Discovery of novel feruloyl esterase activity of BioH in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

Objectives

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.

Results

The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K _m values of 0.48, 6.3, and 1.9 mM, respectively, and k _cat /K _m values of 9.3, 3.8, and 3.8 mM^?1 s^?1, respectively.

Conclusions

Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.

     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

Heterologous expression of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> serotype 1 O-antigen analog in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

Objective

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster.

Results

The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached.

Conclusions

Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

     

Secretory expression of a novel human spermatozoa antigen in <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> and its application to a protein chip

Objective

To produce a recombinant spermatozoa antigen peptide using the E. coli: PhoA system on a protein chip for screening anti-sperm antibodies (ASA).

Results

The purity of the recombinant spermatozoa antigen exceeded 95% after two-step purification, as assessed using SDS-PAGE and HPLC. The diagnostic performance of a protein chip coated with the recombinant antigen peptide was evaluated by examining ASA in 51 infertile patients in comparison with a commercial ELISA kit. The area under the receiver operating characteristic curve (AUC) was 0.944, which indicated that the protein chip coated with recombinant spermatozoa antigen peptide was consistent with ELISA for ASA detection.

Conclusion

A recombinant spermatozoa antigen was expressed in the E. coli PhoA secretory expression system and its potential application for clinical ASA detection was validated.

     

Autodisplay of an avidin with biotin-binding activity on the surface of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli.

Results

Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and β domains, whereas the second consisted of a 314 amino acid from α and truncated β domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA).

Conclusions

Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

     

Characterization of a flavin-containing monooxygenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application to production of indigo and indirubin

Objective

To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments.

Results

The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu²⁺ and recovered by adding 1–10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l^?1 and 103 mg indirubin l^?1 from 2.5 g l-tryptophan l^?1.

Conclusion

The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.

     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

     

Enhancement of crystallinity of cellulose produced by <Emphasis Type="Italic">Escherichia coli</Emphasis> through heterologous expression of <Emphasis Type="Italic">bcsD</Emphasis> gene from <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Objectives

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.

Results

The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.

Conclusion

The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

     

Evaluation of NfsA-like nitroreductases from <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> and <Emphasis Type="Italic">Bartonella henselae</Emphasis> for enzyme-prodrug therapy,targeted cellular ablation,and dinitrotoluene bioremediation

Objectives

To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene.

Results

NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His₆-tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O₂-insensitive nitroreductase E. coli NfsB (NfsB_Ec), NfsA_Nm exhibiting comparable levels of activity to the leading nitroreductase candidate E. coli NfsA (NfsA_Ec). NfsA_Nm is also the first NfsA-family nitroreductase shown to produce a substantial proportion of 4-hydroxylamine end-product. NfsA_Nm and PnbA_Bh were again more efficient than NfsB_Ec at aerobic activation of metronidazole to a cytotoxic form, with NfsA_Nm appearing a promising candidate for improving zebrafish-targeted cell ablation models. NfsA_Nm was also more active than either NfsA_Ec or NfsB_Ec with 2,4- or 2,6-dinitrotoluene substrates, whereas PnbA_Bh was relatively inefficient with either substrate.

Conclusions

NfsA_Nm is a promising new nitroreductase candidate for several diverse biotechnological applications.

     

Production of tartaric acid using immobilized recominant <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objective

To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of l-(+)-tartaric acid.

Results

E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg^?1. The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U^?1 h^?1 during the continuous enzymatic production process.

Conclusion

ESH with a histidine tag was immobilized and used for the continuous production of l-(+)-tartaric acid.

     

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

     

Expression and characterisation of neopullulanase from <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis>

Objectives

To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase.

Results

An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni–NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 °C and pH 6.0, producing panose as the major reaction product.

Conclusions

To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.

     

Genetic improvement of <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> for enhanced biological removal of phosphate

Objectives

To improve its phosphate accumulating abilities for phosphate recycling from wastewater, a magnetotactic bacterium, Magnetospirillum gryphiswaldense, was genetically modified to over-express polyphosphate kinase.

Results

Polyphosphate kinase was over-expressed in the bacterium. The recombinant strain accumulated ninefold more polyphosphate from synthetic wastewater compared to original wild type. The magnetic property of the recombinant M. gryphiswaldense strain was retained.

Conclusions

The recombinant M. gryphiswaldense can be used for phosphate removal and recovery in bioremediation.

     

Levansucrase from <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> NTM048 produces a levan exopolysaccharide with immunomodulating activity

Objectives

A levansucrase from Leuconostoc mesenteroides NTM048 was cloned and expressed and its enzymatic product was characterized.

Results

The fructansucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The recombinant enzyme was purified as a single protein and its properties investigated. The polymer produced by the recombinant enzyme was identified as levan by various means including TLC and NMRs, and the enzyme was identified as a GH68 levansucrase. The enzyme was optimal at pH 5.5–6 and 30 °C, and its activity was stimulated by Ca²⁺. The levan produced by this strain induced IgA production in mice.

Conclusion

Leuconostoc mesenteroides, a probiotic strain, possessed levansucrase which catalyzed the produced levan that had immunomodulating activity.

     

Production of an antioxidative peptide from hairy basil seed waste by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To circumvent the time-consuming and costly problems associated with natural product extraction, a potential antioxidative peptide selected from hairy basil waste after oil extraction was produced by recombinant DNA technology.

Results

Because the target peptide is short, the recombinant peptide containing seven repeats of the target sequence, QTFQYSRGWTN, and the DNA fragment coding this sequence was cloned into the pQE-30 Xa expression vector and transformed into Escherichia coli. After 6 h of recombinant peptide expression in E. coli, the target peptide was purified by Ni²⁺ affinity chromatography and gel extraction. The expected 15 kDa recombinant target peptide construct was verified by modified dot blot analysis. Compared with the chemically synthesized peptide, the recombinant peptide revealed significantly higher antioxidant activities (p < 0.05), as determined by DPPH and ABTS radical scavenging assays, and in vitro DNA damage induced by hydroxyl radicals.

Conclusion

This approach provides an alternative to produce an antioxidative peptide that provides a potential scaffold for the further development of antioxidative peptides for industrial applications.

     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

     

Metabolomics reveals immunomodulation as a possible mechanism for the antibiotic effect of <Emphasis Type="Italic">Persicaria capitata</Emphasis> (Buch.-Ham. ex D. Don) H.Gross

Introduction

In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways.

Objective

The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection.

Methods

Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment).

Results

The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage.

Conclusion

Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.

     

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Objectives

To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs.

Results

CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H₂O₂ and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates.

Conclusions

CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

     

Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis>

Background

The λ Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of λ Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli.

Results

In this report, we have identified conditions that optimize the use of λ Red for recombineering in EHEC and EPEC. Using plasmids that contain a P_tac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (~1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC.

Conclusions

The λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of λ Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.