Dataset describing the amino acid catabolism of Thermoanaerobacter pseudethanolicus

The dataset depicts the catabolism of branched-chain amino acids by Thermoanaerobacter pseudethanolicus in the presence of thiosulfate under different culture conditions. The results reveal that the strain can degrade all three branched-chain amino acids resulting in the production of their corresponding branched-chain fatty acids and branched-chain alcohols with the fatty acids always being the dominant product. The highest amounts of 2-methyl-1-butanol from isoleucine were at pH 6.5, liquid-gas ratio of 0.98, and at 20 mM thiosulfate concentration. A kinetic experiment of the branched-chain amino acids was done in the presence of thiosulfate as are data on selected enzyme activities related to alcohols and aldehydes. Finally, an NMR study using 13C1 methyl-1-butyrate during the degradation of leucine in the presence of thiosulfate was done to prove that the 13C1-methyl-1-butanol was indeed from its corresponding fatty acid.


Value of the Data
• The data presents fermentation products from the catabolism of the branched-chain amino acids, namely leucine, isoleucine, and valine, using thiosulfate as an electron scavenging system by Thermoanaerobacter pseudethanolicus .• The data set shows the influence of culture parameters (pH, partial pressure of hydrogen, initial thiosulfate concentration) on the fermentation of a branched-chain amino acid, Lisoleucine, in batch culture, as well as a kinetic experiment showing the formation of branched-chain fatty acids and alcohols from branched-chain amino acids.• The enzyme activity data using alcohols and aldehydes as substrates suggest that the enzymes involved in the production of alcohols as end products from amino acid catabolism have a degree of promiscuity.• The 13 C NMR experiments reveal that a potential route for the production of the corresponding alcohols results from the reduction of the fatty acid to the alcohol under fermentative conditions.• The fermentation end product data provides insight into the catabolism of amino acids in thermophilic environments such as hot springs where sulfur species are often present.• Suggests that Thermoanaerobacter species are important parts of the nitrogen cycle within hot springs and other thermal environments and the use of alternative electron sinks such as fatty acids • The data is useful for comparison of amino acid catabolism of other mesophilic and thermophilic anaerobes producing higher-value linear and branched-chain aliphatic alcohols.

Data Description
Similar to a number of mesophilic Clostridia, Thermoanaerobacter pseudethanolicus (DSM 2355) degrades the branched-chain amino acids (BCAAs) to a mixture of the corresponding branched-chain fatty acids (BCFA; major end product) and alcohols (BCOH; minor end product) in the presence of thiosulfate [1] .The influence of various culture parameters known to result in shifts in end product formation (such as liquid-gas phase ratio, pH, and the concentration of thiosulfate) was investigated in batch culture using isoleucine as a model BCAA.
The dataset contains eight tables (Tables final.docx),seven of which detail fermentation data, namely metabolic end products such as alcohols, fatty acids, and hydrogen, while the remain-ing table contains enzyme activities towards selected alcohol and aldehyde substrates.Each line details the experimental conditions for a given experiment with a data point for the analyte concentration in mmol per L presented as the average ± standard deviation measured at the indicated time.Additionally, one table summarizes the enzymatic activity of crude cell lysates towards alcohol and aldehyde substrates using NAD + or NADP + as a cofactor.Table 1 summarizes the production of fermentation products (BCFAs and BCOHs) from BCAAs (20 mM) with and without the addition of thiosulfate (40 mM).Similarly, Table 2 shows the effect of the initial pH of the fermentation medium on the catabolism of isoleucine while Table 3 details end-product formation when the strain was cultivated at various liquid-gas phase ratios, a proxy for gauging the influence of the partial pressure of hydrogen, ranging from a low L-G ratio with a large headspace to a high L-G ratio with limited headspace.Table 4 shows the impact of initial thiosulfate concentrations between 0 and 60 mM on the fermentation products from isoleucine catabolism.Additionally, detailed kinetic experiments were performed over a period of 7 days using all three of the BCAAs (valine, isoleucine, and leucine) as single substrates with thiosulfate addition as summarized in Tables 5-7 .Finally, the volumetric activities oxidative enzyme reactions of T. pseudethanolicus cultivated on various substrates using NAD + and NADP + as a cofactor during growth of the strain on leucine with thiosulfate, and leucine with and without thiosulfate but in the presence of 3-methyl-1-butyrate (20 mM) were performed ( Table 8 ).

General methods
Yeast extract was obtained from Difco; nicotinamide cofactors were obtained from Megazyme while all other reagents were acquired from Sigma-Aldrich.Nitrogen gas was acquired from AGA and contained less than 5 ppm O 2 .Isotopically labelled substrates were obtained from Cambridge Isotope Laboratories (MA, USA).

Microorganism and cultivation
Thermoanaerobacter pseudethanolicus (DSM 2355) was obtained from DSMZ culture collection and freezer stocks maintained in Basal Mineral (BM) medium supplemented with 30% v/v glycerol and stored at −20 °C.The strain was routinely cultivated in serum bottles or Hungate tubes using the Basal Mineral (BM) medium prepared as previously described [2] using the Hungate technique [3 , 4] .The content of BM, media preparation and sterilization has been described earlier [2] .Vitamins, trace element solution and, substrates were added separately through sterilized filter (0.45 μm, polyethersulfone or cellulose acetate) after autoclaving.Substrates were provided at a final concentration of 20 mM unless explicitly stated otherwise.All cultivations were performed at 65 °C and at pH of 7.0 with a liquid-gas (L-G) ratio of 1:1 without agitation except when stated otherwise.Inoculation of the strain was taken from the exponential growth phase using an inoculation volume of 2% (v/v).All cultivations were performed as triplicates and fermentation products were quantified after five days of cultivation unless stated otherwise.

Fermentation of branched-chain amino acids in presence and absence of thiosulfate
The capability of T. pseudethanolicus to catabolize branched-chain amino acids, namely valine, leucine, and isoleucine, was evaluated at concentration of 20 mM with and without thiosulfate supplementation (40 mM).Cultivations were carried out serum bottles (117.5 mL nominal volume) for 7 days at which time fermentation broth (1 mL) and headspace gas (0.2 mL) was removed for analysis.

Effect of initial pH on isoleucine fermentation
T. pseudethanolicus was cultivated at various initial pH values (4.0 to 9.0 in 0.5 unit increments) using isoleucine (20 mM) as a model BCAA plus the addition of thiosulfate (20 mM) in 16 × 150 mm Hungate tubes for 14 days.Samples were taken for analysis as described in the previous section.

Effect of liquid-gas phase ratio
T. pseudethanolicus was grown in anaerobic bottles (118.5 mL nominal volume) which were filled with specific volumes of BM medium containing isoleucine (20 mM) and thiosulfate (20 mM); final media volume of 10.0, 30.0, 59.25, 80.0, and 100.0 mL of media to afford L-G ratios of 0.09, 0.34, 1.00 2.12, and 5.41, respectively.Fermentation products were analyzed as above after 14 days.

Effect of initial thiosulfate concentration
The impact of thiosulfate concentration on isoleucine (20 mM) fermentation was performed with the addition of between 0 to60 mM of thiosulfate in 10 mM increments.The experiments were performed Hungate tubes (16 × 150 mm).End products were quantified after 14 days as described above.

Kinetic experiments
Time-course studies of the degradation of leucine, isoleucine, and valine (20 mM) with thiosulfate (20 mM) were performed in 118.5 mL anaerobic bottles over 7 days.1 mL aliquots of fermentation broth and headspace gas were periodically for analysis as described above.

Enzyme assays
Cells for enzymatic assays were grown in 1 L serum bottles fitted with butyl rubber septa containing 500 mL of BM media containing Leu (20 mM) and electron acceptor (20 mM, 3methyl-1-butyrate and/or sodium thiosulfate).After 18 h of cultivation, 5 mg of potassium dithionate was added to protect against oxygen and the cells were harvested by centrifugation (4700 rpm, 0-4 °C, 15 min).Cell pellets were transferred to a serum bottle and stored under a nitrogen atmosphere at -80 °C prior to analysis.Cells were lysed and oxidative enzymes assays using C2-C8 alcohols and aldehydes with NAD + and NADP + using an Nitro blue tetrazolium -linked assays in microtiter plates described by [1 , 6] .
Briefly, 50μL of diluted enzyme solution, 135 μL of reagent solution (330 μM NAD + or NADP + , 330 μL NBT, 0.13% w/v gelatine, 5 mM CaCl 2 in 50 mM Tris buffer, pH 8.0) containing 5.5 mM of the relevant alcohol, and 15 μL of 10X phenazine methosulfate solution (80 μM) were dispensed into microplates.Samples were incubated anaerobically at 65 °C in a Bioscreen C (GrowthCurves, Ltd, Finland) and read every 2 min at 580 nm.A standard curve was generated by the use of NADH and NADPH.ADH activity was calculated according to the formula below, where v is the

Analytical methods
Hydrogen in the bottle headspace and carboxylic acids and alcohols were measured via gas chromatography as previously described [2] .Thiosulfate, hydrogen sulfide, protein, and amino acids were quantified colorimetrically as previously communicated [5] .Optical density (OD) was measured at 600 nm with a Shimadzu UV-1800 UV-Visible spectrophotometer with quartz cuvette ( l = 1 cm) against a water blank.

13 C-labled experiments
T. pseudethanolicus was cultivated on BM medium supplemented with 20 mM of 13 C2 leucine with and without the addition of sodium thiosulfate and/or 3-methyl-1-butyrate (20 mM) for two weeks as previously described [1] .Also, the strain was cultivated with 13 C1 glucose as a control for 48 h and similarly analyzed.
sample volume in mL and t is the time in minutes.ADH act i v it y mU mL = nmol NADH v • t = nmol NADHx 2

Table 1
Degradation of BCAA by Thermoanaerobacter pseudethanolicus with (40 mM) and without thiosulfate after 7 days of cultivation.Values represent the average of triplicate measures ± standard deviation.

Table 3 Influence
of liquid-gas phase ratio concentration on the fermentation of isoleucine (20 mM) and thiosulfate (20 mM|) by Thermoanaerobacter pseudethanolicus after 14 days of cultivation.Values represent the average of triplicate measures ± standard deviation.

Table 4 Influence
of thiosulfate concentration on the fermentation of isoleucine (20 mM) by Thermoanaerobacter pseudethanolicus after 14 days of cultivation.Values represent the average of triplicate measures ± standard deviation.[S 2 O 3 ] (mM) Analyte (mmol/L)

Table 5
Fermentation kinetics of valine (20 mM) in the presence of thiosulfate (20 mM) by Thermoanaerobacter pseudethanolicus .Values represent the average of triplicate measures ± standard deviation.

Table 6
Fermentation kinetics of isoleucine (20 mM) in the presence of thiosulfate (20 mM) by Thermoanaerobacter pseudethanolicus .Values represent the average of triplicate measures ± standard deviation.Fermentation kinetics of leucine (20 mM) in the presence of thiosulfate (20 mM) by Thermoanaerobacter pseudethanolicus .Values represent the average of triplicate measures ± standard deviation.

Table 8
Enzyme activities (alcohol and aldehyde dehydrogenase activities) using either NAD + or NADP + as a factor of culture of T. pseudethanolicus grown on leucine (20 mM) supplemented thiosulfate (20 mM), leucine (20 mM) supplemented 3-methyl-1-butyrate (20 mM), and leucine (20 mM) supplemented 3-methyl-1-butyrate (20 mM) and thiosulfate (20 mM) after 20 h at 65 °C.Enzyme activities performed by adding 50 μL of cell lysate at a microplate containing 135 μL of reaction solution (containing 330 μM of NAD + or NADP + , 330 μM of NBT, 0.13% w/v gelatin, and 5.5 mM of substrate in 50 mM of Tris-HCl buffer, pH 8.0) followed by 15 μL of 80 μM.Change in absorbance (580 nm) monitored and used to calculate activity where 1 unit of activity corresponds to 1 μmol/min of turn over.Values represent the average of triplicate determinations ± standard deviation.
a Relative to ethanol.b Relative to acetaldehyde, NA -not applicable.