Exploring small-scale chemostats to scale up microbial processes: 3-hydroxypropionic acid production in S. cerevisiae.

BACKGROUND: The physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed. RESULTS: We report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43 g gCDW-1, respectively, at the lowest set dilution rate of 0.04 h-1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermenters operating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45 g gCDW-1 under C-limiting, as well as 25.6 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. CONCLUSIONS: Small-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04 h-1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.

New approaches in bioprocess-control: Consortium guidance by synthetic cell-cell communication based on fungal pheromones.

Bioconversions in industrial processes are currently dominated by single-strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single-strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium-based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell-cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone-responsive gene expression, thereby creating pheromone-dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone-controlled sensor-actor systems, exploiting their unique metabolic properties for microbial consortia approaches.

Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc.

Light-field-characterization in a continuous hydrogen-producing photobioreactor by optical simulation and computational fluid dynamics.

Externally illuminated photobioreactors (PBRs) are widely used in studies on the use of phototrophic microorganisms as sources of bioenergy and other photobiotechnology research. In this work, straightforward simulation techniques were used to describe effects of varying fluid flow conditions in a continuous hydrogen-producing PBR on the rate of photofermentative hydrogen production (rH2 ) by Rhodobacter sphaeroides DSM 158. A ZEMAX optical ray tracing simulation was performed to quantify the illumination intensity reaching the interior of the cylindrical PBR vessel. 24.2% of the emitted energy was lost through optical effects, or did not reach the PBR surface. In a dense culture of continuously producing bacteria during chemostatic cultivation, the illumination intensity became completely attenuated within the first centimeter of the PBR radius as described by an empirical three-parametric model implemented in Mathcad. The bacterial movement in chemostatic steady-state conditions was influenced by varying the fluid Reynolds number. The "Computational Fluid Dynamics" and "Particle Tracing" tools of COMSOL Multiphysics were used to visualize the fluid flow pattern and cellular trajectories through well-illuminated zones near the PBR periphery and dark zones in the center of the PBR. A moderate turbulence (Reynolds number = 12,600) and fluctuating illumination of 1.5 Hz were found to yield the highest continuous rH2 by R. sphaeroides DSM 158 (170.5 mL L(-1) h(-1) ) in this study.

Hydrogen production by Rhodobacter sphaeroides DSM 158 under intense irradiation.

To identify optimal hydrogen production conditions using growing cultures of Rhodobacter sphaeroides DSM 158 the effects of varying the reactor's volumetric power input (0.01-1.4kWm(-3)) and irradiation intensity (5-2500Wm(-2)) were investigated in batch and continuous production modes. Irradiation intensity had a greater effect on hydrogen production than volumetric power input. Hydrogen production and photofermentative biomass formation were maximized by irradiation at 2250Wm(-2) with a volumetric power input of 0.55kWm(-3). The bacterial dry weight (2.64gL(-1)) and rate of hydrogen production (195mLL(-1)h(-1)) achieved under these conditions were greater than any that have previously been reported for batch-mode hydrogen production by R. sphaeroides. Continuous mode experiments (D=0.1h(-1)) yielded a bacterial dry weight, hydrogen production rate, productivity and hydrogen yield of 2.35±0.18gL(-1), 165±6.2mLL(-1)h(-1), 3.96LL(-1)d(-1) and 36.6%, respectively.

Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization.

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5 years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.

Growth and exopolysaccharide yield of Lactobacillus delbrueckii ssp. bulgaricus DSM 20081 in batch and continuous bioreactor experiments at constant pH.

Some Lactobacillus delbrueckii ssp. bulgaricus strains are able to synthesize exopolysaccharides (EPS) and are therefore highly important for the dairy industry as starter cultures. The aim of this study was to investigate the nutritional requirements for growth and EPS production of Lactobacillus delbrueckii ssp. bulgaricus DSM 20081. A medium was developed from a semi-defined medium (SDM) in which glucose was replaced by lactose and different combinations of supplements (nucleobases, vitamins, salts, sodium formate and orotic acid) were added. Constant pH batch fermentation with the modified medium resulted in an EPS yield of approximately 210 mg glucose equivalents per liter medium. This was a 10-fold increase over flask cultivation of this strain in SDM. Although not affecting cell growth, the mixture of salts enhanced the EPS synthesis. Whereas EPS production was approximately 12 mg/g dry biomass without salt supplementation, a significantly higher yield (approximately 20 mg/g dry biomass) was observed after adding the salt mixture. In continuous fermentation, a maximal EPS concentration was obtained at a dilution rate of 0.31/h (80 mg EPS/L), which corresponded to a specific EPS production of 49 mg/g dry biomass.

Bioactive metabolite production and stress-related hormones in Devil's claw cell suspension cultures grown in bioreactors.

In a previous report, we showed that cell cultures of Harpagophytum procumbens, a South African plant with high medicinal value, accumulate high amounts of anti-inflammatory phenylethanoid glycosides during cultivation in shake-flasks. The aim of the present study was to transfer the phenylethanoid biosynthetic process to a 3-L stirred tank reactor and a 1-L glass-column bioreactor (operated with pulsed aeration). We found that, with stepwise increases in aeration, the stirred tank reactor yielded similar productivities of verbascoside (the major phenylethanoid glycoside in the cells) to those reported for shake-flask cultures (55.68 vs. 54.78 mg verbascoside/L/day, respectively). Transfer in the pulse-aerated column reactor resulted in 165.42 mg verbascoside/L/day, one of the highest yields reported to date. Further, to evaluate the physiological status of the suspended cells in the bioreactors cultures, we examined their hormone levels and compared them to those of cells in shake-flask cultures. While indole-3-acetic acid levels did not differ significantly between the bioreactor and shake-flask cultures, there were considerable differences in their levels of abscisic, jasmonic, and salicylic acids. These results are discussed with respect to relative stress levels in the different cultivation systems.

Devil's claw hairy root culture in flasks and in a 3-L bioreactor: bioactive metabolite accumulation and flow cytometry.

Phenylethanoids are a group of natural water-soluble compounds with high biological value, which could potentially be commercially produced by hairy root cultures. Thus, we have examined the capacity of transformed root cultures of Devil's claw (Harpagophytum procumbens) to accumulate four phenylethanoid glycosides -beta-OH-verbascoside, verbascoside, leucosceptoside A, and martynoside--in shake-flasks and a 3-L stirred tank reactor. Verbascoside was found to be the major phenylethanoid, and its maximal contents were the same (1.12 mg/g dry weight) in both kinds of culture. However, peak leucosceptoside A contents were 1.6-times higher in bioreactor cultures than in shake-flask cultures. Flow cytometry analysis revealed that G0 + G1-phase cells predominated throughout the growth of the cultures, which was in accordance with the very high proportion of quiescent cells in the transformed roots. The results provide the first demonstration of the potential utility of Devil's claw hairy roots as biofactories for producing high-value phenylethanoid glycosides.

Changes in apolar metabolites during in vitro organogenesis of Pancratium maritimum.

Calli, shoot-clumps and regenerated plants were initiated from young fruits of Pancratium maritimum L. Their genetic stability was monitored by flow cytometry before chemical studies. Apolar metabolites (alkaloids extracted at pH > 7, free fatty acids and fatty alcohols, sterols etc.) were qualitatively and quantitatively analyzed by GC-MS. The results clearly demonstrated that alkaloid synthesis in P. maritimum is closely related with tissue differentiation. The highest amounts of alkaloids and presence of homolycorine and tazettine type compounds (end products of the biosynthetic pathway of the Amaryllidaceae alkaloids) were found in highly differentiated tissues. Galanthamine accumulated in the leaves of plantlets. The amount of hordenine, a protoalkaloid, is related with the ability of tissues to synthesize alkaloids. Saturated fatty acids were found in considerably higher levels in undifferentiated callus cultures and partially differentiated shoot-clumps than in regenerated plants. Mono- and dienoic fatty acids were found at higher levels in non-photosynthesizing tissues - calli, and in vitro and intact bulbs, while α-linolenic acid (trienoic acid) was found in higher amounts in the photosynthesizing leaves of shoot-clumps and regenerated plants than in bulbs and calli. Fatty alcohols were found mainly in leaves, while sterols tended to accumulate in photosynthesizing and undifferentiated tissues.

Antioxidant activity and phenolic content of betalain extracts from intact plants and hairy root cultures of the red beetroot Beta vulgaris cv. Detroit dark red.

Betalains are water-soluble plant pigments that are widely used as food colorants, and have a wide range of desirable biological activities, including antioxidant, anti-inflammatory, hepatoprotective, anti-cancer properties. They can be produced from various plants, notably beetroot, but betalain products obtained in this way also have some undesirable properties and are difficult to standardize. A potentially attractive alternative is to use hairy root cultures. In the study reported here, we found that betalain extracts obtained from hairy root cultures of the red beetroot B. vulgaris cv. Detroit Dark Red also had higher antioxidant activity than extracts obtained from mature beetroots: six-fold higher 2,2-dyphenyl-1-picrylhydrazyl radical scavenging ability (90.7% inhibition, EC(50) = 0.11 mg, vs 14.2% inhibition, EC(50) = 0.70 mg) and 3.28-fold higher oxygen radical absorbance capacity (4,100 microM TE/g dry extract, vs 1,250 microM TE/g dry extract). The high antioxidant activity of the hairy root extracts was associated with increased concentrations (more than 20-fold) of total phenolic concomitant compounds, which may have synergistic effects with betalains. The presence of 4-hydroxybenzoic acid, caffeic acid, catechin hydrate, and epicatechin were detected in both types of extract, but at different concentrations. Rutin was only present at high concentration (1.096 mg.g(-1) dry extract) in betalain extracts from the hairy root cultures, whereas chlorogenic acid was only detected at measurable concentrations in extracts from intact plants.

Efficacy and safety of the Accordion stone-trapping device: in vitro results from an artificial ureterolithotripsy model.

One of the challenges of intracorporeal ureterolithotripsy is undesired stone migration. Stone-trapping devices have been designed to prevent this quite common phenomenon. These devices have to be effective in terms of ureteral obstruction and safe in terms of resistance to the action of commonly used lithotriptors. This work was conducted to evaluate the efficacy and safety of the recently approved Accordion stone-trapping device in vitro. In a rigid, submerged ureteral model with two different diameters (8 and 10 mm), artificial stones were positioned in direct contact with the engaged Accordion device. A defined number of pneumatic pulses of the LithoClast master at different performance levels was applied and the migration distance of the stone was measured after each single pulse. As a control, the same series was repeated without the stone-trapping device. Secondly, the Accordion device was exposed to a previously defined number of pneumatic or Ho:YAG-laser pulses, in direct contact with the lithotripsy probe, up to a total activation time of 2 min. At different time points, the device was controlled for damage and functionality. The mean stone migration distance without the Accordion device was between 39.2 and 52.8 mm and between 37.8 and 75.4 mm in the 8 and 10 mm tubes, respectively. In comparison, the stone or fragment travelling distance with the device was in the 0-2 mm range. This difference was highly significant. Both pneumatic and laser lithotriptor did not affect the functionality of the Accordion device. The Ho:YAG laser causes small perforations of the film without affecting the devices' stability. The Accordion device appears to be highly efficient and safe in vitro. Clinical trials will have to assess its value in endourological practice. Randomised comparative trials comparing different stone-trapping devices are needed.

Bioprocessing of plant cell cultures for mass production of targeted compounds.

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered.

Asymmetric division of Hansenula polymorpha reflected by a drop of light scatter intensity measured in batch microtiter plate cultivations at phosphate limitation.

Hansenula polymorpha RB11 pC10-FMD (P(FMD)- GFP) (FMD promoter gfp gene) was simultaneously cultivated in the Respiration Activity Monitoring System (RAMOS) and in the microtiter plate cultivation system "BioLector" under phosphate limitation. The light scatter signal of the BioLector, for the determination of the biomass concentration in the wells, shows a significant decrease with the onset of the phosphate limitation until a stationary level is reached. At lower initial phosphate concentration this effect is more pronounced and longer time is required until the stationary level of the scattered light is achieved. The oxygen transfer rate signal of the RAMOS and the light scatter signal of the BioLector correlate with respect to the points of time where the maxima and the stationary levels of the courses are reached. In order to understand the effect causing this light scatter behavior, the forward and side scatter properties were investigated off line by flow cytometry. The decay in the light scatter of the BioLector seems to correlate with the formation of two subpopulations of different scatter intensities detected by a flow cytometer. With ongoing cultivation the fraction of cells possessing higher light scattering properties decreases until only a population of lower light scattering properties exists. The rate of transition of the yeast from one subpopulation to the other appears to be correlated with the rate of decrease in the BioLector light scatter signal. The formation of the subpopulations may be caused by an increased asymmetry in the cell cycle due to phosphate limitation.

Improved procedure for nucleus extraction for DNA measurements by flow cytometry of red beet (Beta vulgaris L.) hairy roots.

Three often cited systems for the extraction of plant nuclei for flow cytometric measurement (CyStain PI, Partec GmbH, Münster, Germany, the method of Arumuganathan and Earle, and LB01 buffer) failed, when applied to the hairy roots of red beet (Beta vulgaris). By combining these systems and introducing a centrifugation step, the extraction, staining, and analysis of nuclei from this tissue were performed successfully.

Flow cytometry and phytochemical analysis of a sunflower cell suspension culture in a 5-L bioreactor.

A cell suspension culture of sunflower (Helianthus annuus), a producer of immunologically active polysaccharides, was cultivated in a 5-L stirred tank bioreactor, operated in batch mode. After some changes in the internal bioreactor design a stable growth of Helianthus cells was achieved and the accumulated biomass reached 15.2 g/L (only approximately 5% lower compared to the accumulated biomass in shake-flasks). Flow cytometry used for measuring the cell cycle parameters of suspended Helianthus cells did not reveal significant differences between shake-flasks and bioreactor cultivation modes. For both cultivation methods significant enhancement of the percentage of S-phase cells was observed at the beginning of the cultivation process. Concerning the metabolite production the maximum in exopolysaccharides was reached at day 9 of the cultivation period (1.9 g/L), while the highest amounts of alpha-tocopherol were accumulated at the beginning of the cultivation process (day 2 of the cultivation). These finding were related to the respective stress levels caused by the inoculation procedure. The kinetic parameters of growth and polysaccharide production as well as the time course of carbon source utilization were monitored and discussed.

Flow cytometric investigations of diploid and tetraploid plants and in vitro cultures of Datura stramonium and Hyoscyamus niger.

Plant in vitro systems are valuable sources for the production of biological active substances. However, changed profiles of secondary metabolites, and low, variable yields possibly caused by genetic instabilities complicate their industrial implementation. DNA profiling of plant in vitro cultures may provide data for the selection of highly producing in vitro cultures. Diploid and tetraploid Datura stramonium and Hyoscyamus niger plant as well as calli, and hairy root lines derived from them were analyzed by flow cytometry. Plant in vitro cultures undergo several cycles of endoreduplication more than the explants from which they were obtained. The highest cycle values were observed in calli (e.g. 1.19 for diploid H. niger) possibly induced by the growth factors. However, hairy roots cultivated without growth factor exhibited significant degrees of endoreduplication (cycle value 0.88 for diploid H. niger). Sets of five hairy root lines from each plant and ploidy level showed consistent within-set ploidy patterns. The ploidy profiles of investigated plant in vitro and in vivo differ. For the first time we report that hairy roots of two Solanaceae species undergo endoreduplication. Theploidy profiles of in vitro cultures (hairy roots and calli) seem to be influenced by the genome size, the growth factors applied, and the type of in vitro culture. The transformation of several hairy root lines showed no differences in the ploidy patterns.

Effects of caffeine on stereoselectivities of high cell density biotransformations of cyclic beta-keto esters with Saccharomyces cerevisiae.

Caffeine affects the stereoselectivity of microbial high cell density reductions with commercial grade Saccharomyces cerevisiae (Baker's yeast). Cyclic beta-keto esters ethyl 2-oxocyclopentanoate (1) and ethyl 2-oxocyclohexanoate (3) were shown to be reduced with increased diastereoselectivity (1: 90.1 --> 92.1% de, 3: 75.0 --> 90.0% de) after addition of caffeine. Effects on enantioselectivity were less pronounced (1: 97.3 --> 98.5% ee, 3: 90.1 --> 92.1% ee). The observations are ascribed to the action of caffeine on cellular calcium homeostasis. These effects are accompanied by caffeine-induced cell-death, which preferably takes effect on pre-stressed cells which were found to decrease diastereoselectivity.

cAMP is a ligand for the tandem GAF domain of human phosphodiesterase 10 and cGMP for the tandem GAF domain of phosphodiesterase 11.

N-terminal tandem GAF domains are present in 5 out of 11 mammalian phosphodiesterase (PDE) families. The ligand for the GAF domains of PDEs 2, 5, and 6 is cGMP, whereas those for PDEs 10 and 11 remained enigmatic for years. Here we used the cyanobacterial cyaB1 adenylyl cyclase, which has an N-terminal tandem GAF domain closely related to those of the mammalian PDEs, as an assay system to identify the ligands for the human PDEs 10 and 11 GAF domains. We report that a chimera between the PDE10 GAF domain and the cyanobacterial cyclase was 9-fold stimulated by cAMP (EC50= 19.8 microm), whereas cGMP had only low activity. cAMP increased Vmax in a non-cooperative manner and did not affect the Km for ATP of 27 microm. In an analogous chimeric construct with the tandem GAF domain of human PDE11A4, cGMP was identified as an allosteric activator (EC50 = 72.5 microm) that increased Vmax of the cyclase non-cooperatively 4-fold. GAF-B of PDE10 and GAF-A of PDE11A4 contain an invariant NKFDE motif present in all mammalian PDE GAF ensembles. We mutated the aspartates within this motif in both regions and found that intramolecular signaling was considerably reduced or abolished. This was in line with all data concerning GAF domains with an NKFDE motif as far as they have been tested. The data appeared to define those GAF domains as a distinct subclass within the >3100 annotated GAF domains for which we propose a tentative classification scheme.