Glucose-limited Fed-batch Culture Research Articles

Use of ambr®250 to assess mucic acid production in fed-batch cultures of a marine Trichoderma sp. D-221704

Mucic acid, a diacid with potential use in the food, cosmetic, chemical and pharmaceutical industries, can be produced by microbial conversion of D-galacturonic acid, which is abundant in pectin. Using the ambr®250 bioreactor system, we found that a recently generated transformant (D-221704, formerly referred to as T2) of a marine Trichoderma species produced up to 53 g L−1 mucic acid in glucose-limited fed-batch culture with D-galacturonic acid in the feed at pH 4, with a yield of 0.99 g mucic acid per g D-galacturonic acid consumed. Yeast extract was not essential for high production, but increased the initial production rate. Reducing the amount of glucose as the co-substrate reduced the amount of mucic acid produced to 31 g L−1. Mucic acid could also be produced at pH values less than 4.0 (3.5 and 3.0), but the amount produced was less than at pH 4.0. Furthermore, the yield of mucic acid on D-galacturonic acid at the end of the cultivations (0.5 to 0.7 g g−1) at these low pH levels suggested that recovery may be more difficult at lower pH on account of the high level of crystal formation. Another strain engineered to produce mucic acid, Trichoderma reesei D-161646, produced only 31 g L−1 mucic acid under the conditions used with D-221704.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

BackgroundSubstrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains.ResultsTo achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process.ConclusionThe glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

Bioprocess Development for Lantibiotic Ruminococcin-A Production in Escherichia coli and Kinetic Insights Into LanM Enzymes Catalysis.

Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (<1 μg L–1) and difficulties to cultivate the obligately anaerobic organism. We recently reported the reconstruction of the RumA biosynthesis machinery in Escherichia coli where the fully modified and active peptide was expressed as a fusion protein together with GFP. In the current study we developed a scale-up strategy for the biotechnologically relevant heterologous production of RumA, aimed at overproducing the peptide under conditions comparable with those in industrial production settings. To this end, glucose-limited fed-batch cultivation was used. Firstly, parallel cultivations were performed in 24-microwell plates using the enzyme-based automated glucose-delivery cultivation system EnPresso® B to determine optimal conditions for IPTG induction. We combined the bioprocess development with ESI-MS and tandem ESI-MS to monitor modification of the precursor peptide (preRumA) during bioreactor cultivation. Dehydration of threonine and serine residues in the core peptide, catalyzed by RumM, occurs within 1 h after IPTG induction while formation of thioether cross-bridges occur around 2.5 h after induction. Our data also supplies important information on modification kinetics especially with respect to the fluctuations observed in the various dehydrated precursor peptide versions or intermediates produced at different time points during bioreactor cultivation. Overall, protein yields obtained from the bioreactor cultivations were >120 mg L–1 for the chimeric construct and >150 mg L–1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.

Enhanced production of xylitol from xylose by expression of Bacillus subtilis arabinose:H+ symporter and Scheffersomyces stipitis xylose reductase in recombinant Saccharomyces cerevisiae.

Inefficient transport of xylose into Saccharomyces cerevisiae is a major hurdle for production of xylitol, a natural sweetener with five carbons. To facilitate the xylose transport and hence increase xylose conversion to xylitol, the araE gene encoding an arabinose:H+ symporter (AraE) from Bacillus subtilis and the XYL1 gene from Scheffersomyces stipitis were expressed in Saccharomyces cerevisiae EBY.VW4000, a hxt null mutant. The resulting strain of EXHA exhibited 4.1 fold increases in xylose consumption rate and xylitol productivity, relative to the control strain without AraE. Also, overexpression of AraE in wild type S. cerevisiae D452-2 having all hexose transporters and the XYL1 gene increased both xylose consumption and xylitol production considerably. In a glucose-limited fed-batch culture with intermittent addition of xylose, the DXXA strain with multiple copies of araE and XYL1 produced 177.8g/L xylitol with 2.47g/L-h productivity, which were 26.9 and 17.6 times higher than those for a batch culture of the DX strain expressing the XYL1 gene only, respectively. It was concluded that B. subtilis AraE might be a potent xylose transporter and conferred much higher xylose-consuming and xylitol-producing abilities to S. cerevisiae.

Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production.

Process inhomogeneity leads to rapid side product turnover in cultivation of Corynebacterium glutamicum

BackgroundCorynebacterium glutamicum has large scale industrial applications in the production of amino acids and the potential to serve as a platform organism for new products. This means the demand for industrial process development is likely to increase. However, large scale cultivation conditions differ from laboratory bioreactors, mostly due to the formation of concentration gradients at the industrial scale. This leads to an oscillating supply of oxygen and nutrients for microorganisms with uncertain impact on metabolism. Scale-down bioreactors can be applied to study robustness and physiological reactions to oscillating conditions at a laboratory scale.ResultsIn this study, C. glutamicum ATCC13032 was cultivated by glucose limited fed-batch cultivation in a two-compartment bioreactor consisting of an aerobic stirred tank and a connected non-aerated plug flow reactor with optional feeding. Continuous flow through both compartments generated oscillating profiles with estimated residence times of 45 and 87 seconds in the non-aerated plug flow compartment. Oscillation of oxygen supply conditions at substrate excess and oscillation of both substrate and dissolved oxygen concentration were compared to homogeneous reference cultivations. The dynamic metabolic response of cells within the anaerobic plug flow compartment was monitored throughout the processes, detecting high turnover of substrate into metabolic side products and acidification within oxygen depleted zones. It was shown that anaerobic secretion of lactate into the extracellular culture broth, with subsequent reabsorption in the aerobic glucose-limited environment, leads to mixed-substrate growth in fed-batch processes. Apart from this, the oscillations had only a minor impact on growth and intracellular metabolite characteristics.ConclusionsCarbon metabolism of C. glutamicum changes at oscillating oxygen supply conditions, leading to a futile cycle over extracellular side products and back into oxidative pathways. This phenomenon facilitates a dynamic and flexible shift of oxygen uptake at inhomogeneous process conditions. There is no loss of process characteristics at oscillation times in the minute range, which emphasizes the robustness of C. glutamicum in comparison to other industrial microorganisms. Therefore, the metabolic phenotype of C. glutamicum seems to be particularly well-suited for cultivation at inhomogeneous process conditions for large-scale fed-batch application, which is in good accordance with the respective industrial experiences.

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100L scale. At and below the critical specific growth rate (μcrit) of 0.12h−1 at 14L scale, the biomass yield coefficient (Yx/s) remained constant at 0.4gg−1 with no ethanol production, whereas ethanol yields relative to biomass (keth/x) of up to 0.54gg−1 and a steady decrease in Yx/s were observed at μ>0.12h−1. Production of α-glucuronidase was growth associated at a product yield (kα-glu/x) of 0.45mgg−1, with the highest biomass (37.35g/L) and α-glucuronidase (14.03mg/L) concentrations, were recorded during fed-batch culture at or near to μcrit. Scale-up with constant kLa from 14 to 100L resulted in ethanol concentrations of up to 2.5g/L at μ=0.12h−1. At this scale, α-glucuronidase yield could be maximised at growth rates below μcrit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.

Efficient Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase in Glucose-Limited Fed-Batch Cultivations of Escherichia coli by Continuous Lactose Induction Strategy

The production of a thermophilic 2-deoxyribose-5-phosphate aldolases (DERA) in Escherichia coli BL21 under continuous lactose induction strategy was investigated. The process was combined with the exponential feeding method, controlling the feeding rate to maintain the specific growth rate at 0.15 h(-1). The results indicate that the lactose concentration in the feed medium affected directly the expression of the target protein. The use of 50 g/L in the feed medium resulted in the biomass concentration of 39.3 g DCW/L, and an expression level of above 30%, and the maximum final DERA concentration of 16,200 U/L. Furthermore, the acetate concentration remained at a low level in the fed-batch phase, less than 0.5 g/L. In conclusion, combining glucose feeding with lactose induction is a more powerful way to achieve high cell density cultures and to efficiently produce the thermophilic DERA. The results also indicate the potential industrial utility in the scale production of other recombinant proteins.

Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high-cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed-batch cultivations, very high-cell densities were reached (more than 200 g(CDW) L(-1)) resulting in a recombinant protein titer of about 6.5 g L(-1). To investigate the impact of recombinant protein production and high-cell density fermentation on the metabolism of P. pastoris, we used (13)C-tracer-based metabolic flux analysis in batch and fed-batch experiments. At a controlled growth rate of 0.12 h(-1) in fed-batch experiments an increased TCA cycle flux of 1.1 mmol g(-1) h(-1) compared to 0.7 mmol g(-1) h(-1) for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development.

High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor

BackgroundSingle-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.ResultsCultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 of 30 (10 g L-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.ConclusionsRocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

Dynamic flux responses in riboflavin overproducing Bacillus subtilis to increasing glucose limitation in fed‐batch culture

How do intracellular fluxes respond to dynamically increasing glucose limitation when the physiology changes from strong overflow metabolism near to exclusively maintenance metabolism? Here we investigate this question in a typical industrial, glucose-limited fed-batch cultivation with a riboflavin overproducing Bacillus subtilis strain. To resolve dynamic flux changes, a novel approach to (13)C flux analysis was developed that is based on recording (13)C labeling patterns in free intracellular amino acids. Fluxes are then estimated with stationary flux ratio and iterative isotopomer balancing methods, for which a decomposition of the process into quasi-steady states and estimation of isotopic steady state (13)C labeling patterns was necessary. By this approach, we achieve a temporal resolution of 30-60 min that allows us to resolve the slow metabolic transients that typically occur in such cultivations. In the late process phase we found, most prominently, almost exclusive respiratory metabolism, significantly increased pentose phosphate pathway contribution and a strongly decreased futile cycle through the PEP carboxykinase. As a consequence, higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands, suggesting a transhydrogenase-like mechanism to close the balance of reducing equivalents.

Prediction of kinetic parameters from DNA-binding site sequences for modeling global transcription dynamics in Escherichia coli

The majority of dynamic gene regulatory network (GRN) models are comprised of only a few genes and do not take multiple transcription regulation into account. The models are conceived in this way in order to minimize the number of kinetic parameters. In this paper, we propose a new approach for predicting kinetic parameters from DNA-binding site sequences by correlating the protein–DNA-binding affinities with nucleotide sequence conservation. We present the dynamic modeling of the cra modulon transcription in Escherichia coli during glucose-limited fed-batch cultivation. The concentration of the Cra regulator protein inhibitor, fructose 1,6-bis(phosphate), decreases sharply, eventually leading to the repression of transcription. Total RNA concentration data indicate a strong regulation of transcription through the availability of RNA polymerase. A critical assessment of the results of the model simulations supports this finding. This new approach for the prediction of transcription dynamics may improve the metabolic engineering of gene regulation processes.

Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks

BackgroundHere we describe a novel cultivation method, called EnBase™, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries.ResultsHigh cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel.Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method.ConclusionThe novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.

Screening of Bacillus subtilis transposon mutants with altered riboflavin production

To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a “loss of function” analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture.

Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett-Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO(3), K(2)HPO(4), ZnSO(4), and MnCl(2) were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.

Dual modulation of glucose 6-phosphate metabolism to increase NADPH-dependent xylitol production in recombinant Saccharomyces cerevisiae

To increase the metabolic flux toward the NADPH-generating pentose phosphate pathway in a recombinant Saccharomyces cerevisiae strain that harbors the xylose reductase gene from Pichia stipitis, expression of phosphoglucose isomerase (PGI) encoded by the PGI1 gene was modulated by a promoter replacement using the ADH1 promoter. Although the ADH1 promoter down-regulated PGI expression in glucose-limited condition, the decline of PGI activity did not exert a profound influence on xylitol production in a series of glucose-limited fed-batch cultivations. However, simultaneous enforcement of glucose 6-phosphate dehydrogenase (G6PDH) activity and attenuation of phosphoglucose isomerase activity worked in a cooperative manner to increase xylitol production and to reduce utilization of cosubstrate required for xylitol production in a glucose-limited fed-batch cultivation of the PGI mutant strain with an enhanced G6PDH activity. An 1.9-fold increase in specific xylitol productivity of 0.34 ± 0.03 g/g cells h was achieved compared with the control strain containing xylose reductase only.

Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

A recombinant Escherichia coli strain was developed to produce guanosine 5'-diphosphate (GDP)-L-fucose, donor of L-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-D: -mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25 degrees C and 0.1 mM isopropyl-beta-D-thioglucopyranoside. Maximum GDP-L-fucose concentration of 38.9 +/- 0.6 mg l(-1) was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 +/- 0.5 mg l(-1) GDP-L-fucose under the same cultivation condition.

Plasmid system for the intracellular production and purification of affinity‐tagged proteins in Bacillus megaterium

A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His(6)-tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry.

Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium

Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.