Enantioselective Nitrilase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis>: Cloning,Heterologous Expression,and Bioreactor Studies

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

Statistical optimization for production of carboxymethylcellulase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> DL-3 by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/DL-3 from rice bran using response surface method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.     

Degradation of trichloroethylene by recombinant E. coli in continuous culture

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h^–1.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design

Summary The structural gene yqhD from a wild-type Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme and the structural gene dhaB from Citrobacter freundii encoding glycerol dehydratase were amplified by using the PCR method. The temperature control expression vector pHsh harboring the yqhD and dhaB genes was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-yqhD). The response surface method (RSM) was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 1,3-propanediol by recombinant strain E. coli JM109. The model estimated that a maximal yield of 1,3-propanediol (43.86 g/l) could be obtained when the concentrations of glycerol, yeast extract and vitamin B₁₂ were set at 61.8 g/l, 6.2 g/l and 49 mg/l, respectively; and the fermentation time was 30 h. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yield of 1,3-propanediol. The yield and productivity under the optimal parameters and process can reach 43.1 g/l and 1.54 g/l/h. Maximum 1,3-propanediol yield of 41.1 g/l was achieved in a 5-l fermenter using the optimized medium. This makes the engineered strain have potential application in the conversion of glycerol to 1,3-propanediol on an industrial scale.     

Comparison of optimal conditions for mass production of carboxymethylcellulase by <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 with other recombinants in pilot-scale bioreactor

The optimal conditions for mass production of carboxymethylcellulase (CMCase) by E. coli JM109/A-68 were investigated and compared with other E. coli JM109 recombinants producing CMCase. The optimal agitation speed and aeration rate for cell growth of E. coli JM109/A- 68 were 500 rpm and 0.50 vvm in a 7 L bioreactor, whereas those for production of CMCase were 416 rpm and 0.95 vvm. The optimal vessel pressures for cell growth as well as production of CMCase in a 100 L bioreactor were 0.04 MPa. The maximal production of CMCase by E. coli JM109/A-68 under the optimized conditions in a 100 L bioreactor was 11.0 times higher than its wild type, B. velezensis A-68. Optimal conditions for mass production of CMCase by recombinants were different from those for wild strains. The higher production of CMCase by E. coli JM109/A-68 and other recombinant of E. coli seemed to result from its higher cell growth under the optimal conditions for dissolved oxygen and its mixed-growth associated production pattern compared to the growthassociated production of B. velezensis A-68.     

Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran

A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.     

Enhanced production of GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose by overexpression of NADPH regenerator in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP⁺-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l⁻¹, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.     

Cloning,Purification and Characterization of an NAD-Dependent <Emphasis Type="SmallCaps">d</Emphasis>-Arabitol Dehydrogenase from Acetic Acid Bacterium, <Emphasis Type="Italic">Acetobacter suboxydans</Emphasis>

d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa. The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate is observed when d-arabitol is used as the substrate (K _m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis.     

Production of l-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P_LP_R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.     

A novel method of producing the pharmaceutical intermediate (R)-2-chloromandelic acid by bioconversion

(R)-2-Chloromandelic acid (R­CM) is one of the chiral building blocks used in the pharmaceutical industry. As a result of screening for microorganisms that asymmetrically hydrolyze racemic 2­chloromandelic acid methyl ester (CMM), Exophiala dermatitidis NBRC6857 was found to produce R­CM at optical purity of 97% ee. The esterase that produces R­CM, EstE, was purified from E. dermatitidis NBRC6857, and the optimal temperature and pH of EstE were 30°C and 7.0, respectively. The estE gene that encodes EstE was isolated and overexpressed in Escherichia coli JM109. The activity of recombinant E. coli JM109 cells overexpressing estE was 553 times higher than that of E. dermatitidis NBRC6857. R­CM was produced at conversion rate of 49% and at optical purity of 97% ee from 10% CMM with 0.45 mg-dry-cell/L recombinant E. coli JM109 cells. Based on these findings, R­CM production by bioconversion of CMM may be of interest for future industrial applications.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine production by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The l-phenylalanine (l-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 10¹⁰ pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ _set = 0.18 h⁻¹ in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).     

Enantioselective synthesis of (S)-2-cyano-2-methylpentanoic acid by nitrilase

The nitrilase gene of Rhodococcus rhodochrous J1 was expressed in Escherichia coli using the expression vector, pKK223-3. The recombinant E. coli JM109 cells hydrolyzed enantioselectively 2-methyl-2-propylmalononitrile to form (S)-2-cyano-2-methylpentanoic acid (CMPA) with 96 % e.e. Under optimized conditions, 80 g (S)-CMPA l^?1 was produced with a molar yield of 97 % at 30 °C after a 24 h without any by-products.     

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.

     

Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli Penicillin G amidase precursor polypeptide

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26°C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotrophic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.     

Construction of a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, <Emphasis Type="Italic">Bacillus velezensis</Emphasis> A-68 in a pilot-scale bioreactor

A gene encoding carboxymethylcellulase (CMCase) of Bacillus velezensis A-68 had been cloned in Escherichia coli JM109. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to be optimal carbon and nitrogen sources for production of CMCase by E. coli JM109/A-68. The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for production of CMCase were established by the response surface methodology (RSM). The concentrations of four salts in the medium, K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄, for production of CMCase also were optimized. The optimal temperatures for cell growth and production of CMCase were 37°C. The maximal production of CMCase by E. coli JM109/A-68 was 880.2 U/mL, which was 10.5 time higher than its wild type, B. velezensis A-68. The production of CMCase by E. coli JM109/A-68 was compared with that by B. velezensis A-68 in a 100 L pilot-scale bioreactor under the optimized conditions. The production of CMCase by E. coli JM109/A-68 was found to be the mixed-growth associated unlike the growthassociated production of CMCase by B. velezensis A-68.     

Upscale production of (R)-mandelic acid with a stereospecific nitrilase in an aqueous system

(R)-Mandelic acid (R-MA) is a key precursor for the synthesis of semi-synthetic penicillin, cephalosporin, anti-obesity drugs, antitumor agents, and chiral resolving agents for the resolution of racemic alcohols and amines. In this study, an enzymatic method for the large-scale production of R-MA by a stereospecific nitrilase in an aqueous system was developed. The nitrilase activity of the Escherichia coli BL21(DE3)/pET-Nit whole cells reached 138.6 U/g in a 20,000-L fermentor. Using recombinant E. coli cells as catalyst, 500 mM R,S-mandelonitrile (R,S-MN) was resolved into 426 mM (64.85 g/L) R-MA within 8 h, and the enantiomeric excess (ee) value of R-MA reached 99%. During the purification process, pure R-MA with a recovery rate of 78.8% was obtained after concentration and crystallization. This study paved the foundation for the upscale production of R-MA using E. coli whole cells as biocatalyst.

     

l-Ribulose production by an Escherichia coli harboring l-arabinose isomerase from Bacillus licheniformis

Recombinant Escherichia coli harboring the l-arabinose isomerase (BLAI) from Bacillus licheniformis was used as a biocatalyst to produce l-ribulose in the presence of borate. Effects of substrate concentration, the borate to l-arabinose ratio, pH, and temperature on the conversion of l-arabinose to l-ribulose were investigated. l-Ribulose production was efficient when pH was higher than 9 and temperature was higher than 50 °C. Borate addition to the reaction mixture was essential for high conversion of l-arabinose to l-ribulose as it resulted in an equilibrium shift in favor of the product. Under the optimal conditions determined by response surface methodology, the E. coli harboring BLAI produced 375 g l⁻¹ L-ribulose from 500 g l⁻¹ l-arabinose at a reaction time of 60 min, corresponding to a conversion yield of 75% and productivity of 375 g l⁻¹ h⁻¹. When the resting recombinant E. coli cells were recycled, 85% of the yield was obtained even after seven cycles of reuse. The productivity and final concentration of l-ribulose obtained in the present study were the highest yet reported.