Exploiting phenotypic heterogeneity to improve production of glutathione by yeast.

BACKGROUND: Gene expression noise (variation in gene expression among individual cells of a genetically uniform cell population) can result in heterogenous metabolite production by industrial microorganisms, with cultures containing both low- and high-producing cells. The presence of low-producing individuals may be a factor limiting the potential for high yields. This study tested the hypothesis that low-producing variants in yeast cell populations can be continuously counter-selected, to increase net production of glutathione (GSH) as an exemplar product. RESULTS: A counter-selection system was engineered in Saccharomyces cerevisiae based on the known feedback inhibition of gamma-glutamylcysteine synthetase (GSH1) gene expression, which is rate limiting for GSH synthesis: the GSH1 ORF and the counter-selectable marker GAP1 were expressed under control of the TEF1 and GSH-regulated GSH1 promoters, respectively. An 18% increase in the mean cellular GSH level was achieved in cultures of the engineered strain supplemented with D-histidine to counter-select cells with high GAP1 expression (i.e. low GSH-producing cells). The phenotype was non-heritable and did not arise from a generic response to D-histidine, unlike that with certain other test-constructs prepared with alternative markers. CONCLUSIONS: The results corroborate that the system developed here improves GSH production by targeting low-producing cells. This supports the potential for exploiting end-product/promoter interactions to enrich high-producing cells in phenotypically heterogeneous populations, in order to improve metabolite production by yeast.

Harnessing artificial intelligence-driven approach for enhanced indole-3-acetic acid from the newly isolated Streptomyces rutgersensis AW08.

Indole-3-acetic acid (IAA) derived from Actinobacteria fermentations on agro-wastes constitutes a safer and low-cost alternative to synthetic IAA. This study aims to select a high IAA-producing Streptomyces-like strain isolated from Lake Oubeira sediments (El Kala, Algeria) for further investigations (i.e., 16S rRNA gene barcoding and process optimization). Subsequently, artificial intelligence-based approaches were employed to maximize IAA bioproduction on spent coffee grounds as high-value-added feedstock. The specificity was the novel application of the Limited-Memory Broyden-Fletcher-Goldfarb-Shanno Box (L-BFGS-B) optimization algorithm. The new strain AW08 was a significant producer of IAA (26.116 ± 0.61 µg/mL) and was identified as Streptomyces rutgersensis by 16S rRNA gene barcoding and phylogenetic inquiry. The empirical data involved the inoculation of AW08 in various cultural conditions according to a four-factor Box Behnken Design matrix (BBD) of Response surface methodology (RSM). The input parameters and regression equation extracted from the RSM-BBD were the basis for implementing and training the L-BFGS-B algorithm. Upon training the model, the optimal conditions suggested by the BBD and L-BFGS-B algorithm were, respectively, L-Trp (X1) = 0.58 %; 0.57 %; T° (X2) = 26.37 °C; 28.19 °C; pH (X3) = 7.75; 8.59; and carbon source (X4) = 30 %; 33.29 %, with the predicted response IAA (Y) = 152.8; 169.18 µg/mL). Our findings emphasize the potential of the multifunctional S. rutgersensis AW08, isolated and reported for the first time in Algeria, as a robust producer of IAA. Validation investigations using the bioprocess parameters provided by the L-BFGS-B and the BBD-RSM models demonstrate the effectiveness of AI-driven optimization in maximizing IAA output by 5.43-fold and 4.2-fold, respectively. This study constitutes the first paper reporting a novel interdisciplinary approach and providing insights into biotechnological advancements. These results support for the first time a reasonable approach for valorizing spent coffee grounds as feedstock for sustainable and economic IAA production from S. rutgersensis AW08.

Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells.

Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity of endogenously synthesizing cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells' reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.

Stapled NRPS enhances the production of valinomycin in Escherichia coli.

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases-NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.

Enhanced Production of Gamma-Aminobutyric Acid (GABA) from Lactobacillus futsaii CS3 Using Agri-Food Industries By-Products Under Batch and Fed-Batch Fermentation.

Gamma-aminobutyric acid (GABA) has diverse physiological functions, but its production by lactic acid bacteria is costly due to the culture medium. This study aimed to enhance GABA production by L. futsaii CS3 using low-cost substrates and agri-food industries by-products. Optimal culture conditions were determined using response surface methodology with a central composite design (CCD). Batch and fed-batch fermentation techniques were employed. In the MRS medium with 2% (w/v) monosodium glutamate (MSG), L. futsaii CS3 produced 6.84 g/l of GABA. Further optimization revealed that 2% (w/v) cane sugar resulted in a maximum GABA production of 9.6 g/l, while cane molasses yielded 7.4 g/l. The modified MRS medium with 2% (w/v) MSG, 2% (w/v) cane sugar, 3.06% (w/v) tuna condensate, and 2.5% (w/v) surimi washing water exhibited the highest GABA concentration of 11 g/l. Surimi washing water had a lower GABA concentration of 4.12 g/l. Critical factors identified through CCD analysis were cane sugar, tuna condensate, and MSG. The optimized modified MRS medium consisted of 3.48% (w/v) cane sugar, 3.84% (w/v) tuna condensate, and 10.77% (w/v) MSG, resulting in an actual GABA concentration of 18.27 g/l. Under flask-scale and batch fermentation conditions (initial pH 5, temperature 37 °C), GABA concentrations of 20.63 g/l and 17.24 g/l were obtained after 48 h, respectively. In fed-batch fermentation, GABA concentrations reached 23.01 g/l at 72 h. The addition of cane sugar and tuna condensate effectively enhanced GABA production in L. futsaii CS3, highlighting their suitability as cost-effective substrates for industrial-scale GABA production.

Optimization of the production process for the anticancer lead compound illudin M: process development in stirred tank bioreactors.

BACKGROUND: The fungal natural products illudin S and M have been investigated as precursors for the development of semisynthetic anticancer agents such as Irofulven (illudin S derivative) which is currently in phase II clinical trials. Recently, illudin M derivatives have shown improved in vitro selectivity towards cancer cells encouraging further investigation. This requires a stable supply of the precursor which is produced by Basidiomycota of the genus Omphalotus. We have recently reported a robust shake flask process for the production of gram quantities of illudin M from Omphalotus nidiformis aiming to transfer that process into stirred tank bioreactors, which can be used in a commercial production set-up. However, process transfer across different systems is not straightforward and particularly challenging when the producer is morphologically complex. There are only a few reports that address the development of bioprocesses for the production of compounds from Basidiomycota as these organisms have not been extensively studied because of their complex life cycles and often are difficult to cultivate under laboratory conditions. RESULTS: The recently developed shake flask process delivering stable titers of ~ 940 mg L-1 of illudin M was investigated using off-gas analysis to identify critical parameters which facilitated the transfer from shaken into stirred tank bioreactors. Comparable titers to the shake flask process were achieved in 2 L stirred tank bioreactors (1.5 L working volume) by controlling growth of biomass with a carefully timed pH-shift combined with an improved precursor-feeding strategy. A scale-up experiment in a 15 L bioreactor (10 L working volume), resembling the process at 1.5 L resulted in 523 mg L-1 and is the starting point for optimization of the identified parameters at that scale. CONCLUSION: By identifying and controlling key process parameters, the production process for illudin M was transferred from shake flasks into 2 L stirred tank bioreactors reaching a comparable titer (> 900 mg L-1), which is significantly higher than any previously reported. The insights obtained from 10 L scale pave the way towards further scale-up studies that will enable a sustainable supply of illudin M to support preclinical and clinical development programs.

Optimization of the production process for the anticancer lead compound illudin M: improving titers in shake-flasks.

BACKGROUND: The fungal sesquiterpenes Illudin M and S are important base molecules for the development of new anticancer agents due to their strong activity against some resistant tumor cell lines. Due to nonspecific toxicity of the natural compounds, improvement of the pharmacophore is required. A semisynthetic derivative of illudin S (Irofulven) entered phase II clinical trials for the treatment of castration-resistant metastatic prostate cancer. Several semisynthetic illudin M derivatives showed increased in vitro selectivity and improved therapeutic index against certain tumor cell lines, encouraging further investigation. This requires a sustainable supply of the natural compound, which is produced by Basidiomycota of the genus Omphalotus. We aimed to develop a robust biotechnological process to deliver illudin M in quantities sufficient to support medicinal chemistry studies and future preclinical and clinical development. In this study, we report the initial steps towards this goal. RESULTS: After establishing analytical workflows, different culture media and commercially available Omphalotus strains were screened for the production of illudin M.Omphalotus nidiformis cultivated in a medium containing corn steep solids reached ~ 38 mg L-1 setting the starting point for optimization. Improved seed preparation in combination with a simplified medium (glucose 13.5 g L-1; corn steep solids 7.0 g L- 1; Dox broth modified 35 mL), reduced cultivation time and enhanced titers significantly (~ 400 mg L-1). Based on a reproducible cultivation method, a feeding strategy was developed considering potential biosynthetic bottlenecks. Acetate and glucose were fed at 96 h (8.0 g L-1) and 120 h (6.0 g L-1) respectively, which resulted in final illudin M titer of ~ 940 mg L-1 after eight days. This is a 25 fold increase compared to the initial titer. CONCLUSION: After strict standardization of seed-preparation and cultivation parameters, a combination of experimental design, empirical trials and additional supply of limiting biosynthetic precursors, led to a highly reproducible process in shake flasks with high titers of illudin M. These findings are the base for further work towards a scalable biotechnological process for a stable illudin M supply.

Role of heterotrophic nitrifiers and aerobic denitrifiers in simultaneous nitrification and denitrification process: a nonconventional nitrogen removal pathway in wastewater treatment.

Bacterial species capable of performing both nitrification and denitrification in a single vessel under similar conditions have gained significance in the wastewater treatment scenario considering their unique character of performing the above reactions under heterotrophic and aerobic conditions respectively. Such a novel strategy often referred to as simultaneous nitrification and denitrification (SND) has a tremendous potential in dealing with various wastewaters having low C : N content, considering that the process needs very little or no external carbon source and oxygen supply thus adding to its cost-effective and environmentally friendly nature. Though like other micro-organisms, heterotrophic nitrifiers and aerobic denitrifiers convert inorganic or organic nitrogen-containing substances into harmless dinitrogen gas in the wastewater, their ecophysiological role in the global nitrogen cycle is still not fully understood. Attempts to highlight the role played by the heterotrophic nitrifiers and aerobic denitrifiers in dealing with nitrogen pollution under various environmental operating conditions will help in developing a mechanistic understanding of the SND process to address the issues faced by the traditional methods of aerobic autotrophic nitrification-anaerobic heterotrophic denitrification.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures.

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV-1 virus-like particles.

HIV-1 Gag virus-like particles (VLPs) are promising candidates for the development of future vaccines. Recent viral outbreaks have manifested the need of robust vaccine production platforms able to adapt to new challenges while achieving mass production capacity. For the rapid production of VLPs, the method of transient gene expression (TGE) have proved highly efficient. Based on a previous characterization of the HEK293 cell line upon transient transfection using multiplexed quantitative proteomics, molecular production bottlenecks and metabolic pathways likely to be optimized were identified. In this study, these molecular components and metabolic pathways have been explored and modulated via transient metabolic engineering using approaches like design of experiments to fully exploit and optimize VLP production, transfection and budding efficiency. Upon overexpression of endosomal sorting complex required for transport accessory proteins like NEDD4L and CIT, VLP production increased 3.3 and 2.9-fold, respectively. Overexpression of glycosphingolipid precursor enzyme UGCG improved transfection efficiency by 17% and knocking-down the Gag-binding protein CNP improved 2.5-fold VLP specific productivity. Combining CNP inhibition and UGCG overexpression further improved budding efficiency by 37.3%. Modulating VLP production and accessory pathways like intracellular budding, demonstrated the potential of metabolic engineering to optimize and intensify the development of robust production platforms for future vaccines.

High-throughput evolutionary optimization of the induction medium towards recombinant protein production in BY-2 tobacco.

Bright yellow (BY-2) tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production. This offers a range of benefits, including the separation of the cell growth and heterologous gene expression, lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding. However, low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants. A number of molecular, cell culture and down processing approaches have been made to overcome this problem. Media development for the specific nutritional and hormonal requirements of transgenic plant cells is one of the most efficient cell-culture approaches. We optimized the induction medium towards recombinant protein production in BY-2 and demonstrated the usefulness of evolutionary medium optimization for high-yield protein production in liquid plant cultures. A reliable XVE/GFP model, parallel conducting experiments in a microscale on 96-well plates, and dedicated Gene Game evolutionary optimization software allowed for an effective search of 7611 possible solutions of 11-component media. Within the 4608 formulations tested, the Induct X medium was found with a significant 107.14% increase in protein expression in relation to the standard BY-2 medium.

Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network.

A high degree of charge heterogeneity is an unfavorable phenomenon commonly observed for therapeutic monoclonal antibodies (mAbs). Removal of these impurities during manufacturing often comes at the cost of impaired step yields. A wide spectrum of posttranslational and chemical modifications is known to modify mAb charge. However, a deeper understanding of underlying mechanisms triggering charged species would be beneficial for the control of mAb charge variants during bioprocessing. In this study, a comprehensive analytical investigation was carried out to define the root causes and mechanisms inducing acidic variants of an immunoglobulin G1-derived mAb. Characterization of differently charged species by liquid chromatography-mass spectrometry revealed the reduction of disulfide bonds in acidic variants, which is followed by cysteinylation and glutathionylation of cysteines. Importantly, biophysical stability and integrity of the mAb are not affected. By in vitro incubation of the mAb with the reducing agent cysteine, disulfide bond degradation was directly linked to an increase of numerous acidic species. Modifying the concentrations of cysteine during the fermentation of various mAbs illustrated that redox potential is a critical aspect to consider during bioprocess development with respect to charge variant control.

Engineering Escherichia coli for the utilization of ethylene glycol.

BACKGROUND: A considerable challenge in the development of bioprocesses for producing chemicals and fuels has been the high cost of feedstocks relative to oil prices, making it difficult for these processes to compete with their conventional petrochemical counterparts. Hence, in the absence of high oil prices in the near future, there has been a shift in the industry to produce higher value compounds such as fragrances for cosmetics. Yet, there is still a need to address climate change and develop biotechnological approaches for producing large market, lower value chemicals and fuels. RESULTS: In this work, we study ethylene glycol (EG), a novel feedstock that we believe has promise to address this challenge. We engineer Escherichia coli (E. coli) to consume EG and examine glycolate production as a case study for chemical production. Using a combination of modeling and experimental studies, we identify oxygen concentration as an important metabolic valve in the assimilation and use of EG as a substrate. Two oxygen-based strategies are thus developed and tested in fed-batch bioreactors. Ultimately, the best glycolate production strategy employed a target respiratory quotient leading to the highest observed fermentation performance. With this strategy, a glycolate titer of 10.4 g/L was reached after 112 h of production time in a fed-batch bioreactor. Correspondingly, a yield of 0.8 g/g from EG and productivity of 0.1 g/L h were measured during the production stage. Our modeling and experimental results clearly suggest that oxygen concentration is an important factor in the assimilation and use of EG as a substrate. Finally, our use of metabolic modeling also sheds light on the intracellular distribution through central metabolism, implicating flux to 2-phosphoglycerate as the primary route for EG assimilation. CONCLUSION: Overall, our work suggests that EG could provide a renewable starting material for commercial biosynthesis of fuels and chemicals that may achieve economic parity with petrochemical feedstocks while sequestering carbon dioxide.

Hybrid physics-based and data-driven modeling for bioprocess online simulation and optimization.

Model-based online optimization has not been widely applied to bioprocesses due to the challenges of modeling complex biological behaviors, low-quality industrial measurements, and lack of visualization techniques for ongoing processes. This study proposes an innovative hybrid modeling framework which takes advantages of both physics-based and data-driven modeling for bioprocess online monitoring, prediction, and optimization. The framework initially generates high-quality data by correcting raw process measurements via a physics-based noise filter (a generally available simple kinetic model with high fitting but low predictive performance); then constructs a predictive data-driven model to identify optimal control actions and predict discrete future bioprocess behaviors. Continuous future process trajectories are subsequently visualized by re-fitting the simple kinetic model (soft sensor) using the data-driven model predicted discrete future data points, enabling the accurate monitoring of ongoing processes at any operating time. This framework was tested to maximize fed-batch microalgal lutein production by combining with different online optimization schemes and compared against the conventional open-loop optimization technique. The optimal results using the proposed framework were found to be comparable to the theoretically best production, demonstrating its high predictive and flexible capabilities as well as its potential for industrial application.

Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system.

BACKGROUND: Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020. RESULTS: The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h. CONCLUSIONS: Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

Response surface methodology: A non-conventional statistical tool to maximize the throughput of Streptomyces species biomass and their bioactive metabolites.

Among diverse actinobacteria, Streptomyces is a renowned ongoing source for the production of a large number of secondary metabolites, furnishing immeasurable pharmacological and biological activities. Hence, to meet the demand of new lead compounds for human and animal use, research is constantly targeting the bioprospecting of Streptomyces. Optimization of media components and physicochemical parameters is a plausible approach for the exploration of intensified production of novel as well as existing bioactive metabolites from various microbes, which is usually achieved by a range of classical techniques including one factor at a time (OFAT). However, the major drawbacks of conventional optimization methods have directed the use of statistical optimization approaches in fermentation process development. Response surface methodology (RSM) is one of the empirical techniques extensively used for modeling, optimization and analysis of fermentation processes. To date, several researchers have implemented RSM in different bioprocess optimization accountable for the production of assorted natural substances from Streptomyces in which the results are very promising. This review summarizes some of the recent RSM adopted studies for the enhanced production of antibiotics, enzymes and probiotics using Streptomyces with the intention to highlight the significance of Streptomyces as well as RSM to the research community and industries.

Optimization of the biotechnological production of a novel class of anti-MRSA antibiotics from Chitinophaga sancti.

BACKGROUND: Recently, the discovery of the elansolids, a group of macrolides, was reported. The molecules show activity against methicillin-resistant Staphylococcus aureus as well as other gram-positive organisms. This fact renders those substances a promising starting point for future chemical development. The active atropisomers A1/A2 are formed by macrolactonization of the biosynthesis product A3 but are prone to ring opening and subsequent formation of several unwanted side products. Recently it could be shown that addition of different nucleophiles to culture extracts of Chitinophaga sancti enable the formation of new stable elansolid derivatives. Furthermore, addition of such a nucleophile directly into the culture led exclusively to formation of a single active elansolid derivative. Due to low product yields, methods for production of gram amounts of these molecules have to be established to enable further development of this promising compound class. RESULTS: Production of elansolid A2 by C. sancti was enabled using a synthetic medium with sucrose as carbon source to a final concentration of 18.9 mg L-1. A fed-batch fermentation was ensued that resulted in an elansolid A2 concentration of 55.3 mg L-1. When using glucose as carbon source in a fed-batch fermentation only 34.4 mg L-1 elansolid A2 but 223.1 mg L-1 elansolid C1 were produced. This finding was not unexpected since elansolids A1/A2 and A3 have been reported to easily react with nucleophiles like anthranilic acid, a precursor of tryptophan biosynthesis. Due to the fact that nucleophiles can be incorporated in vivo, a fed-batch cultivation under identical conditions, with addition of anthranilic acid was carried out and lead to almost exclusive formation of elansolid C1 (257.5 mg L-1). CONCLUSION: Reproducible elansolid A2 and C1 production is feasible in different synthetic media at relatively high concentrations that will allow further investigation and semi-synthetic optimization. The feeding of anthranilic acid enables the exclusive production of the stable elansolid derivative C1, which reduces product loss by unspecific reactions and eases downstream processing. This derivative shows activity in the same range as the elansolids A1/A2. Hence, the method can possibly serve as a model-process for incorporation of other nucleophiles and biotechnological production of specifically designed molecules.

Analysis of the efficiency of recombinant Escherichia coli strain cultivation in a gas-vortex bioreactor.

The levels of aeration and mass transfer are critical parameters required for an efficient aerobic bioprocess, and directly depend on the design features of exploited bioreactors. A novel apparatus, using gas vortex for aeration and mass transfer processes, was constructed in the Center of Vortex Technologies (Novosibirsk, Russia). In this paper, we compared the efficiency of recombinant Escherichia coli strain cultivation using novel gas-vortex technology with conventional bioprocess technologies such as shake flasks and bioreactors with mechanical stirrers. We demonstrated that the system of aeration and agitation used in gas-vortex bioreactors provides 3.6 times higher volumetric oxygen transfer coefficient in comparison with mechanical bioreactor. The use of gas-vortex bioreactor for recombinant E. coli strain cultivation allows to increase the efficiency of target protein expression at 2.2 times for BL21(DE3)/pFK2 strain and at 3.5 times for auxotrophic C600/pRT strain (in comparison with stirred bioreactor).

Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris.

Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni-NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % ß-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.

Production of chitin deacetylase by Aspergillus flavus in submerged conditions.

Chitosan is a biopolymer obtained by deacetylation of chitin and has been proven to have various applications in industry and biomedicine. Deacetylation of chitin using the enzyme chitin deacetylase (CDA) is favorable in comparison to the hazardous chemical method involving strong alkali and high temperature. A fungal strain producing CDA was isolated from environmental samples collected from coastal regions of South Kerala, India. It was identified as Aspergillus flavus by morphological characteristics and ITS DNA analysis. Nutritional requirement for maximum production of CDA under submerged condition was optimized using statistical methods including Plackett-Burman and response surface methodology central composite design. A 5.98-fold enhancement in CDA production was attained in shake flasks when the fermentation process parameters were used at their optimum levels. The highest CDA activity was 57.69 ± 1.68 U under optimized bioprocess conditions that included 30 g L(-1) glucose, 40 g L(-1) yeast extract, 15 g L(-1) peptone, and 7 g L(-1) MgCl2 at initial media pH of 7 and incubation temperature of 32°C after 48 hr of incubation, while the unoptimized basal medium yielded 9.64 ± 2.04 U.