Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm,Bombyx mori

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5–2.0 and 5.5–6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84 % of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.     

Cloning and High-Level Expression of the Enzymatic Region of Phytase in E. coli

Phytase is an important enzyme poses great nutritional significance in humans and monogastric animals diets. The phytase production yield using wild sources, including micro-organisms, plants, and animals is sorely low. Thus, recombinant expression of phytase has received increasing interest for achieving production rate. Escherichia coli is the most preferred host for expression of heterologous proteins but overexpression of recombinant phytase in E. coli, met with limited success due to the sequestration of the enzyme into inclusion bodies. In the present study, artificial phytases gene with excellent thermostability and activity were designed by detecting the enzymatic region of the E. coli phytase gene by employing bioinformatics tools. Then, the PCR amplified recombinant gene was expressed in E. coli and the active enzyme was recovered from inclusion bodies. Employing cysteine amino acid in the dialysis buffer succeed to the superior activity of the enzyme with a specific activity of 73.8 U/mg. The optimum temperature and pH for enzyme activity were determined at 60 °C and 4, respectively. The novel recombinant enzyme illustrated perfect thermostability up to 70 °C with maintenance 75% of its activity. The enzyme was stable at pH range of 2–10. Moreover, the effects of ions and chemical compounds on enzyme stability and activity were assessed.

     

Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig?, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3′ to the other copies with a 6 kbp deletion in the 5′ promoter region. The overall size of the integrated transgene complex is 46 kbp. During integration a 66 kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11 day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4–6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P < 0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization

The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70–75 °C and pH 6.5–7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca²⁺ ion and exhibited high catalytic efficiency of 8.3 s^?1 μM^?1 towards phytic acid.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC₅₀?=?13–15 μg/ml).     

Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies

Aims: To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. Methods and Results: The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca²⁺ ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg⁻¹ protein; it also revealed broad pH and temperature ranges of 5·0 to 8·0 and 25 to 70°C, respectively. The K_m value of phytase for hydrolysis of sodium phytate has been determined as 0·392 mmol l⁻¹. The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95°C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26·81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1·978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a β-propeller structure of phytase. Conclusions: This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. Significance and Impact of the Study: Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity.     

Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli

In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2–10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.     

Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM

The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19.5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4.5. Optimum pH and temperature were found respectively as pH 9.5 and 40 °C. The K_m values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0.31, 0.62, and 1.16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

Expression of human plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli as a soluble protein comprised of active and latent forms. Isolation and crystallization of latent PAI-1

Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of ‘latent’ PAI-1. Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2–376 of the 379 amino acid serpin. Synthetic DNA linkers were ligated to the 5′ and 3′ ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E. coli trp promoter. Transformation of E. coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1. Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted M_r. Unglycosylated PAI-1 from E. coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37°C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator. Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography. The purified latent PAI-1 was crystallized.     

Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation

A codon-optimized Escherichia coli appA phytase gene was synthesized and expressed in Pichia pastoris. Two residue substitutions (Q258N, Q349N) were sequentially introduced to enhance its glycosylation activity. Secretion of appA-Q258N/Q349N was approx. 0.3 mg ml^?1 and enzyme activity reached 1,030 U ml^?1. Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg^?1 with an MW of approx. 53 kDa. Compared with appA-WT, appA-Q258N/Q349N showed over 40 % enhancement in thermostability (85 °C for 10 min) and 4–5 °C increases in the melting temperatures (T_m). The K_m and K_cat of appA-Q258N/Q349N were 0.43 mM and 3,058 s^?1, respectively, which are similar with that of appA-WT. The mutant appA-Q258N/Q349N obtained in this study could be used for the large-scale commercial production of phytase.     

Distinct Promoters Affect Pyrroloquinoline Quinone Production in Recombinant Escherichia coli and Klebsiella pneumoniae

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters—lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)—were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.     

Cloning,expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) is a valuable chiral intermediate for the synthesis of antiemetic drug Aprepitant and Fosaprepitant. A Leifsonia xyli HS0904-derived carbonyl reductase (LXCAR), an effective biocatalyst for the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to (R)-BTPE, was overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis indicated that the amino acid sequence of recombinant LXCAR showed 89 % similarity to short-chain dehydrogenase/reductase. E. coli recombinant carbonyl reductase crude extract showed a specific activity of 1.54 U/mg, which was 62 times higher than that of L. xyli HS0904 crude extract. By using error-prone polymerase chain reaction and site-directed mutagenesis, the engineered LXCAR demonstrated superior catalytic activity toward BTAP, and the obtained mutant LXCAR-S154Y exhibited nearly 13-fold, 5.4-fold, and 2.3-fold increase in k _cat/K _m value, k _cat value, and specific activity toward BTAP, respectively, compared to the recombinant LXCAR. Additionally, the reduction of BTAP by whole cells of mutant LXCAR-S154Y afforded a best yield of 99.6 % for (R)-BTPE within 2 h at 200 mM BTAP, which was shortened by 28 and 2 h compared to those catalyzed by L. xyli HS0904 cells and recombinant E. coli cells expressing LXCAR, respectively. Moreover, a yield of 82.5 % for (R)-BTPE was achieved within 12 h at an increased BTAP concentration of up to 1,000 mM (256 g/l), representing a 1.9-fold increase over the recombinant LXCAR. Homology modeling and docking analysis revealed the molecular basis for the high catalytic activity of mutant LXCAR-S154Y toward BTAP. The results present here provide a promising alternative for economical and efficient production of chiral alcohols by engineered LXCAR.