Fed-batch Fermentation Conditions Research Articles

Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris

BackgroundThe yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production.ResultsTranscription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed.ConclusionsPichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules.

Biosynthesis of ethylene glycol from d-xylose in recombinant Escherichia coli

ABSTRACTEthylene glycol (EG) is an important chemical used as antifreeze and a raw material in polyester synthesis. The EG biosynthetic pathway from D-xylose with D-xylonate as key intermediate has some advantages, but showed low EG production. Here, we reconstructed and optimized this pathway in Escherichia coli. In view of the greater intracellular prevalence of NADH, an aldehyde reductase FucO using NADH was employed to convert glycoaldehyde into EG, in replacement of NADPH-dependent reductase YqhD. To suppress the accumulation of by-products acetate and glycolate, two genes arcA and aldA were knocked out. The resultant strain Q2843 produced 72 g/L EG under fed-batch fermentation conditions, with the yield of 0.40 g/g D-xylose and EG productivity of 1.38 g/L/h. The use of NADH-dependent enzyme FucO and by-product elimination significantly improved the performance of EG producing strain, which represented the highest titer, yield and productivity of EG reported so far.

Omega-3 production by fermentation of Yarrowia lipolytica: From fed-batch to continuous.

The omega-3 fatty acid, cis-5,8,11,14,17-eicosapentaenoic acid (C20:5; EPA) has wide-ranging benefits in improving heart health, immune function, and mental health. A sustainable source of EPA production through fermentation of metabolically engineered Yarrowia lipolytica has been developed. In this paper, key fed-batch fermentation conditions were identified to achieve 25% EPA in the yeast biomass, which is so far the highest EPA titer reported in the literature. Dynamic models of the EPA fermentation process were established for analyzing, optimizing, and scaling up the fermentation process. In addition, model simulations were used to develop a two-stage continuous process and compare to single-stage continuous and fed- batch processes. The two stage continuous process, which is equipped with a smaller growth fermentor (Stage 1) and a larger production fermentor (Stage 2), was found to be a superior process to achieve high titer, rate, and yield of EPA. A two-stage continuous fermentation experiment with Y. lipolytica strain Z7334 was designed using the model simulation and then tested in a 2 L and 5 L fermentation system for 1,008 h. Compared with the standard 2 L fed-batch process, the two-stage continuous fermentation process improved the overall EPA productivity by 80% and EPA concentration in the fermenter by 40% while achieving comparable EPA titer in biomass and similar conversion yield from glucose. During the long-term experiment it was also found that the Y. lipolytica strain evolved to reduce byproduct and increase lipid production. This is one of the few continuous fermentation examples that demonstrated improved productivity and concentration of a final product with similar conversion yield compared with a fed-batch process. This paper suggests the two-stage continuous fermentation could be an effective process to achieve improved production of omega-3 and other fermentation products where non-growth or partially growth associated kinetics characterize the process. Biotechnol. Bioeng. 2017;114: 798-812. © 2016 Wiley Periodicals, Inc.

Simultaneous saccharification and fermentation of sweet sorghum after acid pretreatment

ABSTRACTThe sweet sorghum bagasse pretreated with 5% (w/w) acetic acid at an accumulated solid concentration of 20% (w/v) during the 96-h fed-batch simultaneous saccharification and fermentation achieved a maximum ethanol concentration of 53.1 g/L and ethanol yield of 88.7%, compared to 25.7 g/L and 86.7% for the 96-h batch simultaneous saccharification and fermentation at a solid concentration of 10% (w/v), respectively. For comparison, the bagasse pretreated with 0.5% (w/w) sulfuric acid and water under the same fed-batch simultaneous saccharification and fermentation conditions produced maximum ethanol concentrations of 44.3 and 36.5 g/L, and ethanol yields of 77.6 and 69.7%, respectively.

Toward intensifying design of experiments in upstream bioprocess development: An industrial Escherichia coli feasibility study.

In this study, step variations in temperature, pH, and carbon substrate feeding rate were performed within five high cell density Escherichia coli fermentations to assess whether intraexperiment step changes, can principally be used to exploit the process operation space in a design of experiment manner. A dynamic process modeling approach was adopted to determine parameter interactions. A bioreactor model was integrated with an artificial neural network that describes biomass and product formation rates as function of varied fed-batch fermentation conditions for heterologous protein production. A model reliability measure was introduced to assess in which process region the model can be expected to predict process states accurately. It was found that the model could accurately predict process states of multiple fermentations performed at fixed conditions within the determined validity domain. The results suggest that intraexperimental variations of process conditions could be used to reduce the number of experiments by a factor, which in limit would be equivalent to the number of intraexperimental variations per experiment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1343-1352, 2016.

Hybrid modeling as a QbD/PAT tool in process development: an industrial E. coli case study.

Process understanding is emphasized in the process analytical technology initiative and the quality by design paradigm to be essential for manufacturing of biopharmaceutical products with consistent high quality. A typical approach to developing a process understanding is applying a combination of design of experiments with statistical data analysis. Hybrid semi-parametric modeling is investigated as an alternative method to pure statistical data analysis. The hybrid model framework provides flexibility to select model complexity based on available data and knowledge. Here, a parametric dynamic bioreactor model is integrated with a nonparametric artificial neural network that describes biomass and product formation rates as function of varied fed-batch fermentation conditions for high cell density heterologous protein production with E. coli. Our model can accurately describe biomass growth and product formation across variations in induction temperature, pH and feed rates. The model indicates that while product expression rate is a function of early induction phase conditions, it is negatively impacted as productivity increases. This could correspond with physiological changes due to cytoplasmic product accumulation. Due to the dynamic nature of the model, rational process timing decisions can be made and the impact of temporal variations in process parameters on product formation and process performance can be assessed, which is central for process understanding.

A novel MVA-mediated pathway for isoprene production in engineered E. coli.

BackgroundTo deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of great significance in that it is an important platform chemical, which has been used in industrial production of synthetic rubber for tires and coatings or aviation fuel.ResultsWe firstly introduced fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species into E. coli to directly convert MVA(mevalonate) into 3-methy-3-buten-1-ol. And then to transform 3-methy-3-buten-1-ol to isoprene, oleate hydratase (OhyAEM) from Elizabethkingia meningoseptica was overexpressed in E. coli. A novel biosynthetic pathway of isoprene in E. coli was established by co-expressing the heterologous mvaE gene encoding acetyl-CoA acetyltransferase/HMG-CoA reductase and mvaS gene encoding HMG-CoA synthase from Enterococcus faecalis, fatty acid decarboxylase (OleTJE) and oleate hydratase (OhyAEM). Furthermore, to enhance isoprene production, a further optimization of expression level of OleTJE, OhyAEM was carried out by using different promoters and copy numbers of plasmids. Thereafter, the fermentation process was also optimized to improve the production of isoprene. The final engineered strain, YJM33, bearing the innovative biosynthetic pathway of isoprene, was found to produce isoprene up to 2.2 mg/L and 620 mg/L under flask and fed-batch fermentation conditions, respectively.ConclusionsIn this study, by using metabolic engineering techniques, the novel MVA-mediated biosynthetic pathway of isoprene was successfully assembled in E. coli BL21(DE3) with the heterologous MVA upper pathway, OleTJE from Jeotgalicoccus species and OhyAEM from Elizabethkingia meningoseptica. Compared with traditional MVA pathway, the novel pathway is shortened by 3 steps. In addition, this is the first report on the reaction of converting MVA into 3-methy-3-buten-1-ol by fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species. In brief, this study provided an alternative method for isoprene biosynthesis, which is largely different from the well-developed MEP pathway or MVA pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0236-2) contains supplementary material, which is available to authorized users.

Improved 1,3-propanediol production by Escherichia coli from glycerol due to Co-expression of glycerol dehydratase reactivation factors and succinate addition

Escherichia coli was engineered to produce 1,3-propanediol (1,3-PDO) from glycerol, an inexpensive carbon source. This was done by introducing a synthetic pathway consisting of glycerol dehydratase, glycerol dehydratase reactivation factor, and 1,3-propanediol oxidoreductase isoenzyme. The JM-30BY15AB harboring pQE30/dhaB123, yqhD and pQE15A/gdrA, gdrB produced 1,3-PDO (7.2 g/L) from glycerol, at a level higher than that produced by JM-30BY harboring pQE30/dhaB123, yqhD (4.1 g/L). When 10mM succinate was added to the medium, the titer of 1,3-PDO and the glycerol consumption increased to 9.9 and 23.84 g/L, respectively. In addition, the ratio of NADH to NAD+ increased by 43%. The titer of 1,3-PDO and glycerol consumption were 145.6 and 86.6% higher, respectively, than those from the control which harbors one vector system without gdrAB and did not include succinate addition. Under fed-batch fermentation conditions, the titer of 1,3-PDO and its conversion yield from glycerol were 13.11 g/L and 0.49 g/g, respectively. This dual-vector system may be a useful approach for the co-expression of recombinant proteins. Further, succinate addition is a promising route for the biotechnological production of NADH-dependent microbial metabolites.

User-friendly optimization approach of fed-batch fermentation conditions for the production of iturin A using artificial neural networks and support vector machine

In the field of microbial fermentation technology, how to optimize the fermentation conditions is of great crucial for practical applications. Here, we use artificial neural networks (ANNs) and support vector machine (SVM) to offer a series of effective optimization methods for the production of iturin A. The concentration levels of asparagine (Asn), glutamic acid (Glu) and proline (Pro) (mg/L) were set as independent variables, while the iturin A titer (U/mL) was set as dependent variable. General regression neural network (GRNN), multilayer feed-forward neural networks (MLFNs) and the SVM were developed. Comparisons were made among different ANNs and the SVM. The GRNN has the lowest RMS error (457.88) and the shortest training time (1 s), with a steady fluctuation during repeated experiments, whereas the MLFNs have comparatively higher RMS errors and longer training times, which have a significant fluctuation with the change of nodes. In terms of the SVM, it also has a relatively low RMS error (466.13), with a short training time (1 s). According to the modeling results, the GRNN is considered as the most suitable ANN model for the design of the fed-batch fermentation conditions for the production of iturin A because of its high robustness and precision, and the SVM is also considered as a very suitable alternative model. Under the tolerance of 30%, the prediction accuracies of the GRNN and SVM are both 100% respectively in repeated experiments.

Recent advances in production of succinic acid from lignocellulosic biomass

Production of succinic acid via separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are alternatives and are environmentally friendly processes. These processes have attained considerable positions in the industry with their own share of challenges and problems. The high-value succinic acid is extensively used in chemical, food, pharmaceutical, leather and textile industries and can be efficiently produced via several methods. Previously, succinic acid production via chemical synthesis from petrochemical or refined sugar has been the focus of interest of most reviewers. However, these expensive substrates have been recently replaced by alternative sustainable raw materials such as lignocellulosic biomass, which is cheap and abundantly available. Thus, this review focuses on succinic acid production utilizing lignocellulosic material as a potential substrate for SSF and SHF. SSF is an economical single-step process which can be a substitute for SHF - a two-step process where biomass is hydrolyzed in the first step and fermented in the second step. SSF of lignocellulosic biomass under optimum temperature and pH conditions results in the controlled release of sugar and simultaneous conversion into succinic acid by specific microorganisms, reducing reaction time and costs and increasing productivity. In addition, main process parameters which influence SHF and SSF processes such as batch and fed-batch fermentation conditions using different microbial strains are discussed in detail.

Metabolic engineering of Escherichia colifor the biosynthesis of alpha-pinene

Backgroundα-Pinene is an important natural product that is widely used in flavorings, fragrances, medicines, fine chemicals and high-density renewable fuels. Currently, α-Pinene used in industry is mainly produced either by tapping trees (gum turpentine) or as a byproduct of paper pulping (crude sulfate turpentine, CST). However, the extraction of it from trees is tedious and inefficient and requires substantial expenditure of natural resources. Therefore, it is necessary to seek sustainable technologies for α-pinene production.ResultsTo construct the microbial synthetic pathway of α-pinene in E. coli, we co-expressed native geranyl diphosphate synthase (IspA) from E. coli and α-pinene synthase (Pt30) from Pinus taeda, and then to increase the geranyl diphosphate (GPP) content in the cells, a suitable geranyl diphosphate synthase (GPPS2) was selected from two different origins. Furthermore, to enhance α-pinene production, a novel biosynthetic pathway of α-pinene was assembled in E. coli BL21(DE3) with the heterologous hybrid mevalonate (MVA) pathway, GPPS2 and α-pinene synthase (Pt30). The final genetic strain, YJM28, harboring the above novel biosynthetic pathway of α-pinene, accumulated α-pinene up to 5.44 mg/L and 0.97 g/L under flask and fed-batch fermentation conditions, respectively. The conversion efficiency of glucose to α-pinene (gram to gram) in the metabolically engineered strain reached 2.61%.ConclusionsIn this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of α-pinene was successfully assembled in E. coli BL21(DE3) with the heterologous hybrid MVA pathway, GPPS2 and α-pinene synthase (Pt30). In addition, this is the first report on α-pinene fed-batch fermentation, and our results represent improvements over previous reports.

The role of aldehyde/alcohol dehydrogenase (AdhE) in ethanol production from glycerol by Klebsiella pneumoniae

Transcriptome analysis of a K. pneumoniae GEM167 mutant strain derived by irradiation with gamma rays, which exhibited high-level production of ethanol from glycerol, showed that the mutant expressed AdhE at a high level. Ethanol production decreased significantly, from 8.8 to 0.5gl(-1), when an adhE-deficient derivative of that strain was grown on glycerol. Bacterial growth was also reduced under such conditions, showing that AdhE plays a critical role in maintenance of redox balance by catalyzing ethanol production. Overexpression of AdhE enhanced ethanol production, from pure or crude glycerol, to a maximal level of 31.9gl(-1) under fed-batch fermentation conditions; this is the highest level of ethanol production from glycerol reported to date.

Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization

Hirudin variant III (HV3) is potentially useful in the prevention and treatment of cataracts. To prepare sufficient amounts of rHV3 for further preclinical studies, we developed an effective process for robust preparative-scale extracellular production of rHV3 in Escherichia coli. In a 7-l bioreactor, under the optimal fed-batch fermentation conditions, rHV3 was excreted into the culture supernatant and yielded up to 915 mg l(-1). Then, a four-step purification procedure was applied to the product, which included ultrafiltration, hydrophobic chromatography, anion-exchange chromatography, and preparative reversed-phase fast protein liquid chromatography (FPLC). The overall maximum recovery attained was 56 %, the purity reached at least 99 % as evaluated by HPLC analysis, the molecular weight was determined to be 7,011.10 Da by matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and the pI was 4.46 as analyzed by isoelectric focusing. The N- and C-terminal sequence analysis confirmed the product homogeneity. The final product contained at most 10 pg of residual DNA per dose (0.2 mg) of rHV3 by high-sensitivity hybridization assay and at most 3 EU endotoxin protein/mg by limulus amebocyte lysate assay. Taken together, the rHV3 produced in multigram quantities in E. coli by this bioprocess meets the regulatory criteria for biopharmaceuticals and can be used as a drug candidate for preclinical studies.

Bio-isoprene production using exogenous MVA pathway and isoprene synthase in Escherichia coli

In this paper, an original strategy is employed to biosynthesize the isoprene by heterologously co-expressing the Saccharomyces cerevisiae MVA pathway and isoprene synthase (IspS) from Populus alba in the Escherichia coli BL21 (DE3) strain, which was screened from three different IspS enzymes. The finally genetic strain YJM13 harboring the MVA pathway and ispSPa gene could accumulate isoprene up to 2.48mg/l and 532mg/l under the flask and fed-batch fermentation conditions, respectively, which is about three times and five times to the control strain. The result proves to be higher than that in the report documents. In this way, a potential production system for isoprene from renewable sources via the MVA pathway in E. coli has been provided.

High yield Rhizopus chinenisis prolipase production in Pichia pastoris: Impact of methanol concentration

Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5 to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L, with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol fed-batch phase was very important for cell viability and protease activity.

WITHDRAWN: Production of bioethanol from Typha latifolia enzymatic hydrolysates under batch and fed-batch fermentation conditions

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Global expression profiling of Bacillus subtilis cells during industrial‐close fed‐batch fermentations with different nitrogen sources

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation.

Efficient use of lactose for the lac promotercontrolled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions

Efficient use of lactose for the lac promotercontrolled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions

Efficient use of lactose for the lac promoter-controlled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions.

Derivatives of the lac promoter (tac, pac, rac) belong to the strongest bacterial promoters which are frequently used for the induced overexpression of foreign genes in Escherichia coli. However, their use in fermentation processes is strongly restricted because of the high cost of the inducer iso-propyl-beta-D-thiogalactopyranoside (IPTG). The aim of this work was to investigate the possibility of using lac-derived promoters in high cell density processes resulting in a high yield of the induced recombinant protein if glucose is the main carbon and energy source. Lactose is tested as inducer of the main antigenic coat protein (VP1) of the foot and mouth disease (FMD) virus in a T7-RNA polymerase expression system. It was shown that lactose is able to induce the expression of the recombinant gene to an amount of the VP1 protein corresponding to 20% of the total cell protein.

The production of the enzyme dextransucrase using nonaerated fermentation techniques

High yields of the enzyme dextransucrase have been produced repeatedly by fed-batch fermentation techniques. Activities in excess of 21.9 U/cm(3) have been obtained by culturing Leuconostoc mesenteroides NRRL B-512(F) under nonaerated fed-batch fermentation conditions. Aerobic fermentations carried out under identical conditions have consistently produced enzyme of less than 17 U/cm(3), but with no difference in the final cell concentration in the broth. Different types of yeast extract have been found to have significant effect on the final cell concentration and more especially on the enzyme activity with enzyme yields varying by as much as 50% when different types of yeast extracts were used.