A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli.

BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. CONCLUSION: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

Set-up of a pharmaceutical cell bank of Magnetospirillum gryphiswaldense MSR1 magnetotactic bacteria producing highly pure magnetosomes.

We report the successful fabrication of a pharmaceutical cellular bank (PCB) containing magnetotactic bacteria (MTB), which belong to the Magnetospirillum gryphiswaldense MSR1 species. To produce such PCB, we amplified MTB in a minimal growth medium essentially devoid of other heavy metals than iron and of CMR (Carcinogenic, mutagenic and reprotoxic) products. The PCB enabled to acclimate MTB to such minimal growth conditions and then to produce highly pure magnetosomes composed of more than 99.9% of iron. The qualification of the bank as a PCB relies first on a preserved identity of the MTB compared with the original strain, second on genetic bacterial stability observed over 100 generations or under cryo-preservation for 16 months, third on a high level of purity highlighted by an absence of contaminating microorganisms in the PCB. Furthermore, the PCB was prepared under high-cell load conditions (9.108 cells/mL), allowing large-scale bacterial amplification and magnetosome production. In the future, the PCB could therefore be considered for commercial as well as research orientated applications in nanomedicine. We describe for the first-time conditions for setting-up an effective pharmaceutical cellular bank preserving over time the ability of certain specific cells, i.e. Magnetospirillum gryphiswaldense MSR1 MTB, to produce nano-minerals, i.e. magnetosomes, within a pharmaceutical setting.

Enhancement of Biomass Production of Diatom Nitzschia sp. S5 through Optimisation of Growth Medium Composition and Fed-Batch Cultivation.

The growing commercial application of microalgae in different industry sectors, including the production of bioenergy, pharmaceuticals, nutraceuticals, chemicals, feed, and food, demands large quantities of microalgal biomass with specific compositions produced at reasonable prices. Extensive studies have been carried out on the design of new and improvement of current cultivation systems and the optimisation of growth medium composition for high productivity of microalgal biomass. In this study, the concentrations of the main macronutrients, silicon, nitrogen and phosphorus, essential for the growth of diatom Nitzschia sp. S5 were optimised to obtain a high biomass concentration. The effect of main macronutrients on growth kinetics and cell composition was also studied. Silicon had the most significant effect on diatom growth during batch cultivation. The concentration of biomass increased 5.45-fold (0.49 g L-1) at 1 mM silicon concentration in modified growth medium compared to the original Guillard f/2 medium. Optimisation of silicon, nitrogen, and phosphorus quantities and ratios further increased biomass concentration. The molar ratio of Si:N:P = 7:23:1 mol:mol:mol yielded the highest biomass concentration of 0.73 g L-1. Finally, the fed-batch diatom cultivation of diatom using an optimised Guillard f/2 growth medium with four additions of concentrated macronutrient solution resulted in 1.63 g L-1 of microalgal biomass. The proteins were the most abundant macromolecules in microalgal biomass, with a lower content of carbohydrates and lipids under all studied conditions.

Systematic metabolic engineering of Escherichia coli for the enhanced production of cinnamaldehyde.

Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.

Enhancement of phycocyanin productivity and thermostability from Arthrospira platensis using organic acids.

Intracellular hyperaccumulation of phycocyanin (PC) and its high susceptibility to degradation at higher temperatures are major challenging problems associated with its production from cyanobacteria. The present study evaluated different concentrations of organic acids (1, 2, and 3 mM) (citric acid, acetic acid, succinic acid, fumaric acid, and oxalic acid) under fed-batch mode on the biomass and phycobiliproteins' production from Arthrospira platensis. Besides they were evaluated at 2.5-7.5 mM as preservative to stabilize PC at high temperatures. The incorporation of 3 mM of succinic acid into the cultivation medium enhanced the biomass and PC productivity to 164.05 and 26.70 mg L-1 day-1, which was ~ 2- and threefold higher than control, respectively. The produced PC in this treatment was food-grade with a 2.2 purity ratio. The use of organic acids also enhanced the thermal stability of PC. Citric acid (7.5 mM) markedly promoted the half-life values of PC to 189.44 min compared to 71.84 min in the control. The thermodynamic analysis confirmed higher thermostability of PC in the presence of organic acids and indicated the endothermic and non-spontaneity of the thermal denaturation process. The findings of the present study confirmed that organic acids could be utilized as cost effective and sustainable compounds for promoting not only phycobiliproteins' production but also the thermostability of PC for potential application in food industry.

Utilization of delactosed whey permeate for the synthesis of ethyl acetate with Kluyveromyces marxianus.

Ethyl acetate is an important organic solvent and currently produced from fossil carbon resources. Microbial synthesis of this ester from sugar-rich waste could be an interesting alternative. Therefore, synthesis of ethyl acetate by Kluyveromyces marxinanus DSM 5422 from delactosed whey permeate (DWP) was studied in an aerated stirred bioreactor at 40 °C. DWP is mainly composed of residual lactose and minerals. The minerals inhibited yeast growth, as witnessed by an increased lag period, a reduced growth rate, and an extended process duration. All experiments were therefore carried out with diluted DWP. In a series of batch experiments, the pH of iron-deficient DWP medium varied between 4.8 and 5.9. The pH of the cultivation medium significantly influenced cell growth and product syntheses, with the highest ethyl acetate yield of 0.347 g g-1 and lowest by-product formation achieved at pH 5.1. It is likely that this effect is due to pH-dependent iron chelation, which affects the iron bioavailability and the intracellular iron content, thus affecting growth and metabolite synthesis. The viability of yeast cells was always high despite the harsh conditions in DWP medium, which enabled extended usage of the biomass in repeated-batch and fed-batch cultivations. These two culture techniques increased the volume of DWP processed per time by 32 and 84% for the repeated-batch and the fed-batch cultivation, respectively, without a drop of the ester yield. KEY POINTS: • Delactosed whey permeate was converted to ethyl acetate with a high rate and yield. • The formation of ethyl acetate in DWP medium at iron limitation is pH-dependent. • Highly active yeasts from batch processes enabled extension as fed and repeated batch.

Enhanced production of Bacopa saponins by repeated batch strategy in bioreactor.

Cultivation of cell suspension culture of Bacopa monnieri targeting the production of bacosides was explored in a 5-l stirred tank reactor using statistically optimized conditions. The bioreactor cultivation conditions were modified and this led to profuse biomass growth (2.81 ± 0.20 g/l) and total bacosides (1.26 ± 0.23 mg/g in cells and 0.60 ± 0.11 mg/l in fermenter broth) production in 9 days. The values of static volumetric mass transfer coefficient (kLa), dimensionless mixing time (Nm) were measured in the bioreactor. The culture grew efficiently and produced enhanced amount of bacoside A (5.59 ± 0.41 mg/g total bacosides in cells and 3.12 ± 0.13 mg/l in the fermenter broth) using one cycle of repeated batch strategy adopted in the bioreactor for 15 days. The intracellular concentration of bacoside A3 (1.18 ± 0.11 mg/g), bacopaside II (2.09 ± 0.35 mg/g), bacopaside X (0.79 ± 0.17 mg/g) and bacopasaponin C (2.24 ± 0.23 mg/g) were significantly higher in repeated batch as compared to batch bioreactor cultivation. The yield of total bacosides in the fermenter broth was 5-times higher in repeated batch as compared to batch cultivation. This strategy can be helpful for the enhanced production of other valuable triterpenoid saponins.

Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli.

Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.

Batch fermentation kinetics of acetoin produced by Bacillus subtilis HB-32.

OBJECTIVES: The aim of this work was to study the changes of bacterial cell growth, acetion formation and glucose consumption with fermentation time during batch cultivation. RESULTS: A mathematical model of cell growth, product synthesis, and substrate consumption changes with time during the batch cultivation of acetion was established. By analyzing the fitting curve of the kinetic model, it is found that the calculated value of the model fits well with the experimental value, and the fitting model R2 is greater than 0.98. CONCLUSIONS: The kinetic model established in this experiment can better reflect the batch cultivation process of acetion.

Cultivation technology development of Rhodothermus marinus DSM 16675.

This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.

Biosynthesis of isocitric acid in repeated-batch culture and testing of its stress-protective activity.

Biosynthesis of Ds(+)-threo-isocitric acid from ethanol in the Yarrowia lipolytica batch and repeated-batch cultures was studied. Repeated-batch cultivation was found to provide for a good biosynthetic efficiency of the producer for as long as 748 h, probably due to maintenance of high activities of enzymes involved in the biosynthesis of isocitric acid. Under optimal repeated-batch cultivation conditions, the producer accumulated 109.6 g/L Ds(+)-threo-isocitric acid with a production rate of 1.346 g/L h. The monopotassium salt of isocitric acid isolated from the culture liquid and purified to 99.9% was found to remove neurointoxication, to restore memory, and to improve the learning of laboratory rats intoxicated with lead and molybdenum salts. Taking into account the fact that the neurotoxic effect of heavy metals is mainly determined by oxidative stress, the aforementioned favorable action of isocitric acid on the intoxicated rats can be explained by its antioxidant activity among other pharmacological effects.

Phototrophic cultivation of NaCl-tolerant mutant of Spirulina platensis for enhanced C-phycocyanin production under optimized culture conditions and its dynamic modeling.

Commercial cultivation of Spirulina sp. is highly popular due to the presence of high amount of C-phycocyanin (C-PC) and other valuable chemicals like carotenoids and γ-linolenic acid. In this study, the pH and the concentrations of nitrogen and carbon source were manipulated to achieve improved cell growth and C-PC production in NaCl-tolerant mutant of Spirulina platensis. In this study, highest C-PC (147 mg · L-1 ) and biomass (2.83 g · L-1 ) production was achieved when a NaCl-tolerant mutant of S. platensis was cultivated in a nitrate and bicarbonate sufficient medium (40 and 60 mM, respectively) at pH 9.0 under phototrophic conditions. Kinetic study of wildtype S. platensis and its NaCl-tolerant mutant was also done to determine optimum nitrate concentrations for maximum growth and C-PC production. Kinetic parameter of inhibition (Haldane model) was fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that the maximum specific growth rate (µmax ) for NaCl-tolerant mutant increased by 17.94% as compared to its wildtype counterpart, with a slight increase in half-saturation constant (Ks ), indicating that this strain could grow well at high concentration of NaNO3 . C-PC production rate (Cmax ) in mutant cells increased by 12.2% at almost half the value of Ks as compared to its wildtype counterpart. Moreover, the inhibition constant (Ki ) value was 207.85% higher in NaCl-tolerant mutant as compared to its wildtype strain, suggesting its ability to produce C-PC even at high concentrations of NaNO3 .

Importance of the cultivation history for the response of Escherichia coli to oscillations in scale-down experiments.

Large-scale bioreactors are inhomogeneous systems, in which the fluid phase expresses concentration gradients. They depend on the mass transfer and fluid dynamics in the reactor, the feeding strategy, the cell-specific substrate uptake parameters, and the cell density. As high cell densities are only obtained at low specific growth rates, it is necessary to investigate the cellular responses to oscillations in particular under such conditions, an issue which is mostly neglected. Instead, the feed oscillations are often started directly after the batch phase, when the specific growth rate is close to the maximum. We show here that the cultivation mode before oscillations are started has a tremendous effect on the metabolic responses. In difference to cells, which were pre-grown under batch conditions at a high growth rate, Escherichia coli cells that were pre-grown under glucose limitation at a low growth rate accumulate short-chain fatty acids (acetate, lactate, succinate) and glycolysis-related amino acids to a higher extent in a two-compartment scale-down bioreactor. Thus, cells which enter oscillations from a lower specific growth rate seem to react more sensitive to oscillations than cells that are subjected to oscillations directly after a batch phase. These results are interesting in designing reliable scale-down systems, which better reflect large-scale bioprocesses.

Biomarkers allow detection of nutrient limitations and respective supplementation for elimination in Pichia pastoris fed-batch cultures.

BACKGROUND: Industrial processes for recombinant protein production challenge production hosts, such as the yeast Pichia pastoris, on multiple levels. During a common P. pastoris fed-batch process, cells experience strong adaptations to different metabolic states or suffer from environmental stresses due to high cell density cultivation. Additionally, recombinant protein production and nutrient limitations are challenging in these processes. RESULTS: Pichia pastoris producing porcine carboxypeptidase B (CpB) was cultivated in glucose or methanol-limited fed-batch mode, and the cellular response was analyzed using microarrays. Thereby, strong transcriptional regulations in transport-, regulatory- and metabolic processes connected to sulfur, phosphorus and nitrogen metabolism became obvious. The induction of these genes was observed in both glucose- and methanol- limited fed batch cultivations, but were stronger in the latter condition. As the transcriptional pattern was indicative for nutrient limitations, we performed fed-batch cultivations where we added the respective nutrients and compared them to non-supplemented cultures regarding cell growth, productivity and expression levels of selected biomarker genes. In the non-supplemented reference cultures we observed a strong increase in transcript levels of up to 89-fold for phosphorus limitation marker genes in the late fed-batch phase. Transcript levels of sulfur limitation marker genes were up to 35-fold increased. By addition of (NH4)2SO4 or (NH4)2HPO4, respectively, we were able to suppress the transcriptional response of the marker genes to levels initially observed at the start of the fed batch. Additionally, supplementation had also a positive impact on biomass generation and recombinant protein production. Supplementation with (NH4)2SO4 led to 5% increase in biomass and 52% higher CpB activity in the supernatant, compared to the non-supplemented reference cultivations. In (NH4)2HPO4 supplemented cultures 9% higher biomass concentrations and 60% more CpB activity were reached. CONCLUSIONS: Transcriptional analysis of P. pastoris fed-batch cultivations led to the identification of nutrient limitations in the later phases, and respective biomarker genes for indication of limitations. Supplementation of the cultivation media with those nutrients eliminated the limitations on the transcriptional level, and was also shown to enhance productivity of a recombinant protein. The biomarker genes are versatily applicable to media and process optimization approaches, where tailor-made solutions are envisioned.

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

Fed-batch production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in soluble form in Escherichia coli and its purification and characterization.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple two-step chromatographic procedure. To improve the soluble yield, expression of TRAIL in Escherichia coli was induced with low IPTG concentration (0.1 mM) at low temperature (28 °C) supplemented with ZnSO4 (0.5 mM), using glycerol as carbon source. Under the optimized conditions, 4.14 ± 0.19 g/L of TRAIL in soluble form was achieved at 19 h without pure oxygen. To purify the recombinant TRAIL, we developed an efficient two-step chromatographic procedure including affinity chromatography and cation-exchange chromatography, especially improved the cation-exchange chromatography using a combination of pH and NaCl gradients strategy. Consequently, 4313.5 mg of target protein with high purity (98.1%) was obtained from 2.3 L of cell broth. Our results also showed that the purified TRAIL was with ordered secondary and tertiary structures, in homogeneous form and with strong cytotoxicity.

Rewiring a secondary metabolite pathway towards itaconic acid production in Aspergillus niger.

BACKGROUND: The industrially relevant filamentous fungus Aspergillus niger is widely used in industry for its secretion capabilities of enzymes and organic acids. Biotechnologically produced organic acids promise to be an attractive alternative for the chemical industry to replace petrochemicals. Itaconic acid (IA) has been identified as one of the top twelve building block chemicals which have high potential to be produced by biotechnological means. The IA biosynthesis cluster (cadA, mttA and mfsA) has been elucidated in its natural producer Aspergillus terreus and transferred to A. niger to enable IA production. Here we report the rewiring of a secondary metabolite pathway towards further improved IA production through the overexpression of a putative cytosolic citrate synthase citB in a A. niger strain carrying the IA biosynthesis cluster. RESULTS: We have previously shown that expression of cadA from A. terreus results in itaconic acid production in A. niger AB1.13, albeit at low levels. This low-level production is boosted fivefold by the overexpression of mttA and mfsA in itaconic acid producing AB1.13 CAD background strains. Controlled batch cultivations with AB1.13 CAD + MFS + MTT strains showed increased production of itaconic acid compared with AB1.13 CAD strain. Moreover, preliminary RNA-Seq analysis of an itaconic acid producing AB1.13 CAD strain has led to the identification of the putative cytosolic citrate synthase citB which was induced in an IA producing strain. We have overexpressed citB in a AB1.13 CAD + MFS + MTT strain and by doing so hypothesize to have targeted itaconic acid production to the cytosolic compartment. By overexpressing citB in AB1.13 CAD + MFS + MTT strains in controlled batch cultivations we have achieved highly increased titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h while no CA was produced. CONCLUSIONS: Expression of the IA biosynthesis cluster in Aspergillus niger AB1.13 strain enables IA production. Moreover, in the AB1.13 CAD strain IA production resulted in overexpression of a putative cytosolic citrate synthase citB. Upon overexpression of citB we have achieved titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h in controlled batch cultivations. By overexpressing citB we have also diminished side product formation and optimized the production pathway towards IA.

Production of extracellular fructans by Gluconobacter nephelii P1464.

UNLABELLED: Bacterial extracellular fructans, known as levans, have potential applications in food, pharmaceutical and cosmetic industries and high fructan producing strains could contribute into the cost reduction and more extensive commercial usage of them. An acetic acid bacteria (AAB) isolate P1464 was obtained from the Microbial Strain Collection of Institute of Microbiology and Biotechnology, University of Latvia and identified as Gluconobacter nephelii by DNA-DNA hybridization and the formation of extracellular fructans by this strain was confirmed. Isolated extracellular fructose polymers were characterized using FT-IR spectroscopy and the structural features of fructan appeared as similar to the reference sample of bacterial levan. Molecular mass estimates showed that the isolated G. nephelii P1464 fructose polymer has a relatively small molecular weight (Mw 1122·939 ± 153·453 kDa) and a sizeable polydispersity (Mw/Mn = 21·57 ± 1·60), as compared with other AAB, which could promote their physiological activity, including the prebiotic effects. Obtained at different cultivation conditions characteristics of fructan production, including the biotechnological indices such as the productivity (Qp) and yield (Yp/s) ranging from 0·774 to 1·244 g l(-1)  h and from 0·181 to 0·436 g g(-1) , respectively, confirmed, that G. nephelii P1464 could be used as promising strain for commercial production of levan. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial fructans, known as levans, have extensive options for practical usage, however, actually limited due to high production costs. Therefore, the searches for efficient producer strains should be an urgent task to reduce costs. This study is the first report on the formation of fructans by a novel strain of acetic acid bacteria (AAB) Gluconobacter nephelii P1464. Characteristics obtained at different cultivation conditions confirmed the operation of a competitive and perspective producer strain. Isolated extracellular fructans are characterized by a lower molecular weight as compared with other AAB which could promote their physiological activity, including the prebiotic effects.

Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation.

The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett-Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.

Capsular polysaccharide production by Streptococcus pneumoniae serotype 1: from strain selection to fed-batch cultivation.

Streptococcus pneumoniae is a human pathogen largely transmitted by aerosols. Vaccines are the main strategy against this pathogen, and the capsular polysaccharide (PS) is its major antigen. S. pneumoniae serotype 1 is associated with large outbreaks and epidemics of invasive diseases. The aims of this work were to screen serotype 1 strains to identify the best PS1 producer, evaluate three peptones for PS1 production, investigate the effects of culture medium components using a design of experiments (DoE), a statistic tool for optimization, and propose a new medium/cultivation strategy. After flask cultivation of nine strains, two that produced high PS1 and biomass values were chosen for further evaluation in the bioreactor, and ST595/01 was chosen as the best PS1 producer strain. Among the peptones tested (Casamino acids, Soytone, and Phytone), the highest PS1 production (298 mg/L) was reached with Phytone. Next, DoE (2(4-1)) was performed to evaluate the effects of yeast extract (YE), Phytone, L-asparagine (Asn), and L-glutamine (Gln), yielding the following results: Phytone presented positive effects (p < 0.05) for maximum production of biomass, PS1, acetate, and lactate; YE showed positive effects for biomass and acid production (p < 0.05); Gln exerted a minor positive effect on PS1 yield factor on glucose (p < 0.1); and Asn presented only an effect on acetate production (p < 0.1). Hence, a new culture medium was formulated based on Phytone, YE, and glucose, and batch and fed-batch cultivations were evaluated. The fed-batch cultivation showed almost 2 times the biomass and 2.5 times the PS1 production as the batch culture, and 8-10 times higher PS1 production than has been previously reported.