Towards renewable flavors,fragrances, and beyond

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Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli

In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a β-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn²⁺-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml⁻¹ PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher β-glucanase concentration (65.6 kU ml⁻¹), i.e. 1.5-fold compared to the internal adsorbent system. An erratum to this article can be found at     

Integrated bioprocess for the simultaneous production of lactic acid and dairy sewage treatment

An integrated biological process was developed for the conversion of whey lactose to lactic acid. We report about the achievement of maximum COD reduction and thus a substantial unburdening of the environment, combined with the economic production of lactic acid, appropriate for industrial scale. The process – designed for continuous operation – consists of four main steps: (i) Protein recovery by ultrafiltration leading to the first product: protein concentrate. The resulting filtrate is the fermentation substrate acid whey permeate. (ii) Adjustment of the composition of the permeate in the medium preparation step in order to ensure the proper function of the following process steps. (iii) Conversion of the lactose to lactate by fermentation with lactic acid bacteria in a cell recycle reactor, using ceramic microfiltration membranes. (iiii) Conversion of the lactate in the cell-free permeate stream of the fermentation to free lactic acid by bipolar electrodialysis. A stable operation of the process was attained up to more than 2000?hours. Using a new selected strain of lactic acid bacteria, a lactic acid productivity of 17?g?l^?1?h^?1 is achieved at total lactose conversion without any nitrogen supplements like yeast extract. A lactic acid concentration of 190?g?l^?1 is obtained in the acidic cell of the electrodialysis unit and the COD of the remaining sewage is diminished by 92%. As an additional cost reduction item, the neutralization agent of the fermentation is recovered in the caustic cell of the bipolar electrodialysis unit. A cost evaluation for an industrial scale process (100?000?t of whey per year) resulted in a price of 0.66 $ per kg of lactic acid, which under present terms hits the goal of making this process economic for the large scale production of lactic acid as an attractive building block for various purposes in chemical industry.     

利用Pichia pastoris生产S-腺苷甲硫氨酸的发酵工艺

在摇瓶中考察了重组Pichia pastoris发酵的诱导剂量,L-甲硫氨酸,以及pH对腺苷甲硫氨酸产量的影响.放大到3.7 L发酵罐和30 L发酵罐后,研究了重组细胞的发酵过程变化,对S-腺苷甲硫氨酸初步纯化.摇瓶中优化后的发酵条件是:每天添加1%甲醇诱导,L-甲硫氨酸为50mmol/L,培养基pH 5.0.培养144 h后SAM产量达到2.32 g/L.3.7 L发酵罐中发酵251 h后细胞浓度为120 g/L,SAM总量为15.18 g.放大到30 L发酵罐中,发酵225.5 h后细胞浓度约为120 g/L,SAM总量为145.05 g.纯化后SAM的纯度为93.5%,回收率为84.5%.     

Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger

A comprehensive approach of bioprocess design at various levels was used to optimize microbial production of extracellular fructofuranosidase, important as biocatalyst to derive fructooligosaccharides with broad application in food or pharmaceutical industry. For production, the recombinant strain Aspergillus niger SKAn1015 was used, which expresses the fructofuranosidase encoding gene suc1 under control of a strong constitutive promoter. In a first screening towards an optimized medium, glucose, nitrate, Fe²⁺, and Mn²⁺ were identified as beneficial for production. A minimal medium with optimized concentration of these key nutrients, obtained by central composite design experiments and quadratic modelling, provided a threefold increased fructofuranosidase activity in the culture supernatant (400 U/mL) as compared to the originally described medium. Utilizing the optimized medium, the process was then transferred from shake flask into a fed-batch-operated bioreactor. Hereby, the intended addition of talc microparticles allowed engineering the morphology of A. niger into a highly active mycelial form, which strongly boosted production. Fructofuranosidase production was highly specific as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The secreted enzyme activity of 2,800 U/mL, corresponding to about 3 g/L of fructofuranosidase, achieved by the microparticle-enhanced fed-batch process, is tenfold higher than that of any other process reported so far, so that the presented bioprocess strategy appears as a milestone towards future industrial fructofuranosidase production.     

Biocatalytic preparation of natural flavours and fragrances

During the past years biocatalytic production of fine chemicals has been expanding rapidly. Flavours and fragrances belong to many different structural classes and therefore represent a challenging target for academic and industrial research. Here, we present a condensed overview of the potential offered by biocatalysis for the synthesis of natural and natural-identical odorants, highlighting relevant biotransformations using microorganisms and isolated enzymes. The industrial processes based on biocatalytic methods are discussed in terms of their advantages over classical chemical synthesis and extraction from natural sources. Recent applications of the biocatalytic approach to the preparation of the most important fine odorants are comprehensively covered.     

Biotechnological production of flavours and fragrances

The biotechnological generation of natural aroma compounds is rapidly expanding. Aroma chemicals, such as vanillin, benzaldehyde (bitter almond, cherry) and 4-(R)-decanolide (fruity–fatty) are marketed on a scale of several thousand tons per year. Their possible production by single-step biotransformations, bioconversions and de novo synthesis using microorganisms, plant cells or isolated enzymes is shown. The perspectives of bioprocesses for the oxifunctionalisation of lower terpenes by genetically modified organisms and economic aspects are discussed. Received: 27 May 1997 / Received revision: 25 September 1997 / Accepted: 28 September 1997     

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.     

Integrated bioprocess for the stereospecific production of linalool oxides from linalool with Corynespora cassiicola DSM 62475

Linalool oxides are of interest to the flavour industry because of their lavender notes. Corynespora cassiicola DSM 62475 has been identified recently as a production organism because of high stereoselectivity and promising productivities [Mirata et al. (2008) J Agric Food Chem 56(9):3287–3296]. In this work, the stereochemistry of this biotransformation was further investigated. Predominantly (2R)-configured linalool oxide enantiomers were produced from (R)-(?)-linalool. Comparative investigations with racemic linalool suggest that predominantly (2S)-configured derivatives can be expected by using (S)-(+)-configured substrate. Substrate and product inhibited growth even at low concentrations (200?mg?l^?1). To avoid toxic effects and supply sufficient substrates, a substrate feeding product removal (SFPR) system based on hydrophobic adsorbers was established. Applying SFPR, productivity on the shake flask scale was increased from 80 to 490?mg?l^?1?day^?1. Process optimisation increased productivity to 920?mg?l^?1?day^?1 in a bioreactor with an overall product concentration of 4.600?mg?l^?1 linalool oxides.     

Automated production support for the bioprocess industry

This paper describes the application of Artificial Intelligence and Multivariate Statistical Techniques to two industrial fermentation systems. In the first example, an Expert System is shown to provide tighter control of an important process parameter. This is shown to lead to improved consistency of operation. In the second application, Principal Component Analysis is applied to a final stage fermentation production facility. The results presented indicate that the algorithm can provide concise indicators of process faults that can be presented to the operators to assist them in taking suitable corrective actions.     

A continuous and adsorptive bioprocess for efficient production of the natural aroma chemical 2-phenylethanol with yeast

Natural 2-phenylethanol is a high value aroma chemical and can be produced from l-phenylalanine via Ehrlich pathway by yeasts. Due to serious product inhibition, the space-time yield is usually low. A continuous approach using macroporous resin as in situ adsorbent made it possible that the quantity and viability of the cells were improved simultaneously. With Saccharomyces cerevisiae sp. strain R-UV3, the highest space-time yield of 0.90 gL(-1)h(-1) reported so far was obtained.     

Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein

An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.     

Combining genetic engineering and bioprocess concepts for improved phenylpropanoid production

The group of natural aromatic compounds known as phenylpropanoids has diverse applications, but current methods of production which are largely based on synthesis from petrochemicals or extraction from agricultural biomass are unsustainable. Bioprocessing is a promising alternative, but improvements in production titers and rates are required to make this method profitable. Here the recent advances in genetic engineering and bioprocess concepts for the production of phenylpropanoids are presented for the purpose of identifying successful strategies, including adaptive laboratory evolution, enzyme engineering, in-situ product removal, and biocatalysis. The pros and cons of bacterial and yeast hosts for phenylpropanoid production are discussed, also in the context of different phenylpropanoid targets and bioprocess concepts. Finally, some broad recommendations are made regarding targets for continued improvement and areas requiring specific attention from researchers to further improve production titers and rates.     

Short Communication: Optimization of bioprocess conditions for exopolysaccharide production by Klebsiella oxytoca

Optimization of bioprocess conditions increased exopolysaccharide production by a strain of Klebsiella oxytoca from 6g/l to 15g/l; this corresponded to an increase in medium viscosity from 36cP at 12s–1 to 20,000 cP at 0.6 s–1. A combination of equal proportions of tryptone nitrogen and urea nitrogen proved to be the best nitrogen source. Lactose was shown to be the preferred carbon source. At an optimum temperature of 25°C, a pH of 7 was found to be the best for exopolysaccharide production. The concentration of exopolysaccharide produced on whey, enriched whey, enriched whey permeate and lactose-rich medium was comparable.     

Applications of modelling for bioprocess design and control in industrial production

The on-line measurement of the relevant parameters and the control conception for three production processes for fine chemicals by fermentation and biotransformation at the 15 m³ scale were developed. The models describe the bioprocesses which successfully result in fully automated manufacturing steps. Modelling also proved to be a valuable tool for a better insight into biochemical fundamentals of the processes. Moreover, proper use of data logging, modelling and process control was important for quality, since two processes were controlled on-line and quality relevant deviations were registered early. Finally, combining modelling with simulation, we could drastically reduce both development time and cost.List of Symbols F l/h flux - V l volume - U ₀ g/l nicotinonitrile concentration influx - U g/l actual nicotinonitrile concentration - q _ug/gh specific educt (=nicotinonitrile) transformation rate - x g/l biocatalyst concentration - p ₀ g/l nicotinamide concentration influx - p g/l actual nicotinamide concentration - q _pg/gh specific product (=nicotinamide) formation rate - k parameter loss of activity - q _{u, max}g/gh max. specific educt transformation rate - K _ug/l saturation constant for nicotinonitrile - K _ig/l inhibition constant for nicotinonitrile - K _iig/l inhibition constant for nicotinamide - MW _Ag/mol molecular weight for nicotinonitrile - MW _Bg/mol molecular weight for nicotinamide - NS Nicotinic acid - 6-HNS 6-Hydroxynicotinic acid - r _{NS, 6HNS} g/lh 6-HNS production rate - r _{6HNS, X} g/lh biomass production rate - r _{NS, 6HNS, max} g/lh max. 6-HNS production rate - S _NS g/l actual NS concentration - K _{S, NS} g/l saturation constant for NS - K _{i, 6HNS} g/l inhibition constant for 6-HNS - K _o2 g/l saturation constant for oxygen - r _{6HNS, X, max} g/lh max. biomass production rate - S _6HNS g/l actual 6-HNS concentration - K _{ii, NS} g/l inhibition constant for NS - RQ mol/mol respiration quotient - S _xylg/l actual xylene concentration - K _{i, xyl}g/ inhibition constant for xylene - K _{i, DMPY}g/ inhibition constant for 2,5-dimethylpyrazine - r _Xg/lh biomass production rate - r _{X, max}g/lh max. biomass production rate - K _{s, xyl}g/l saturation constant for xylene - S _DMPYg/l actual concentration of DMPY - K _{i, MPCA}g/ inhibition constant for MPCA - K _O2g/ saturation constant for oxygen - S _MPCAg/l actual MPCA concentration - S _O2g/l actual oxygen concentration - r _MPCAg/lh MPCA production rate - r _{MPCA, max}g/lh max. MPCA production rate - k _lgl inhibition constant for the intermediates - k _{s, DMPY}gl saturation constant for DMPY     

Integrated system for bioprocess monitoring and diagnosis using a local area network

Bioprocess monitoring and data analysis utilizing a local area network (LAN) is described in which an integrated computer environment provides for real-time monitoring from several remote personal computers with easy evaluation of the current process status and providing a common utilization of fermentation data. The computer network also enhances the decision-making process in the management of the production plant. Bioprocess control utilizing a LAN environment is expected to promote better utilization of fermentation data accumulated through repeated operations and to realize advanced control of fermentation processes. © Rapid Science Ltd. 1998     

Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.     

Monitoring a bioprocess for ethanol production using FT-MIR and FT-Raman spectroscopy

The application of Fourier transform mid-infrared (FT-MIR) spectroscopy and Fourier transform Raman (FT-Raman) spectroscopy for process and quality control of fermentative production of ethanol was investigated. FT-MIR and FT-Raman spectroscopy along with multivariate techniques were used to determine simultaneously glucose, ethanol, and optical cell density of Saccharomyces cerevisiae during ethanol fermentation. Spectroscopic measurement of glucose and ethanol were compared and validated with the high-performance liquid chromatography (HPLC) method. Spectral wave number regions were selected for partial least-squares (PLS) regression and principal component regression (PCR) and calibration models for glucose, ethanol, and optical cell density were developed for culture samples. Correlation coefficient (R ²) value for the prediction for glucose and ethanol was more than 0.9 using various calibration methods. The standard error of prediction for the PLS first-derivative calibration models for glucose, ethanol, and optical cell density were 1.938 g/l, 1.150 g/l, and 0.507, respectively. Prediction errors were high with FT-Raman because the Raman scattering of the cultures was weak. Results indicated that FT-MIR spectroscopy could be used for rapid detection of glucose, ethanol, and optical cell density in S. cerevisiae culture during ethanol fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 185–190. Received 16 November 2000/ Accepted in revised form 12 January 2001