Biosynthesis of antibiotic prodiginines in the marine bacterium Hahella chejuensis KCTC 2396

AIMS: Hahella chejuensis KCTC 2396 produces red pigments, showing antibacterial and algicidal activities. The main red-coloured metabolite of the pigments was identified as antibiotic prodigiosin. With the expectation that the red pigments are a mixture of a series of close relatives, the aim of the present study is to detect new antibiotic prodigiosin analogues and to analyse the biosynthetic pattern for prodiginines in KCTC 2396. METHODS AND RESULTS: Except prodigiosin, the other constituents in the red pigments were confirmed as well-known dipyrrolyldipyrromethene prodigiosin, norprodigiosin, and undecylprodiginine. Additionally, four new prodigiosin analogues, each of which was distinguished from prodigiosin (C(5)), according to differences in alkyl chain length (C(3)-C(7)), were detected in small quantities by liquid chromatography mass spectrometry/mass spectrometry spectroscopy. Owing to the presence of a cytotoxic methoxy group, it is expected that all the new prodigiosin analogues are bioactive. CONCLUSIONS: Four characterized prodiginines, including prodigiosin and four new prodigiosin analogues are produced in different ratio in KCTC 2396. All of the prodiginines possess a common linear tripyrrolyl structure and a cytotoxic methoxy group. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that KCTC 2396 is able to produce antibiotic prodigiosin, undecylprodiginine and new prodigiosin analogues in a mixture of pigments. It is also shown that KCTC 2396 possesses a novel system for the simultaneous production of multiple prodiginines in a single micro-organism.     

Cellotriose-hydrolyzing activity conferred by truncating the carbohydrate-binding modules of Cel5 from Hahella chejuensis

Bioprocess and Biosystems Engineering - Processivity is a typical characteristic of cellobiohydrolases (CBHs); it enables the enzyme to successively hydrolyze the ends of cellulose chains and to...     

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.     

Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-d-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.     

An expansin-like protein from Hahella chejuensis binds cellulose and enhances cellulase activity

Molecular function of the expansin superfamily has been highlighted for cellulosic biomass conversion. In this report, we identified a new bacterial expansin subfamily by analysis of related bacterial sequences and biochemically examined a member of this new subfamily from Hahella chejuensis (HcEXLX2). Among the various complex polysaccharides tested, HcEXLX2 bound most efficiently to cellulose. The relative binding constant (K _r ) against Avicel was 2.1 L g⁻¹ at pH 6.0 and 4°C. HcEXLX2 enhanced the activity of cellulase, producing about 4.6 times more hydrolysis product after a 36 h reaction relative to when only cellulase was used. The extension strength test on filter paper indicated that HcEXLX2 has a texture loosening effect on filter paper, which was 53% of that observed for 8 M urea treatment. These activities, compared with a cellulose binding domain from Clostridium thermocellum, implied that the synergistic effect of HcEXLX2 comes from not only binding to cellulose but also disrupting the hydrogen bonds in cellulose. Based on these results, we suggest that the new bacterial expansin subfamily functions by binding to cell wall polysaccharides and increasing the accessibility of cell wall degrading enzymes.     

Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co⁺ and Co²⁺ can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.     

Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent

Harmful algal blooms, caused by rapid growth and accumulation of certain microalgae in the ocean, pose considerable impacts on marine environments, aquatic industries and even public health. Here, we present the 7.2-megabase genome of the marine bacterium Hahella chejuensis including genes responsible for the biosynthesis of a pigment which has the lytic activity against a red-tide dinoflagellate. H.chejuensis is the first sequenced species in the Oceanospiralles clade, and sequence analysis revealed its distant relationship to the Pseudomonas group. The genome was well equipped with genes for basic metabolic capabilities and contained a large number of genes involved in regulation or transport as well as with characteristics as a marine heterotroph. Sequence analysis also revealed a multitude of genes of functional equivalence or of possible foreign origin. Functions encoded in the genomic islands include biosynthesis of exopolysacchrides, toxins, polyketides or non-ribosomal peptides, iron utilization, motility, type III protein secretion and pigmentation. Molecular structure of the algicidal pigment, which was determined through LC-ESI-MS/MS and NMR analyses, indicated that it is prodigiosin. In conclusion, our work provides new insights into mitigating algal blooms in addition to genetic make-up, physiology, biotic interactions and biological roles in the community of a marine bacterium.     

Expression, reconstruction and characterization of codon-optimized carbonic anhydrase from Hahella chejuensis for CO2 sequestration application

The high production of functional carbonic anhydrase (CA) is required for practical CO₂ sequestration application mediated by CA. Here, the synthetic gene based on Escherichia coli codon usage of new α-type CA (HC-aCA) of Hahella chejuensis, a Korea marine microorganism, was highly expressed in E. coli. We obtained a high yield of functional HC-aCA by denaturing/refolding process and incorporating zinc ion into its active site. The refolded HC-aCA displayed a half-deactivation temperature of 60 °C with maximal activity at 50 °C, and had high pH stability in alkali condition with maximal activity at pH 10.0. The esterase activity of HC-aCA almost doubled at high salt concentration ranging from 0.67 to 2.0 M NaCl. HC-aCA catalyzed the conversion of CO₂ to CaCO₃ as calcites form in the presence of Ca²⁺. The refolded HC-aCA could be a promising candidate for the development of efficient CA-based CO₂ sequestration processes.     

Mutant selection of <Emphasis Type="Italic">Hahella chejuensis</Emphasis> KCTC 2396 and statistical optimization of medium components for prodigiosin yield-up

Prodigiosin is a natural red pigment with algicidal activity against Cochlodinium polykrikoides, a major harmful red-tide microalga. To increase the yield of prodigiosin, a mutant of Hahella chejuenesis KCTC 2396, assigned M3349, was developed by an antibiotic mutagenesis using chloramphenicol. When cultured in Sucrose-based Marine Broth medium (SMB), M3349 could produce prodigiosin at 1.628+/-0.06 g/L, while wild type producing at 0.658+/-0.12 g/L under the same conditions. To increase the yield of prodigiosin production by M3349, significant medium components were determined using a two-level Plackett-Burman statistical design technique. Among fourteen components included in SMB medium, NaCl, Na2SiO3, MgCl2, H3BO3, Na2HPO4, Na2SO4, and CaCl2 were determined to be important for prodigiosin production. The medium formulation was finally optimized using a Box-Behnken design as follows: sucrose 10.0, peptone 8.0, yeast extract 2.0, NaCl 10.0, Na2SO4 12.0, CaCl2 1.8, MgCl2 0.7 g/L; and H3BO3 22.0, Na2HPO4 20.0, Na2SiO3 8.0 mg/L. The predicted maximum yield of prodigiosin in the optimized medium was 2.43 g/L by the Box-Behnken design, while the practical production was 2.60+/-0.176 g/L, which was 3.9 times higher than wild type with SMB Medium (0.658 g/L).     

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80 °C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135 ± 6% and 193 ± 8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50 °C and about 45 °C respectively, while 65 °C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.     

Cloning and characterization of thermostable endoglucanase (Cel8Y) from the hyperthermophilic Aquifex aeolicus VF5

Aquifex aeolicus is the hyperthermophilic bacterium known, with growth-temperature maxima near 95 degrees C. The cel8Y gene, encoding a thermostable endoglucanase (Cel8Y) from Aquifex aeolicus VF5, was cloned into a vector for expression and expressed in Escherichia coli XL1-Blue. A clone of 1.7 kb fragment containing endoglucanase activity, designated pKYCY100, was sequenced and found to contain an ORF of 978 bp encoding a protein of 325 amino acid residues, with a calculated molecular mass of 38,831 Da. This endoglucanase was designated cel8Y gene. The endoglucanase has an 18-amino-acid signal peptide but not cellulose-binding domain. The endoglucanase of A. aeolicus VF5 had significant amino acid sequence similarities with endoglucanases from glycosyl hydrolase family 8. The predicted amino acid sequence of the Cel8Y protein was similar to that of CMCase of Cellulomonas uda, BcsC of Escherichia coli, CelY of Erwinia chrysanthemi, and CMCase of Acetobacter xylinum. The molecular mass of Cel8Y was calculated to be 36,750 Da, which is consistent with the value obtained from result of CMC-SDS-PAGE of the purified enzyme. Cel8Y was thermostable, exhibiting maximal activity at 80 degrees C and pH optima of 7.0 and with half-lives of 2 h at 100 degrees C, 4 h at 90 degrees C.     

Overexpression, on-column refolding and isotopic labeling of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily

A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of βγ-crystallin superfamily. This hypothetical protein contains a putative βγ-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (¹⁵N/¹³C) labeled protein with high yield for NMR studies is reported. The uniformly ¹⁵N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [¹⁵N]–[¹H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.     

Engineering a family 9 processive endoglucanase from Paenibacillus barcinonensis displaying a novel architecture

Cel9B from Paenibacillus barcinonensis is a modular endoglucanase with a novel molecular architecture among family 9 enzymes that comprises a catalytic domain (GH9), a family 3c cellulose-binding domain (CBM3c), a fibronectin III-like domain repeat (Fn3_1,2), and a C-terminal family 3b cellulose-binding domain (CBM3b). A series of truncated derivatives of endoglucanase Cel9B have been constructed and characterized. Deletion of CBM3c produced a notable reduction in hydrolytic activity, while it did not affect the cellulose-binding properties as CBM3c did not show the ability to bind to cellulose. On the contrary, CBM3b exhibited binding to cellulose. The truncated forms devoid of CBM3b lost cellulose-binding ability and showed a reduced activity on crystalline cellulose, although activity on amorphous celluloses was not affected. Endoglucanase Cel9B produced only a small ratio of insoluble products from filter paper, while most of the reducing ends produced by the enzyme were released as soluble sugars (91%), indicating that it is a processive enzyme. Processivity of Cel9B resides in traits contained in the tandem of domains GH9–CBM3c, although the slightly reduced processivity of truncated form GH9–CBM3c suggests a minor contribution of domains Fn3_1,2 or CBM3b, not contained in it, on processivity of endoglucanase Cel9B.     

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Background

The non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.

Results

Eight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.

Conclusions

Our results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.

     

Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.     

Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.     

Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96 A. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a beta-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu(186) and Glu(359), are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a (1)H NMR experiment using the reduced cellooligosaccharide as a substrate.