Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

Influence of bacterial density during preculture on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato

An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD_600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and resuspended in a liquid cocultivation medium-I, adjusted to OD_600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency is stable and highly efficient.     

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low <Emphasis Type="Italic">Agrobacterium</Emphasis> density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD₆₀₀) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD₆₀₀ being more effective for the transformation of Crimson Seedless and 0.2 OD₆₀₀ for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.     

Scale-up of recombinant hirudin production fromSaccharomyces cerevisiae

Scale-up of hirudin production fromSaccharomyces cerevisiae from bench-scale to pilot-scale was carried out based on constant volumetric oxygen transfer coefficient (K _L a). Fed-batch mode of cultivation using step-wise feeding strategy of galactose was employed for the production of hirudin in a 30-L and a 300-L pilot-scale fermentor. The final hirudin concentrations were achieved 390 mg/L and 286.1 mg/L, and the volumetric productivities were 80.4% and 90.7% with the 30-L and 300-L fermentors, respectively, compared to the productivity of the 5-L bench-scale fermentor.     

丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

[目的]探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能.[方法]使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因(trxB)进行插入失活,得到突变株,通过Southern杂交方法验证插入内含子的拷贝数;在基本培养基中进行分批发酵,比较并分析突变株的生长特点;通过pH控制,利用限磷的连续发酵方法使...     

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD₆₀₀ of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD₆₀₀ of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD₆₀₀ of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.     

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.     

Induction of a Futile Embden-Meyerhof-Parnas Pathway in Deinococcus radiodurans by Mn: Possible Role of the Pentose Phosphate Pathway in Cell Survival

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO₂. Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.     

Overexpression of NAD kinase in recombinant <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> harboring the <Emphasis Type="BoldItalic">phbCAB</Emphasis> operon improves poly(3-hydroxybutyrate) production

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y _PHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.     

Impact of different pH control agents on biopesticidal activity of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> during the fermentation of starch industry wastewater

Different pH control agents (NaOH/H₂SO₄—SodSulp, NaOH/CH₃COOH—SodAcet, NH₄OH/CH₃COOH—AmmoAcet and NH₄OH/H₂SO₄—AmmoSulp) were used to investigate their effects on growth, enzyme production (alkaline protease and amylase), and entomotoxicity of Bacillus thuringiensis var. kurstaki HD-1 (Btk) against eastern spruce budworm larvae (Choristoneura fumiferana) using starch industry wastewater (SIW) as a raw material in a 15-l fermentor. AmmoSulp and SodSulp were found to be the best pH control agents for alkaline protease and amylase production, respectively; whereas, the fermented broth obtained by using SodAcet as pH control agents recorded the highest delta-endotoxin production of 1043.0 mg/l and entomotoxicity value 18.4 × 10⁹ SBU/l. Entomotoxicity of re-suspended centrifuged pellet in one-tenth of original volume in case of SodAcet as pH control agents was 26.7 × 10⁹ SBU/l and was the highest value compared to three other pH control agents.     

Evaluation of parameters affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of citrus

An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars. It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 μM acetosyringone were required for improvement of transformation efficiency. Co-cultivation duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency as did OD₆₀₀ value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for maximum transgenic production are required for each individual cultivar.     

Fed-batch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures ofEscherichia coliYK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression, was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically defined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of 0.25 gg⁻¹h⁻¹, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.     

Effect of temperature on growth parameters of psychrophilic bacteria and yeasts

Three bacterial (Pedobacter heparinus, Pedobacter piscium, Pedobacter cryoconitis) and three yeast strains (Saccharomyces cerevisiae, Leucosporidiella creatinivora, Rhodotorula glacialis) of different thermal classes (mesophiles and psychrophiles) were tested for the effect of temperature on a range of growth parameters, including optical density, viable cell numbers, and cell dry mass, in order to determine the temperature conditions under which maximum biomass formation is obtained. Maximum values of growth parameters obtained at the stationary growth phase of the strains were used for statistical calculation. Temperature had a significant (P ≤ 0.05) effect on all growth parameters for each strain; correlations between the growth parameters were significant (P ≤ 0.05–0.01). The maximum growth temperature or the temperature at which microbial growth was fastest was in no case the temperature at which the investigated strains produced the highest amount of biomass. All tested psychrophilic bacteria and yeast strains produced highest amounts of cells (as calculated per mg cell dry mass or per OD₆₀₀ unit) at 1°C, while cell numbers of mesophiles were highest at 20°C. Thus, cultivation temperatures close to the maximum growth temperature are not appropriate for studying psychrophiles.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Strategies for high titre plasmid DNA production in Escherichia coli DH5α

The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD₆₀₀ 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD₆₀₀ 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD₆₀₀ 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 °C), low dO₂) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °C, glucose-/dO₂-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD₆₀₀ of 80.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.