, rather than the most thermodynamically favorable solution. We therefore developed a novel optimization method termed as thermodynamic optimum searching (TOS) to calculate the thermodynamically optimal solution, based on the second law of thermodynamics, the minimum magnitude of the Gibbs free energy change and the maximum entropy production principle (MEPP). Then, TOS was applied to five physiological conditions of Escherichia coli to evaluate its effectiveness. The resulting prediction accuracy was found significantly improved (10.7-48.5%) by comparing with the 13C-fluxome data, indicating that TOS can be considered an advanced calculation and prediction tool in metabolic engineering. Biotechnol. Bioeng. 2013; 110: 914-923. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Summary Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions. [ABSTRACT FROM AUTHOR]

AaManA, a β-1,4-mannanase from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31, was expressed in Escherichia coli and purified in a form suitable for X-ray crystallographic analysis. Crystals were obtained using the hanging-drop vapour-diffusion method at 291 K using ammonium dihydrogen phosphate as a precipitant. Data were collected from native mannanase and from a selenomethionyl derivative to 1.90 and 1.99 Å, respectively, at 100 K. The native crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 44.34, b = 75.55, c = 88.02 Å. The derivative crystal belonged to the same space group as native AaManA, with unit-cell parameters a = 44.55, b = 75.70, c = 92.66 Å [ABSTRACT FROM AUTHOR]

Alanine racemase (DadXOF4), a dimeric endogenous PLP-dependent alkaline enzyme from alkaliphilic Bacillus pseudofirmus OF4, was expressed in Escherichia coli and purified with a His6 tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 291 K using a solution containing 1.4 M sodium/potassium phosphate pH 8.2. The protein crystallized in space group P212121, with two protein molecules in the asymmetric unit. [ABSTRACT FROM AUTHOR]

The article presents a diagram related to the article "Structure and Function of a "Head-to-Middle" Prenyltransferase: Lavandulyl Diphosphate Synthase" by Y. Ma and colleagues in the current issue.

Die irreguläre Terpensynthase Lavandulyldiphosphatsynthase (LPPS) produziert die Vorstufe des kommerziell wichtigen Lavendelduftstoffs Lavandulol durch einen ungewöhnlichen “Kopf ‐ zur ‐ Mitte” ‐ Mechanismus. E. Oldfield, R. ‐ T. Guo, Y. Ma et al. präsentieren in der Zuschrift auf S. 4799 ff. die erste Struktur von LPPS im Komplex mit seinem Substrat. Die schmetterlingsförmige Struktur ähnelt der von Proteinen, die an der Biosynthese bakterieller Zellwände beteiligt sind, und führt zu einem einzigartigen Kondensationsmechanismus, der von allgemeinem Interesse im Kontext der Terpensynthese ist. [ABSTRACT FROM AUTHOR]

N α -acetyl-α-lysine was found as a new type of compatible solutes that acted as an organic cytoprotectant in the strain of Salinicoccus halodurans H3B36. A novel lysine N α -acetyltransferase gene (shkat), encoding an enzyme that catalysed the acetylation of lysine exclusively at α position, was identified from this moderate halophilic strain and expressed in Escherichia coli. Sequence analysis indicated ShKAT contained a highly conserved pyrophosphate-binding loop (Arg-Gly-Asn-Gly-Asn-Gly), which was a signature of the GNAT superfamily. ShKAT exclusively recognized free amino acids as substrate, including lysine and other basic amino acids. The enzyme showed a wide range of optimal pH value and was tolerant to high-alkali and high-salinity conditions. As a new member of the GNAT superfamily, the ShKAT was the first enzyme recognized free lysine as substrate. We believe this work gives an expanded perspective of the GNAT superfamily, and reveals great potential of the shkat gene to be applied in genetic engineering for resisting extreme conditions., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The catalytic domain of an alkaline beta-mannanase from the alkaliphilic Bacillus sp. N16-5 has been expressed and purified. The recombinant enzyme was crystallized using the hanging-drop vapour-diffusion method at 298 K. X-ray diffraction data were collected to 1.6 A resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.03, b = 63.31, c = 83.34 A. Initial phasing was carried out by molecular replacement using the three-dimensional structure of a mannanase from the alkaliphilic Bacillus sp. JAMB602 as a search model.

AaManA, a beta-1,4-mannanase from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31, was expressed in Escherichia coli and purified in a form suitable for X-ray crystallographic analysis. Crystals were obtained using the hanging-drop vapour-diffusion method at 291 K using ammonium dihydrogen phosphate as a precipitant. Data were collected from native mannanase and from a selenomethionyl derivative to 1.90 and 1.99 A, respectively, at 100 K. The native crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.34, b = 75.55, c = 88.02 A. The derivative crystal belonged to the same space group as native AaManA, with unit-cell parameters a = 44.55, b = 75.70, c = 92.66 A.