Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region.

Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.

A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria.

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The Michaelis-Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 µM and 1.55 × 10(-)(4 )µmol min(-1), respectively, and the enzyme was optimally active at pH 5.0 and 37°C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-ß-d-cellobioside (0.3 U mg(-1)), CMC (105.9 U mg(-1)), birch wood xylan (132.3 U mg(-1)), oat spelt xylan (67.9 U mg(-1)), and 2-hydroxyethyl-cellulose (26.3 U mg(-1)). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil.

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium.

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 x 10⁻² U mg⁻¹, respectively. The Michaelis-Menten constant (K (m) values) and the maximal reaction velocities (V(max) values) of CelEdx16 were 47.1 µM and 9.6 x 10⁻³ µmole min⁻¹ when endocellulase activity was measured and 106.3 µM and 2.1 x 10⁻5 µmol min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40 °C.

Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal ß-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal ß-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded ß-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

Hesperidin a flavanoglycone protects against gamma-irradiation induced hepatocellular damage and oxidative stress in Sprague-Dawley rats.

Oxidative stress plays a pivotal role in the pathogenesis and progression of gamma-irradiation induced cellular damage and the administration of dietary antioxidants has been suggested to protect against the subsequent tissue damage. Here, we present the data to explore the hepatoprotective and antioxidant effect of hesperidin, a naturally occurring citrus flavanoglycone, against gamma-irradiation induced oxidative damage in the liver of rats. Healthy male Sprague-Dawley rats were exposed to gamma-irradiation (1 Gy, 3 Gy and 5 Gy) and were administered hesperidin (50 mg/kg and 100 mg/kg, b.w, orally) for 7 days post irradiation. The changes in body weight, liver weight, spleen index, serum and liver aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (gamma-GT) and serum ceruloplasmin levels were determined along with differences in the liver histopathology. Liver thiobarbuturic acid reactive substance as an index for lipid peroxidation and the levels of enzymatic antioxidants like superoxide dismutase, catalase, glutathione peroxidase and the status of non-enzymatic antioxidants as an index for oxidative stress were also determined. Exposure to gamma-irradiation resulted in hepatocellular damage in a dose-dependent manner, featuring a significantly decreased body weight and liver weight and higher levels of serum AST, ALT, ALP, LDH and gamma-GT levels and a simultaneous decrease in their levels in the liver tissue. Oxidative stress was evidenced by elevated levels of lipid peroxidation and a decrease in the levels of key enzymatic and non-enzymatic antioxidants in the liver. However, the gamma-irradiation induced toxic effects were dramatically and dose-dependently inhibited by hesperidin treatment as observed by the restoration in the altered levels of the marker enzymes, lipid peroxidation, enzymatic and non-enzymatic antioxidants. The results of the biochemical observations were supported by the histopathological findings. Thus, oral administration of hesperidin was found to offer protection against gamma-irradiation induced hepatocellular damage and oxidative stress in rats, probably by exerting a protective effect against hepatocellular necrosis via its free radical scavenging and membrane stabilizing ability.

Occurrence of agmatine pathway for putrescine synthesis in Selenomonas ruminatium.

Selenomonas ruminantium synthesizes cadaverine and putrescine from L-lysine and L-ornithine as the essential constituents of its peptidoglycan by a constitutive lysine/ornithine decarboxylase (LDC/ODC). S. ruminantium grew normally in the presence of the specific inhibitor for LDC/ODC, DL-alpha-difluoromethylornithine, when arginine was supplied in the medium. In this study, we discovered the presence of arginine decarboxylase (ADC), the key enzyme in agmatine pathway for putrescine synthesis, in S. ruminantium. We purified and characterized ADC and cloned its gene (adc) from S. ruminantium chromosomal DNA. ADC showed more than 60% identity with those of LDC/ODC/ADCs from Gram-positive bacteria, but no similarity to that from Gram-negative bacteria. In this study, we also cloned the aguA and aguB genes, encoding agmatine deiminase (AguA) and N-carbamoyl-putrescine amidohydrolase (AguB), both of which are involved in conversion from agmatine into putrescine. AguA and AguB were expressed in S. ruminantium. Hence, we concluded that S. ruminantium has both ornithine and agmatine pathways for the synthesis of putrescine.

Anti-tumor activity of an immunotoxin (TGFα-PE38) delivered by attenuated Salmonella typhimurium.

The anticancer strategy underlying the use of immunotoxins is as follows: the cancer-binding domain delivers the toxin to a cancer cell, after which the toxin enters and kills the cell. TGFα-PE38 is an immunotoxin comprising transforming growth factor alpha (TGFα), a natural ligand of epidermal growth factor receptor (EGFR), and a modified Pseudomonas exotoxin A (PE38) lacking N terminal cell-binding domain, a highly potent cytotoxic protein moiety. Tumor cells with high level of EGFR undergo apoptosis upon treatment with TGFα-PE38. However, clinical trials demonstrated that this immunotoxin delivered by an intracerebral infusion technique has only a limited inhibitory effect on intracranial tumors mainly due to inconsistent drug delivery. To circumvent this problem, we turned to tumor-seeking bacterial system. Here, we engineered Salmonella typhimurium to selectively express and release TGFα-PE38. Engineered bacteria were administered to mice implanted with mouse colon or breast tumor cells expressing high level of EGFR. We observed that controlled expression and release of TGFα-PE38 from intra-tumoral Salmonellae by either an engineered phage lysis system or by a bacterial membrane transport signal led to significant inhibition of solid tumor growth. These results demonstrated that delivery by tumor-seeking bacteria would greatly augment efficacy of immunotoxin in cancer therapeutics.

Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource.

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system.

Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system. Cell growth in 96-well plates was measured by monitoring optical density over 11-14h at 37°C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-ß-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 10(4) clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.

A rapid and simple method for preparing an insoluble substrate for screening of microbial xylanase.

Several types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.

Protective effect of hesperidin, a citrus flavanoglycone, against γ-radiation-induced tissue damage in Sprague-Dawley rats.

The present study was designed to evaluate the radioprotective effect of hesperidin, a citrus flavanoglycone, against γ-radiation-induced cellular damage in the liver, heart, and kidney of rats. Whole-body γ-radiation exposure (5 Gy) of healthy adult rats resulted in cellular damage and oxidative stress manifested as increased levels of serum marker enzymes, lipid peroxidation, and fibrosis in the tissues, accompanied by depletion of cellular glutathione and abnormal alteration in the levels of lysosomal enzymes. Treatment with hesperidin (50 and 100 mg/kg, p.o.) for 7 days was found to offer significant protection against γ-radiation-induced toxicity in the tissues, which was evident by the improved status of most of the parameters investigated. Further, the histological examination of periodic acid-Schiff-stained tissue sections of animals treated with hesperidin following radiation exposure showed minimal necrotic damage with a recovery pattern in a dose-dependent manner compared with radiation-exposed animals. The results of our study show that administration of hesperidin offers effective protection against γ-radiation-induced cellular damage and oxidative stress in rats.