Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.     

Introduction of the carbohydrate-activated promoter P(malK) for recombinant protein production

A production protocol for the use of the malK promoter was established. The protocol includes two phases: an initial fed-batch phase on glucose to reach a high cell density and a fed-batch phase on maltose for production of the desired recombinant protein. It is suggested that this cultivation scheme could be used for all promoters that are catabolite repressed by glucose and where growth and production need to be separated. The specific feature of this system is shown by its ability to control the rate of synthesis of the product protein, ss-galactosidase. In the production phase with a constant feed or an exponential feeding of 0.1 h(-1) it took 4 h longer to reach the maximum specific production rate than with the higher dilution rates of 0.25 h(-1) and 0.4 h(-1), respectively. In the above experiments a dilution rate of 0.3 h(-1) in the growth phase was used. The volumetric production of this system could furthermore be extended to 40 h. All protocol procedures so far tested resulted in the same maximum production rate, but reached in different lengths of time. It is argued that this system is particularly well suited for the production of proteins that have a complex structure and/or need to be produced in a soluble form or to be exported to the periplasm.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid alpha-ligase

In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.     

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.     

Influence of scale-up on the quality of recombinant human growth hormone

The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.     

Synergetic utilization of glucose and glycerol for efficient myo-inositol biosynthesis

myo-Inositol (MI) as a dietary supplement can provide various health benefits. One major challenge to its efficient biosynthesis is to achieve proper distribution of carbon flux between growth and production. Herein, this challenge was overcome by synergetic utilization of glucose and glycerol. Specifically, glycerol was catabolized to support cell growth while glucose was conserved as the building block for MI production. Growth and production were coupled via the phosphotransferase system, and both modules were optimized to achieve efficient production. First, the optimal enzyme combination was established for the production module. It was observed that enhancing the production module resulted in both increased MI production and better cell growth. In addition, glucose was shown to inhibit glycerol utilization via carbon catabolite repression and the inhibition was released by over-expressing glycerol kinase. Furthermore, the inducible promoter was replaced by strong constitutive promoters to avoid inducer use. With these efforts, the final strain produced MI with both high titer and yield. In fed-batch cultivation, 76 g/L of MI was produced, showing scale-up potential. This study provides a promising strategy to achieve rational distribution of carbon flux.     

Novel electrochemical-enzymatic model which quantifies the effect of the solution Eh on the kinetics of ferrous iron oxidation with Acidithiobacillus ferrooxidans

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.     

Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies

Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.     

Detection of sialoglycomolecules in five plant trypanosomatids and in an insect phytophagous isolate

A mutation in the cell division gene ftsK causes super-induction of sigma(70)-dependent stress defense genes, such as uspA, during entry of cells into stationary phase. In contrast, we report here that stationary phase induction of sigma(S)-dependent genes, uspB and cfa, is attenuated and that sigma(S) accumulates at a lower rate in ftsK1 cells. Ectopic overexpression of rpoS restored induction of the rpoS regulon in the ftsK mutant, as did a deletion in the recA gene. Thus, a mutation in the cell division gene, ftsK, uncouples the otherwise coordinated induction of sigma(S)-dependent genes and the universal stress response gene, uspA, during entry into stationary phase.     

高密度发酵制备重组人OCIF活性域多肽融合蛋白

以补料-分批发酵方式在3．7L发酵罐上实现了人破骨细胞形成抑制因子活性域多肽(OCIF AD)的高密度发酵融合表达。通过参数控制。发酵液最终OD600达12．5(相当于35g湿菌体,L),表达量占菌体总蛋白40％左右,含量超过0．6g/L,并且90％,呈可溶性而直接具有生物学活性。直链淀粉树脂亲和层析法一步纯化,纯度达90％以上。     

Biotechnological aspects of the production of the anticancer drug podophyllotoxin

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l^–1 in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l^–1 day^–1, when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.     

Effects of overproducing the universal stress protein, UspA, in Escherichia coli K-12.

A plasmid with the structural uspA gene under the control of a tac promoter was used to study the effects of altering uspA expression levels under various growth conditions. We found that increasing UspA synthesis to levels corresponding to physiologically induced levels decreased the cell growth rate in minimal medium and reduced or abolished the cells' capacity to adapt to upshift conditions. As was demonstrated by two-dimensional gel electrophoresis, increased uspA expression caused global changes in the pattern of protein synthesis. In addition, electrophoretic analysis together with V8 protease peptide mapping demonstrated that the pIs of some specific proteins became more acidic as a result of the elevation of the levels of UspA.     

Transferability of miRNA-technology to bioprocessing: Influence of cultivation mode and media

The biopharmaceutical industry strives for improvement of their production processes. In recent years, miRNAs have been shown to positively impact the production capacity of recombinant CHO cells, especially with regard to difficult to express proteins. Effective and reliable gene regulation of process relevant target genes by miRNAs is a prerequisite for integrating them into the toolbox of industrial cell engineering strategies. However, most studies rely on transient transfection of miRNA mimics; there is low standardization in evaluation of miRNA function and little knowledge on transferability of effects found during transient expression to stable expression during industry relevant fed-batch cultivation. In order to provide more insight into this topic, we used the pcDNA6.2 vector for stable miRNA overexpression during batch and fed-batch cultivation in CHO DG44 cells, optimized the vector, and compared the miRNA levels and effects with those achieved by transfection of miRNA mimics. We found that miR-1 downregulated TWF1 mRNA in different recombinant CHO DG44 clones in a dose-dependent manner during transient batch cultivation. Cells stably overexpressing miR-1 also showed a TWF1 mRNA downregulation when cultivated in batch mode using in-house medium 1. However, when the cells stably overexpressing miR-1 were cultivated in fed-batch mode using in-house medium 2. Consequently, a change of cultivation mode and medium seems to have an impact on target gene regulation by miRNA. Taken together, our findings highlight the importance to standardize miRNA evaluations and test miRNAs in the final application environment.