Fed-batch Culture Mode Research Articles

Highly-efficient co-production of microbial lipid and magnesium ammonium phosphate from N-acetyl-D-glucosamine

The valorization of chitin-rich wastes into chemicals and biofuels holds immense economic and environmental benefits. Here, N-acetyl-D-glucosamine (GlcNAc), the basic structural unit of chitin, was firstly described for co-producing microbial lipid and magnesium ammonium phosphate (MAP). Due to the strong substrate inhibition of GlcNAc, a fed-batch culture mode was successfully adopted to achieve high cell density by Cutaneotrichosporon oleaginosum. When a phosphate limitation strategy was applied, cell mass, lipid titer, content, yield, and productivity were 102.7 g/L, 74.2 g/L, 72.2 %, 21.4 g/100 g, and 0.69 g/L/h, respectively. The ammonium ion was efficiently precipitated by forming MAP with a removal rate around 95.4 %. The lipid samples showed high similarity to vegetable oil, which emerged as high-quality precursor for biodiesel production. This study offers a promising strategy for full conversion of GlcNAc into lipid and slow-release fertilizer, which provides an attractive technical route for turning the chitin-rich materials into valuable products.

Self-controlled in silico gene knockdown strategies to enhance the sustainable production of heterologous terpenoid by Saccharomyces cerevisiae

Microbial bioengineering is a growing field for producing plant natural products (PNPs) in recent decades, using heterologous metabolic pathways in host cells. Once heterologous metabolic pathways have been introduced into host cells, traditional metabolic engineering techniques are employed to enhance the productivity and yield of PNP biosynthetic routes, as well as to manage competing pathways. The advent of computational biology has marked the beginning of a novel epoch in strain design through in silico methods. These methods utilize genome-scale metabolic models (GEMs) and flux optimization algorithms to facilitate rational design across the entire cellular metabolic network. However, the implementation of in silico strategies can often result in an uneven distribution of metabolic fluxes due to the rigid knocking out of endogenous genes, which can impede cell growth and ultimately impact the accumulation of target products. In this study, we creatively utilized synthetic biology to refine in silico strain design for efficient PNPs production. OptKnock simulation was performed on the GEM of Saccharomyces cerevisiae OA07, an engineered strain for oleanolic acid (OA) bioproduction that has been reported previously. The simulation predicted that the single deletion of fol1, fol2, fol3, abz1, and abz2, or a combined knockout of hfd1, ald2 and ald3 could improve its OA production. Consequently, strains EK1∼EK7 were constructed and cultivated. EK3 (OA07△fol3), EK5 (OA07△abz1), and EK6 (OA07△abz2) had significantly higher OA titers in a batch cultivation compared to the original strain OA07. However, these increases were less pronounced in the fed-batch mode, indicating that gene deletion did not support sustainable OA production. To address this, we designed a negative feedback circuit regulated by malonyl-CoA, a growth-associated intermediate whose synthesis served as a bypass to OA synthesis, at fol3, abz1, abz2, and at acetyl-CoA carboxylase-encoding gene acc1, to dynamically and autonomously regulate the expression of these genes in OA07. The constructed strains R_3A, R_5A and R_6A had significantly higher OA titers than the initial strain and the responding gene-knockout mutants in either batch or fed-batch culture modes. Among them, strain R_3A stand out with the highest OA titer reported to date. Its OA titer doubled that of the initial strain in the flask-level fed-batch cultivation, and achieved at 1.23 ± 0.04 g L−1 in 96 h in the fermenter-level fed-batch mode. This indicated that the integration of optimization algorithm and synthetic biology approaches was efficiently rational for PNP-producing strain design.

Effective CO2 capture by the fed-batch culture of Chlorella vulgaris

Microalgae are promising candidates for CO2 capture and concomitant biomass production. Chlorella vulgaris was grown in the batch and fed-batch cultures to develop a culture strategy targeting maximum CO2 capture and conversion to biomass. Growth at five different dissolved CO2 (dCO2) concentrations in the feeding media (atmospheric, 1.55, 1.62, 1.75 and 1.88 g L−1) was tested to assess the effect of dCO2 concentration on the growth, biomass productivity, and nutrients removal efficiency of microalgae. Results suggest that fed-batch culture outperformed “standard” batch cultivation with a higher algal growth rate (2.3 times). As expected, the algal growth was limited at low (atmospheric) dCO2 concentrations and inhibited at high levels (1.75 and 1.88 g L−1). C. vulgaris grown with medium containing 1.62 g L−1 dCO2 showed the highest growth rate (0.094 h−1) with the shortest doubling time (7.4 h), maximum biomass productivity, nitrogen and phosphorus uptake rates (222, 7.5 and 1.6 mg L−1 d−1, respectively). By pre-dissolving CO2 into the feeding media and using the fed-batch culture mode, a high CO2 removal efficiency could be achieved.

Scale-up and fed-batch cultivation strategy for the enhanced co-production of microbial lipids and carotenoids using renewable waste feedstock

Agro-industrial by-product valorization as a feedstock for the bioproduction of high-value products has demonstrated a feasible alternative to handle the environmental impact of waste. Oleaginous yeasts are promising cell factories for the industrial production of lipids and carotenoids. Since oleaginous yeasts are aerobic microorganisms, studying the volumetric mass transfer (kLa) could facilitate the scale-up and operation of bioreactors to grant the industrial availability of biocompounds. Scale-up experiments were performed to assess the simultaneous production of lipids and carotenoids using the yeast Sporobolomyces roseus CFGU-S005 and comparing the yields in batch and fed-batch mode cultivation using agro-waste hydrolysate in a 7 L bench-top bioreactor. The results indicate that oxygen availability in the fermentation affected the simultaneous production of metabolites. The highest production of lipids (3.4 g/L) was attained using the kLa value of 22.44 h−1, while higher carotenoid accumulation of 2.58 mg/L resulted when agitation speed was increased to 350 rpm (kLa 32.16 h−1). The adapted fed-batch mode in the fermentation increased the production yields two times. The fatty acid profile was affected according to supplied aeration and after the fed-batch cultivation mode. This study showed the scale-up potential of the bioprocess using the strain S. roseus in the obtention of microbial oil and carotenoids by the valorization of agro-industrial byproducts as a carbon source.

Highly efficient fed-batch modes for enzymatic hydrolysis and microbial lipogenesis from alkaline organosolv pretreated corn stover for biodiesel production

High-density culture for microbial lipid preparation from low-cost lignocellulosic feedstocks is crucial for commercial-scale biodiesel production. Herein, fed-batch saccharification of alkaline organosolv pretreatment (AOP) of corn stover at an extremely high solids content of 47% (w/v) released 299.5 g/L of lignocellulosic sugars including 18.3% of soluble oligosaccharides. Three types of liquid hydrolysates for seed culture, fermentation, and feeding during fed-batch culture were obtained from the hydrolysate slurry using a two-step washing strategy with 99.3% of sugars recovery. Cutaneotrichosporon oleaginosum showed excellent capacity for assimilating both monosaccharides and oligosaccharides for lipid production using the fed-batch culture mode. Lipid concentration, content, and yield gained 42.3 g/L, 64.6%, and 20.4 g/100 g, respectively. Turbid hydrolysate collected with high recovery of high-concentration sugars and simplified process could be directly served as feeding medium. In general, the overall hydrolysis yield and lipid yield using fed-batch mode accounted for 93.2% and 97.6% of those using batch mode, respectively, resulting in a lipid output of 102.8 g/kg raw corn stover. The fatty acid composition and the prediction of biodiesel properties of lipid samples indicated the suitability for high-quality fuel production. This study provided valuable information for designing highly efficient lignocelluloses-to-biodiesel routes.

Process optimization for recycling of bread waste into bioethanol and biomethane: A circular economy approach

Bread is the second most wasted food in the UK with annual wastage of 292,000 tons. In the present work, bread waste (BW) was utilized for fermentative production of ethanol by Saccharomyces cerevisiae KL17. Acidic and enzymatic saccharification of BW was carried out resulting in the highest glucose release of 75 and 97.9 g/L which is 73.5 and 95.9% of theoretical yield, respectively. The obtained sugars were fermented into ethanol initially in shake flask followed by scale up in bioreactor in batch and fed-batch mode. In the fed-batch mode of cultivation, the maximum ethanol titers of 111.3, 106.9, and 114.9 g/L with conversion yield and productivity of 0.48, 0.47, and 0.49 g/g, and 3.1, 3.0, and 3.2 g/L.h was achieved from pure glucose, glucose-rich acidic and enzymatic hydrolysates, respectively. Further to improve the process economics, the solid residues after acidic (ABW) and enzymatic (EBW) hydrolysis of BW along with respective fermentation residues (FR) obtained after the ethanol production were pooled and subjected to anaerobic digestion. The solid residue from ABW + FR, and EBW + FR yielded a biochemical methanation potential (BMP) of 345 and 379 mL CH4/g VS, respectively. Life cycle assessment of the process showed that the total emissions for ethanol production from BW were comparable to the emissions from more established feedstocks such as sugarcane and maize grain and much lower when compared to wheat and sweet potato. The current work demonstrates BW as promising feedstock for sustainable biofuel production with the aid of circular biorefining strategy. To the authors knowledge, this is the first time, such a sequential system has been investigated with BW for ethanol and biomethane production. Further work will be aimed at ethanol production at pilot scale and BMP will be accessed in a commercial anaerobic digester.

A fermentation process for the production of poly(3-hydroxybutyrate) using waste cooking oil or waste fish oil as inexpensive carbon substrate

The utilization of waste cooking oil (WCO) or waste fish oil (WFO) as inexpensive carbon substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 was investigated. Fed-batch cultivation mode in bioreactor was applied in this study. High cell dry weight (CDW) of 135.1 g/L, PHB content of 76.9 wt%, PHB productivity of 1.73 g/L/h, and PHB yield of 0.8 g/g were obtained from WCO. In the case of WFO, the CDW, PHB content, PHB productivity, and PHB yield were 114.8 g/L, 72.5 wt%, 1.73 g/L/h, and 0.92 g/g, respectively. The PHB productivity and yield obtained in the current study from WCO or WFO are among the highest reported so far for PHA production using oils as sole carbon substrate, suggesting that both WCO and WFO can be used as inexpensive carbon substrates for the production of PHA on an industrial scale.

Streptomyces tsukubensis VKM Aс-2618D-an Effective Producer of Tacrolimus.

The Streptomyces sp. VKM Ac-2618D strain has been identified, and its morphological and physiological features have been studied in relation to the production of the immunosuppressant tacrolimus. The phenotypic variability of the strain was analyzed, and a dissociant with a high level of tacrolimus production was selected. Based on a comprehensive study of morphological, physiological, and chemotaxonomic properties and on phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis are shown. The results broaden the understanding of the characteristics of polyketide biosynthesis and can be used in the development of technology for tacrolimus production.

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells.

Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a high amount of culture feed results in the production of metabolic byproducts. In this work, we used a continuous feeding strategy to reduce metabolic byproducts and improve mouse–human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 expression in Chinese hamster ovary-K1 cells. Moreover, the effects of the feeding strategy on the cell culture and monoclonal antibody production were evaluated in chemically defined suspension cultures of recombinant CHO-K1 cells. Compared with bolus feeding methods, the continuous feeding method did not have any advantages when the feeding amount was low, but with a high feeding amount, the continuous feeding method significantly reduced the concentrations of lactate and NH4+ in the later culture stage. At the end of the culture stage, compared with bolus feeding methods, the lactate and NH4+ concentrations under the continuous feeding mode were reduced by approximately 45% and 80%, respectively. In addition, the antibody C12 expression level was also increased by almost 10%. Compared to the bolus feeding method, the antibody C12 produced by the continuous feeding method had a lower content of high-mannose glycoforms. Further analysis found that the osmolality of the continuous feeding method was lower than that of the typical fed-batch bolus feeding method. Conclusively, these results indicate that the continuous feeding method is very useful for reducing metabolic byproducts and achieving higher levels of mAb production.

Streptomyces tsukubensis ВКМ Ac-2618Д - эффективный продуцент такролимуса

The identification of the Streptomyces sp. VKM Ac-2618D strain had been carried out and its morphological and physiological features have been studied in relation to the immunosuppressant tacrolimus production. The phenotypic variability of the strain was analyzed and the dissociant with a high level of tacrolimus production was identified. Based on a comprehensive study of morphological, physiological and chemotaxonomic properties, as well as phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis were shown. The results obtained expand the understanding of the peculiarities of polyketide biosynthesis and can be used in the development of a tacrolimus production technology. Key words: Streptomyces tsukubensis, tacrolimus, FK-506, dissociant, genome, biosynthesis The research was carried out within the framework of the state assignment АААА-А16-116062110077-6.

Advances in the research and applications of orange fluorescent protein

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

Outdoor cultivation of the green microalga Chlorella minutissima in mini pond system under batch and fed-batch modes integrating low-dose sequential phosphate addition (LDSPA) strategy for biodiesel production

Microalgae are appraised as the renewable feedstock for biodiesel production. With this view, the current study examines the possibility of a locally isolated microalga, Chlorella minutissima for production of biodiesel, under batch and fed-batch cultivation modes, being subjected to seasonal variations. Effects of different culture depths (5, 10, 15, and 20 cm) on the biomass yield and lipid accumulation were investigated using the mini pond system, and alterations in different physicochemical factors were documented. Both the batch as well as the fed-batch cultivations recorded the 15 cm culture depth to be optimum for obtaining the maximum biomass and lipid production. Consequently, this optimal depth was chosen for further experimentation integrating low-dose sequential phosphate addition (LDSPA) under both batch and fed-batch modes. For batch cultivation under LDSPA mode, the estimated annual biomass productivity was 10.04 ton hectare−1 year−1, with the lipid productivity of 1.37 ton hectare−1 year−1, considering 11 cycles of cultivation per year. On the other hand, the fed-batch cultivation under LDSPA mode projected the annual biomass productivity of 15.4 ton hectare−1 year−1 and lipid productivity of 2.29 ton hectare−1 year−1, even with 10 cycles of cultivation yearly, thus, certifying the supremacy of the fed-batch cultivation with LDSPA for obtaining a higher productivity of lipid. The biodiesel generated from the produced biomass demonstrated a greater prevalence of monounsaturated and saturated fatty acid methyl esters. Additionally, the fuel characteristics of the biodiesel samples were analyzed and recorded to be at par with the national (Indian) as well as international (European and American) biodiesel standards, hence, indicating its suitability as an alternative to petroleum-based fuel.

Rich production media as a platform for CHO cell line development

Recent cell culture media for mammalian cells can be abundantly formulated with nutrients supporting production, but such media can be limited to use in host cell culture, transfection, cell cloning, and cell growth under the low cell density conditions. In many cases, appropriate platform media are used for cell line development, and then replaced with rich media for production. In this study, we demonstrate rich chemically defined media for Chinese hamster ovary (CHO) cells that are suitable as basal media both for cell line development and for final production of culture process. Set up for transfection, semi-solid media optimization, mini-pool screening, and single cell cloning media development were performed, and final clones were obtained with higher productivity in fed-batch culture mode using rich formulated media comparing with lean formulated media. Developed methods may remove the requirements for cell adaptation to production media after cell line development, and relieve the clonality issues associated with changing the culture media. Furthermore, established methods have advantages over traditional approaches, including saving resources and decreasing the time and the effort required to optimize the production process.

The growth and lutein accumulation in heterotrophic Chlorella protothecoides provoked by waste Monascus fermentation broth feeding.

Although the potential of heterotrophic microalgae served as a sustainable source for lutein, it was still crucial to formulate a suitable medium to offset the cost involved in algal biomass cultivation while improve inherent lutein productivity. The objective of this study was to investigate the feasibilities of waste Monascus fermentation broth medium (MFBM) toward heterotrophic Chlorella protothecoides-enriched lutein. The results indicated that C. protothecoides subjected to MFBM batch feeding achieved 7.1g/L biomass and 7.27mg/g lutein. The resulting lutein productivity (7.34mg/L/day) represented 1.54-fold more than that of frequently used Basal medium. Concurrently, the effective metabolism and absorption of carbon, nitrogen, and phosphorus in MFBM by C. subellipsoidea cultivation make it easily complied with the permissible dischargeable limits for fermentation broth. When response to fed-batch culture mode, the biomass and lutein productivity peaked 20.4g/L and 9.11mg/L/day with concentrated MFBM feeding. Transcriptomics data hinted that MFBM feeding manipulated lutein biosynthesis key checkpoints (e.g., lycopene β-cyclase and lycopene ε-cyclase) while accelerated energy pathways (e.g., glycolysis and TCA cycle) to contribute such high lutein productivity in C. protothecoides. These encouraging findings not only provided indications in applying nutrient-rich fermentation broth for affordable microalgae cultivation but also presented possibilities in linking algal high value-added products like lutein with high-efficient biological nutrition removal from industrial fermentation processing.

Comparison of downstream processing methods in purification of highly active laccase

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Modeling the physicochemical properties of DNA:PEI polyplexes on transient gene expression: A DoE-based approach

Cell-based production of viral particles has gain interest in the last years due to promising therapeutic applications. In the field of vaccination, novel vaccines are required to face the new pathogen outbreaks, and rapid and more efficient processes are required to respond as fast as possible to the vaccination demand worldwide. On the other hand, viral vector-driven gene therapies have demonstrated to be efficient and safe in numerous clinical trials, and three of them are already approved for commercialization. However, viral vector production is still a bottleneck in the road to the clinic. Although batch and fed-batch culture modes are preferred in industry, continuous culture strategies have demonstrated to improve viral titers and to reduce the bioprocessing costs. Therefore, there is increasing interest in exploring and optimizing continuous strategies in order to intensify viral vector production bioprocesses. This review is a summary of how continuous cultures have been applied to viral particle production (viral vaccines and viral vectors), the improvements achieved so far and the future perspectives in this field.

Physiology and methane productivity of Methanobacterium thermaggregans

Accumulation of carbon dioxide (CO2), associated with global temperature rise, and drastically decreasing fossil fuels necessitate the development of improved renewable and sustainable energy production processes. A possible route for CO2 recycling is to employ autotrophic and hydrogenotrophic methanogens for CO2-based biological methane (CH4) production (CO2-BMP). In this study, the physiology and productivity of Methanobacterium thermaggregans was investigated in fed-batch cultivation mode. It is shown that M. thermaggregans can be reproducibly adapted to high agitation speeds for an improved CH4 productivity. Moreover, inoculum size, sulfide feeding, pH, and temperature were optimized. Optimization of growth and CH4 productivity revealed that M. thermaggregans is a slightly alkaliphilic and thermophilic methanogen. Hitherto, it was only possible to grow seven autotrophic, hydrogenotrophic methanogenic strains in fed-batch cultivation mode. Here, we show that after a series of optimization and growth improvement attempts another methanogen, M. thermaggregas could be adapted to be grown in fed-batch cultivation mode to cell densities of up to 1.56 g L−1. Moreover, the CH4 evolution rate (MER) of M. thermaggregans was compared to Methanothermobacter marburgensis, the CO2-BMP model organism. Under optimized cultivation conditions, a maximum MER of 96.1 ± 10.9 mmol L−1 h−1 was obtained with M. thermaggregans—97% of the maximum MER that was obtained utilizing M. marburgensis in a reference experiment. Therefore, M. thermaggregans can be regarded as a CH4 cell factory highly suited to be applicable for CO2-BMP.

Yarrowia lipolytica application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L−1) and was stimulated by moderate pH (4.5) and agitation (800 rev min−1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L−1 of PA with volumetric productivity of 0.90 g L−1 h−1. The yield of PA biosynthesis reached a high level of 1.03 g g−1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.

A pilot-scale bioprocess to produce amphidinols from the marine microalga Amphidinium carterae: Isolation of a novel analogue

Marine dinoflagellate microalgae belonging to the genus Amphidinium are a key source of an interesting group of polyketide metabolites with potent bioactivities, wide-ranging functional diversity and stereochemical complexity, but low natural availabilities. The feasibility of a microalgae dinoflagellate-based sustainable bioprocess for producing amphidinols (APDNs) by photoautotrophic culture of Amphidinium carterae in a pilot-scale LED-illuminated bubble column photobioreactor (PBR) was therefore investigated. A fed-batch culture mode with pulse feeding strategy provided a growth pattern strongly limited by the availability of phosphate content in the culture medium that stimulated the production of cellular APDNs. Since A. carterae was found to be much more shear-sensitive than other shear-tolerant non-dinoflagellate microalgae, the culture height and air flow rate were established to ensure the absence of damaging levels of hydrodynamic stress. The biomass capacity yielded by the PBR at the end of the culture (0.540 g d.w. L−1 equivalent to 1.70 × 106 cell mL−1) corresponded to that estimated stoichiometrically from the experimentally determined biomass P-molar formula (C329 O126 H732 N69 S3 P1) and the total phosphorus and nitrogen balances. The downstream processing section was initially conceived to recover APDNs excreted by cells into the supernatant. A dry APDN-enriched extract concentration of 49 mg per liter of supernatant was obtained. This purification process led to partitioning of the extract into several fractions and sub-fractions thereof. Only two sub-fractions were studied, yielding thereof highly pure (>95% pure) luteophanol D and lingshuiol A, and a new, roughly purified (>80% pure) APDN, namely amphidinol 20. The percentages of luteophanol D, lingshuiol A and amphidinol 20 by dry weight of the APDN-enriched extract obtained were 1%, 0.39% and 0.31%, respectively, thus representing a concentration in the culture supernatant of 490, 191 and 152 μg L−1, respectively.

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein.

BackgroundThe increase of the protein expression via ribosomal manipulation is one of the suggested cellular mechanisms involved in EnBase fed-batch mode of cultivation. However, this system has not been implemented for cytotoxic proteins.ObjectivesHere, the expression pattern of α-Luffin, a ribosome inactivation protein (RIP) with an innate toxicity, was investigated in EnBase system and the effect of low temperature cultivation on the increase of α-Luffin solubility was determined.Materials and MethodsThe encoding cDNA for mature α-Luffin was synthesized and subcloned into pET28a plasmid under the control of T7 promoter. The E. coli expression yield in EnBase® Flo fed-batch system was compared with traditional batch mode at two temperatures: 25 °C and 30 °C. Sampling was performed at several time intervals and solubility of recombinant-protein was checked on SDS-PAGE in pellet and supernatant samples. The purification of recombinant protein was performed by Ni-NTA column.ResultsIn fed-batch cultivation mode, the early incubation time was desirable at 30 °C whereas the maximum amount of soluble α-Luffin was achieved from the extended protein synthesis period (12 and 24h post induction) at 25 °C.ConclusionsOur founding showed that EnBase had a greater efficacy in producing higher soluble protein ratios compared to batch cultivation growth rate, however for cytotoxic proteins, incubation temperature and time need to be optimized. Owing to the advantages of natural toxins from RIP family for producing anticancer immune-conjugates, well optimization of this protein expression is of importance regarding industrial aspects. The optimized condition proposed here is promising in terms of large scale soluble production of α-Luffin without the need for refolding.