RESUMEN
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.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Escherichia coli , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Fosfatos/metabolismo , Plásmidos/genética , Proteínas Recombinantes/metabolismoRESUMEN
Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality attributes in fed-batch cultures of recombinant Chinese hamster ovary (rCHO) cells. However, the relation between tyrosine and these aspects is not yet fully defined. In order to further elucidate such a relation, two groups of fed-batch experiments with high tyrosine (H-T) or low tyrosine (L-T) additions producing an IgG1 monoclonal antibody against CD20 were implemented to investigate the intracellular and extracellular effects of tyrosine on the culture performance. It was found that the scarcity of tyrosine led to the distinctive reduction in both viable cell density and antibody specific production rate, hence the sharply reduced titer, possibly related to the impaired translation efficiency caused by the substrate limitation of tyrosine. In addition, alterations to the critical quality attributes were detected in the L-T group, compared to those in the H-T condition. Notable decrease in the contents of intact antibody was found under the L-T condition because of the elevated reductive level in the supernatant. Moreover, the aggregate content in the L-T condition was also reduced, probably resulting from the accumulation of extracellular cystine. In particular, the lysine variant content noticeably increased with tyrosine limitation owing to the downregulation of two carboxypeptidases, i.e., CpB and CpH. Overall, understanding the role of tyrosine in these aspects is fundamental to the increase of product titers and control of critical quality attributes in the monoclonal antibody production of rCHO cell fed-batch cultures. KEY POINTS: ⢠Tyrosine is essential in the maintenance of product titers and the control of product qualities in high cell density cultivations in rCHO cell. ⢠This study revealed the bottleneck of decreased qmAbupon the deficiency of tyrosine. ⢠The impact of tyrosine on the critical product qualities and the underlying mechanisms were also thoroughly assessed.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Medios de Cultivo/química , Tirosina/farmacología , Animales , Antígenos CD20/inmunología , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Células CHO , Cricetulus , Inmunoglobulina G/biosíntesisRESUMEN
The aim of the present work was to study the growth of two nitrifying bacteria. For modelling the nitrifying subsystem of the MELiSSA loop, Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25931 were grown separately and in cocultures. The kinetic parameters of a stoichiometric mass balanced Pirt model were identified: µmax=0.054h(-1), decay rate b=0.003h(-1) and maintenance rate m=0.135gN-NH4(+)·gX(-1)·h(-1) for Nitrosomonas europaea; µmax=0.024h(-1), b=0.001h(-1) and m=0.467gN-NO2(-)·gX(-1)·h(-1) for Nitrobacter winogradskyi. A predictive structured model of nitrification in co-culture was developed. The online evolution of the addition of KOH is correlated to the nitritation; the dissolved oxygen concentration is correlated to both nitritation and nitratation. The model suitably represents these two variables so that transient partial nitrification is assessed. This is a clue for avoiding partial nitrification by predictive functional control.
Asunto(s)
Modelos Teóricos , Nitrobacter/crecimiento & desarrollo , Nitrosomonas europaea/crecimiento & desarrollo , Bacterias , Reactores Biológicos , Técnicas de Cocultivo , Cinética , Nitrificación , Nitrobacter/metabolismo , Nitrosomonas/crecimiento & desarrollo , Nitrosomonas europaea/metabolismoRESUMEN
A nonpathogenic bacterial strain Bacillus amyloliquefaciens TUL 308 synthesized minor 2,3-butanediol (2,3-BD) amounts from glucose, fructose, sucrose, and glycerol, and efficiently produced the diol from molasses and hydrolysates of food processing residues. Batch fermentations yielded 16.53, 10.72, and 5 g/L 2,3-BD from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp (at initial concentrations equivalent to 45, 20, and 30 g/L glucose, respectively), and 25.3 g/L 2,3-BD from molasses (at its initial concentration equivalent to 60 g/L saccharose). Fed-batch fermentations in the molasses-based medium with four feedings with either glucose or sucrose (in doses increasing their concentration by 25 g/L) resulted in around twice higher maximum 2,3-BD concentration (of about 60 and 50 g/L, respectively). The GRAS Bacillus strain is an efficient 2,3-BD producer from food industry byproducts.
Asunto(s)
Bacillus amyloliquefaciens/metabolismo , Butileno Glicoles/metabolismo , Manipulación de Alimentos , Beta vulgaris/metabolismo , Biomasa , Reactores Biológicos , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Fermentación , MelazaRESUMEN
Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity â¼100-fold and the heparin/heparan sulfate yield â¼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in â¼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of â¼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.
Asunto(s)
Anticoagulantes , Células CHO , Heparina/biosíntesis , Ingeniería Metabólica , Animales , Reactores Biológicos , Vías Biosintéticas , Cricetinae , Cricetulus , Heparina/metabolismoRESUMEN
Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min-1. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O2) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O2 control, varying glucose concentration from 1.0 to 3.0 g l -1, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O 2 control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O2 control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.