Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

Production of yeast chitin–glucan complex from biodiesel industry byproduct

Crude glycerol from the biodiesel industry was used as carbon source for high cell density fed-batch cultivation of Pichia pastoris aiming at producing a chitin–glucan complex (CGC). More than 100 g L^?1 biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g g^?1 during the batch phase and 0.63 g g^?1 during the fed-batch phase. The chitin–glucan complex was recovered from the yeast cell wall by hot alkaline extraction. CGC content in the cell wall was found to be relatively constant throughout the cultivation (18–26%) with a volumetric productivity of 1.28 g L^?1 h^?1 at the end of the fed-batch phase. The molar ratio of chitin:β-glucan in the extracted biopolymer was 16:84, close to other CGC extracted from Aspergillus biomass. The extracted polymer was characterized by Differential Scanning Calorimetry (DCS) and solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and compared with commercial biopolymers, namely, crab shell chitin and/or chitosan, algal β-glucan (laminarin) and fungal chitin–glucan complex (kiOsmetine).     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ_crit) of 0.12 h⁻¹ at 14 L scale, the biomass yield coefficient (Y_x/s) remained constant at 0.4 g g⁻¹ with no ethanol production, whereas ethanol yields relative to biomass (k_eth/x) of up to 0.54 g g⁻¹ and a steady decrease in Y_x/s were observed at μ > 0.12 h⁻¹. Production of α-glucuronidase was growth associated at a product yield (k_α-glu/x) of 0.45 mg g⁻¹, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ_crit. Scale-up with constant k_La from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ = 0.12 h⁻¹. At this scale, α-glucuronidase yield could be maximised at growth rates below μ_crit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Optimization of fermentation parameters for the biomass and DHA production of Schizochytrium limacinum OUC88 using response surface methodology

Fermentation parameters for biomass and DHA production of Schizochytrium limacinum OUC88 in a fermenter (working volume 7 L) were optimized using Plackett–Burman and central composite rotatable design. Out of 10 factors studied by Plackett–Burman design, 4 influenced the biomass production significantly. Central composite rotatable design was used to optimize the significant factors and response surface plots were generated. Using these response surface plots and point prediction, optimized values of the factors were determined as follows temperature (°C) 23 °C, aeration rate 1.48 L min⁻¹ L⁻¹, agitation 250 rpm and inoculum cells in mid-exponential phase, the maximum yield of DCW and DHA were 24.1 and 4.7 g L⁻¹, respectively. These predicted values were also verified by validation experiments.     

Bioconversion of spent coffee grounds into carotenoids and other valuable metabolites by selected red yeast strains

Spent coffee grounds (SCG) represent the main coffee industry residues with a great potential to be reutilized in various biotechnological processes. In this study, several carotenogenic yeasts strains were exploited for the production of vitamin-enriched biomass, cultivating in SCG-based media. The fermentation was firstly carried out in Erlenmeyer flasks in order to select the best biomass and pigment producer. Among four tested strains, Sporobolomyces roseus showed the highest potential for the accumulation of carotenoids. Maximum pigment concentration and yield was obtained when cultivating in SCG-based media, 12.59 mg l⁻¹ and 1.26 mg g⁻¹, respectively. Comparing both, the batch and the fed-batch cultivation modes, the strategy of sequential addition of pre-concentrated SCG media in the bioreactor gave higher biomass yield (maximum 41 g l⁻¹ during 41–48 h after the beginning of fermentation). Thus, SCG can be considered as potentially promising industrial waste stream for economically feasible production of enriched yeasts biomass.     

Impacts of monosodium glutamate industrial wastewater on plant growth and soil characteristics

Research into utilization of monosodium glutamate industrial wastewater (MSGW) as a plant nutrient source was undertaken. The physico-chemical and microbiological characteristics of MSGW were analyzed in detail. Effect of MSGW on early growth of Chinese cabbage (Brassica rapa L. cv. Pekinensis) and maize (Zea mays L. cv. Bright Jean) was tested by the seed germination bioassay. Subsequently, in a greenhouse pot experiment using the same plant species, effects of MSGW application rates on the plant biomass yield, nitrogen content and soil properties were analyzed. The MSGW was characterized by high levels of N (56.7 g l^?1), organic C (344.6 g l^?1), total solids (600 g l^?1) and other minerals. At MSGW concentrations below 1%, germination indices for both the plant species were significantly (p < 0.01) higher than the control. Further, the greenhouse study results indicated significant increase in the plant biomass yield at MSGW application rates of 5000 and 7500 l ha^?1. As the MSGW dose increased, the biomass yield decreased, decreasing the N-use efficiency. Maize showed significantly higher wastewater N-use efficiency compared to the Chinese cabbage. Although the total culturable bacterial and fungal counts in the raw MSGW were low, addition of MSGW to the soil increased the soil microbial activities and soil respiration. Soil organic C was also increased by the addition of MSGW, due to the presence of significant amounts of organic C in the wastewater. This preliminary study demonstrates that by proper management of the pH and optimization of application rate, MSGW can be utilized as a nutrient source for plant growth. Further long-term field studies to evaluate the environmental impact of MSGW usage in agriculture are being designed to reduce the environmental risks associated with the reuse of this underutilized wastewater in the agriculture.     

Removal of Cu(II) ions by biosorption onto powdered waste sludge (PWS) prior to biological treatment in an activated sludge unit: A statistical design approach

Biological treatment of synthetic wastewater containing Cu(II) ions was realized in an activated sludge unit with pre-adsorption of Cu(II) onto powdered waste sludge (PWS). Box-Behnken experimental design method was used to investigate Cu(II), chemical oxygen demand (COD) and toxicity removal performance of the activated sludge unit under different operating conditions. The independent variables were the solids retention time (SRT, 5–30 d), hydraulic residence time (HRT, 5–25 h), feed Cu(II) concentration (0–50 mg L^?1) and PWS loading rate (0–4 g h^?1) while percent Cu(II), COD, toxicity (TOX) removals and the sludge volume index (SVI) were the objective functions. The data were correlated with a quadratic response function (R² = 0.99). Cu(II), COD and toxicity removals increased with increasing PWS loading rate and SRT while decreasing with the increasing feed Cu(II) concentration and HRT. Optimum conditions resulting in maximum Cu(II), COD, toxicity removals and SVI values were found to be SRT of 30 d, HRT 15 h, PWS loading rate 3 g h^?1 and feed Cu(II) concentration of less than 30 mg L^?1.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).     

An annual cycle of biomass and productivity of Vallisneria americana in a subtropical spring-fed estuary

An annual cycle of biomass and productivity of wild celery (Vallisneria americana) was studied in Kings Bay, FL, USA. In situ growth rates were measured monthly between March 2001 and June 2002 in high-density stands, using a modified hole-punching technique, and applied to shoot density data to obtain areal estimates of production. Mean shoot density varied greatly over the study period, ranging between 200 and 800 shoots m⁻². Mean total biomass ranged between 162 and 1013 g m⁻², with aboveground material comprising, on average, 70% of total biomass. Total annual estimated production of new attached shoots was 519 g m⁻². Leaf growth rates peaked at >50 mg shoot⁻¹ d⁻¹, and mass-specific leaf growth ranged 0.6–1.8% d⁻¹. Annually, individual shoots produced 7.4 g of leaf material and completely replaced standing leaf biomass 3.5 times. Areal leaf production was highest in late spring/summer of 2001, and ranged between 3.6 and 23.0 g m⁻² d⁻¹. Annual total leaf production was 2704 g m⁻². Seasonality was not apparent in most variables monitored monthly; only 1 of the 64 relationships we examined between environmental variables (nutrients, chlorophyll a, and irradiance) and Vallisneria biological variables were significant, with relative growth rate increasing linearly with irradiance. Peak biomass and productivity of Vallisneria in Kings Bay were high compared to literature values for other Vallisneria populations as well as global averages for well-studied seagrasses, emphasizing the potential importance of Vallisneria to whole ecosystem functioning in springs, lakes, and oligohaline reaches of many estuaries.     

High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor

Haematococcus pluvialis was cultivated under photoautotrophic conditions in a bubble column with fed-batch addition of nutrients, especially nitrate, and a cell number above 5 × 10⁶ cells mL⁻¹ was attained after 300 h.The reduction of nutrient concentrations accompanied by dilution of the fermentation broth and an increase in the light intensity enhanced accumulation of astaxanthin. The final astaxanthin concentration of 390 mg L⁻¹ was several times higher than ever reported. This combination of fed-batch addition of nutrients and dilution of broth for nutrient deficiency is a promising method for attainment of high cell and astaxanthin concentrations in a bubble column photobioreactor.     

New integrated bioprocess for the continuous production,extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor

Recently, a bubbleless membrane bioreactor (BMBR) has been successfully developed for biosurfactant production by Bacillus subtilis [1]. In this study, for the first time, continuous culture were carried out for the production of surfactin in a BMBR, both with or without a coupled microfiltration membrane. Results from continuous culture showed that a significant part of biomass was immobilized onto the air/liquid membrane contactor. Immobilized biomass activity onto the air/liquid membrane contactor was monitored using a respirometric analysis. Kinetics of growth, surfactin and primary metabolites production were investigated. Planktonic biomass, immobilized biomass and surfactin production and productivity obtained in batch culture (3 L) of 1.5 days of culture were 4.5 g DW, 1.3 g DW, 1.8 g and 17.4 mg L^?1 h^?1, respectively. In continuous culture without total cell recycling (TCR), the planktonic biomass was leached, but immobilized biomass reached a steady state at an estimated 6.6 g DW. 11.5 g of surfactin was produced after 3 days of culture, this gave an average surfactin productivity of 54.7 mg L^?1 h^?1 for the continuous culture, which presented a surfactin productivity of 30 mg L^?1 h^?1 at the steady state. TCR was then investigated for the continuous production, extraction and purification of surfactin using a coupled ultrafiltration step. In continuous culture with TCR at a dilution rate of 0.1 h^?1, planktonic biomass, immobilized biomass, surfactin production and productivity reached 7.5 g DW, 5.5 g DW, 7.1 g and 41.6 mg L^?1 h^?1 respectively, after 2 days of culture. After this time, biomass and surfactin productions stopped. Increasing dilution rate to 0.2 h^?1 led to the resumption of biomass and surfactin production and these values reached 11.1 g DW, 10.5 g DW, 7.9 g and 110.1 mg L^?1 h^?1, respectively, after 3 days of culture. This study has therefore shown that with this new integrated bioprocess, it was possible to continuously extract and purify several grams of biosurfactant, with purity up to 95%.     

Effects of phosphorus and nitrogen amendments on the growth of Egeria najas

The effect of nutrient addition on the growth of E. najas was evaluated in a dose response experiment using sand amended with phosphorus (P) and nitrogen (N), and in enrichment trials with N and P amendments to natural sediments. Plants, water and sediment came from lagoons of the Upper Paraná River Floodplain and from Itaipu Reservoir (Brazil). Relative growth rates (RGRs) of E. najas shoots, based on dry mass (DM), varied from 0.03 to 0.060 d⁻¹ for both nutrients. Root:shoot biomass ratios were related to sediment exchangeable P (r = −0.419; P = 0.03) and N (r = −0.54; P = 0.006), however root RGR was not related to sediment nutrient concentrations. When natural sediments were amended with N and P, neither shoot nor root RGRs differed among treatments for substrata from either the reservoir or the floodplain lagoons (P > 0.05). Comparison of nutrient concentrations measured in natural sediments collected from several sites in both the Upper Paraná River Floodplain (range 49–213 μg P g⁻¹ DM; 36–373 μg N g⁻¹ DM) and Itaipu Reservoir (range 43–402 μg P g⁻¹ DM; 7.9–238 μg N g⁻¹ DM) showed that sediment N and P from these systems usually exceeded minimum requirements necessary for E. najas growth, as measured in the dose response experiment. Together, these results indicate that E. najas, at least in early stages of development, responds to sediment nutrient amendments and relies upon bottom sediments to meet its N and P requirements and that for at least two Brazilian ecosystems, growth of this species is not limited by insufficient sediment N or P. Thus, reducing N and P in water is not enough to control E. najas growth in short time periods in these ecosystems.     

Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000 mM) in a space-time yield per gram of biomass (DCW) of 22.9 mmol L⁻¹ h⁻¹ g DCW⁻¹ at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.     

Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h⁻¹, 1.4 g PCI L⁻¹ were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h⁻¹, and 2.0-fold compared to μ = 0.25 h⁻¹. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.     

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(β1-3)Gal(β1-4)Glc).In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L⁻¹ culture) was 3.6-times higher compared to cultivation on D-glucose.Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (μ = 0.05) was maintained by an exponential galactose feed. In total, 16 g L⁻¹ lactose were fed and resulted in final yields of 12.72 ± 0.21 g L⁻¹ lacto-N-tetraose and 13.70 ± 0.10 g L⁻¹ lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L⁻¹ h⁻¹.