Evaluation of Carbon and Electron Flow in Lactobacillus brevis as a Potential Host for Heterologous 1-Butanol Biosynthesis

Heterofermentative lactic acid bacterium Lactobacillus brevis may be considered as a promising host for heterologous butanol synthesis because of tolerance to butanol and ability to ferment pentose and hexose sugars from wood hydrolysates that are cheap and renewable carbohydrate source. Carbon and electron flow was evaluated in two L. brevis strains in order to assess metabolic potential of these bacteria for heterologous butanol synthesis. Conditions required for generation of acetyl-CoA and NADH which are necessary for butanol biosynthesis have been determined. Key enzymes controlling direction of metabolic fluxes in L. brevis in various redox conditions were defined. In anaerobic glucose fermentation, the carbon flow through acetyl-CoA is regulated by aldehyde dehydrogenase ALDH possessing low affinity to NADH and activity ( = 200 μM, Vmax= 0.03 U/mg of total cell protein). Aerobically, the NADHoxidase NOX ( = 25 μM, Vmax = 1.7 U/mg) efficiently competes with ALDH for NADH that results in formation of acetate instead of acetyl-CoA. In general, external electron acceptors (oxygen, fructose) and pentoses decrease NADH availability for native ethanol and recombinant butanol enzymes and therefore reduce carbon flux through acetyl-CoA. Pyruvate metabolism was studied in order to reveal redirection possibilities of competitive carbon fluxes towards butanol synthesis. The study provides a basis for the rational development of L. brevis strains producing butanol from wood hydrolysate. NADH m K


Introduction
A number of studies have been carried out on the production of liquid fuels from renewable plant sources by microbial synthesis.A special attention is paid to alcohols, mainly ethanol and 1-butanol as an effective substitute for gasoline and diesel in combustion engines [1,2].1-butanol has an advantage over ethanol due to its higher energy content, lower volatility, less ignition problems, better miscibility with gasoline and the possibility to use it without modification of engines and infrastructure for supplying and distribution [3].Anaerobic bacteria of the genus Clostridium are the natural producers of 1-butanol [4].However, the application of existing Clostridium strains for large scale industry is not feasible in the current economic conditions because of low 1-butanol titer (<15 g/l), two-phase metabolism (formation of acids precedes the formation of solvents) hampering organization of a continuous fermentation process, synthesis of a significant amount of by-products (acetone, ethanol, acetate, butyrate) and expensive fermentation substrates.
Economically viable production of biofuels should be based on an inexpensive, renewable raw material like plant biomass.Recently developed SEW (SO 2 -ethanolwater) pulping is a promising fractionation process for lignocellulosic biomass.Its advantages over conventional pulping methods include simplified chemical recovery, lower capital costs and rapid impregnation of the feed stocks [12,13].SEW-hydrolysate from spruce chips contains mannose, glucose, galactose, xylose, and arabinose which can be used for microbial fermentation [14].However, hydrolysis by-products such as formic acid, acetic acid, levulinic acid, furfural and hydroxymethyl furfural inhibit fermentation of many industrial microorganisms, including E. coli [15,16].
Heterofermentative lactic acid bacterium Lactobacillus brevis is able to ferment pentose as well as hexose sugars from various plant sources [17] including acid hydrolyzate of hemicellulose [18].In addition L. brevis is tolerant to 3% butanol and may be easily adapted to increased butanol concentration [9,19].Thus it can be considered as a potential platform for heterologous butanol synthesis from cheap renewable plant sources.The first objective of the present work is to investigate the ability of wild-type L. brevis strains to ferment sugars of SEWhydrolyzate from spruce chips that are abundant waste of woodworking and pulp and paper industry.
In the previous study we constructed a L. brevis butanol-producing strain by cloning thl, hbd, crt, bcd, etfB, and etfA genes from Clostridium acetobutylicum [9].However, the butanol titer in recombinant strain did not exceed ~300 mg•l −1 .Expression of the butanol pathway genes did not change the level and the ratio of native L. brevis end-products: the spectra of metabolites other than butanol were similar in recombinant and wild-type L. brevis strains that indicated the inability of the recombinant pathway to compete with native ones.The present study is focused on evaluation of carbon and electron flow in wild-type L. brevis strains in order to assess metabolic potentialities of these bacteria for heterologous butanol synthesis and reveal bottlenecks preventing efficient butanol synthesis by recombinant L. brevis strains.

Bacterial Strains and Growth Conditions
Lactobacillus brevis ATCC 367, Lactobacillus brevis ATCC 8287, and Clostridium acetobutylicum ATCC 824 were used for metabolic studies.

Chromatograph
Metabolic end-products of L. brevis were identified and quantified by high-performance liquid chromatography (HPLC).Waters 2695 Separations module was equipped with Waters 2414 Refractive Index Detector, Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm × 9 µm), and Micro-Guard Cation H + -Cartridge (Bio-Rad, Hercules, CA, US).The column was heated at 65˚C; the eluent (5 mM H 2 SO 4 ) was circulated at a flow rate of 0.60 mL•min −1 .The sugars were determined by Waters 2690 Separations module with Waters 2414 Refractive Index Detector, equipped with a Bio-Rad HPX-87P column (300 mm  7.8 mm  9 µm) and two Micro-Guard Deashing Cartridges (Bio-Rad, Hercules, CA, US) at 70˚C with a flow rate of 0.60 mL•min −1 using deionized water as eluent.Cellobiose (Roth, Karlsruhe, Germany) was added to the samples as an internal standard.Sugar and metabolite concentrations were calculated from at least three experiments, the accuracy was within ±5%.

Enzyme Assays
Cell-free extracts were prepared as described previously [9].The protein content was determined with bicinchoninic acid [21] or by Bradford [22] after removal of the cell debris.Bovine serum albumine (Sigma) was used as a standard.
Lactate dehydrogenase, NADH:H 2 O oxidase, alcohol dehydrogenase, aldehyde dehydrogenase, and 3-hydroxybutyryl-CoA dehydrogenase activities were measured by NADH oxidation at 340 nm ( = 6.22) in accordance with the published procedures [9,23,24].ADH activity was measured by NAD + reduction as described by Berezina et al. [9].All activities were assayed at 30˚C and pH 6.7 that corresponded to intracellular conditions in logarithmic growth phase [25].
NADH mM  Enzyme activity of pyruvate dehydrogenase was assayed as described by Yahui et al. [26] from the extracts of aerobically or semi-anaerobically grown cells collected at logarithmic growth phase, at pH 7.1 (enzyme optimum) and 6.7 (intracellular pH).Pyruvate formate lyase activity was measured as described by Asanuma et al. [27], in an anaerobic chamber.The cell extracts for PFL assay were prepared anaerobically as described by Berezina et al. [9].Pyruvate oxidase, and pyruvate decarboxylase activities were assayed as described previously [28,29].One unit of activity was defined as 1 µmol of substrate utilized or product formed in a minute per mg of total cell protein (U/mg).

Measurement of Intracellular NAD + and
NADH Levels A freshly grown cell culture was concentrated by centrifugation up to 30 mg•ml −1 of total cell protein.For NAD + extraction, 0.1 ml of perchloric acid was mixed with 0.4 ml of cell suspension, incubated for 5 min at 60˚C and immediately chilled on ice.The extract was neutralized to pH 7.0 with 0.5 ml of 2 M KOH containing 1.2 M Tris-HCl (pH 9.0) and 1.36 M semicarbazide.The precipitate was removed by centrifugation, and the supernatant was used for NAD + measurements.For NADH extraction, 0.1 ml of 2 M KOH was mixed with 0.3 ml of cell suspension, incubated for 5 min at 60˚C and immediately chilled on ice.The extract was neutralized on ice to pH 8.0 with 0.4 ml of 1M HEPES, pH 5.0.The precipitate was removed by centrifugation and the supernatant was used for NADH measurements.NAD + and NADH concentrations in the extract were determined by enzyme assay with commercial alcohol and lactate dehydrogenases (Sigma-Aldrich) according to Bergmeyer [30].Absorbance at 340 nm was measured by a Cintra 404 spectrophotometer.Total intracellular [NAD + + NADH] and [NADH] concentrations were calculated assuming that 1 mg of total cell protein binds 3.7 μl of cytosole [31].

Effect of Aeration and Sugar Composition on Growth Characteristics of Two L. brevis Strains
Although L. brevis lacks cytochromes, porphyrins, and respiratory enzymes and is generally recognized as an anaerobic bacteria [32], the aeration positively influenced growth rate of both strains (Figure 1).In aerobic incubation in MRS medium the ATCC 8287 had a higher growth rate than the ATCC 367, while in semi-anaerobic conditions the difference between the strains was very small (Figure 1(a)).
In HM medium the ATCC 8287 strain had a higher growth rate than the ATCC 367 strain both in aerobic and semi-anaerobic conditions (Figure 1(b)).
Both strains were able to ferment glucose, xylose, arabinose and galactose of SEW-hydrolysate, but not mannose (Figure 2).The 8287 strain fermented xylose better than the 367 strain that correlates with faster growth of the 8287 strain in semi-anaerobic conditions (Figure 1

(b)).
In semi-anaerobic fermentation of MRS medium lactate, ethanol and mannitol were produced from glucose and fructose.The level of acetate at first went down, and  then came back to initial value (Figure 3(a)).In aerobic MRS fermentation by L. brevis 8287, lactate and acetate were produced in equimolar amounts during exponential growth phase.In stationary growth phase the decrease in lactate concentration was equal to increase in acetate concentration.Ethanol was not produced aerobically and mannitol only in minute amounts (Figure 3(b)).Aerobic and semi-anaerobic fermentations patterns of L. brevis 367 were similar to L. brevis 8287.
The time-dependent profiles of sugar utilization and metabolite formation during semi-anaerobic and aerobic fermentation of HM medium containing hexoses, pentoses, and uronic acids (Table 1) by L. brevis 8287 are shown in Figures 3(c) and (d).Lactate, ethanol and acetate were produced semi-anaerobically whereas only lactate and acetate were produced aerobically.In stationary growth phase of aerobic incubation, the concentration of lactate decreased while the acetate concentration increased correspondingly (Figure 3(d)).For L. brevis ATCC 367 the pattern of metabolic end-products was similar.Both strains accumulated biomass in HM medium better than in MRS medium, and the largest difference was observed in semi-anaerobic ATCC 8287 culture (Figure 1).
Fermentation balance of the ATCC 8287 strain cultivated in semi-anaerobic and aerobic conditions on MRS and HM media is shown in Table 2.
Overall lactate dehydrogenase activity was higher than the other detected enzyme activities: V max 37.0 and 26.5 U/mg of total cell protein in the 367 and 8287 strains correspondingly.ALDH activity was 0.02 and 0.03 U/mg in 367 and 8287 strains correspondingly (Table 4).ADH activity following the ALDH in the ethanol-forming pathway was 10.8 U/mg in both strains.No reverse ADH activity was detected in cell extracts at pH 6.7 or pH 6.3.This indicates that the process of ethanol formation is irreversible in physiological conditions.values for LDH and ALDH were comparable and about 2 -2.5 times higher than of ADH (Table 4).NOX has higher activity compared to ALDH and the highest affinity to NADH among all the tested enzymes: V max (NOX) = 1.7 U/mg, K m (NOX) = 25 µM versus V max (ALDH) = 0.03 U/mg, K m (ALDH) = 200 µM for ATCC 8287 strain (Table 4).
In the recombinant butanol-producing L. brevis strains [7], the 3-hydroxybutyryl-CoA dehydrogenase (HBD, EC 1.1.1.35)is the first NADH-dependent enzyme of the heterologous butanol pathway originated from acetyl-CoA.The of HBD is 178 µM, which is slightly lower than of ALDH but sufficiently higher than of NOX (Table 4).

K m
Intracellular NAD + /NADH ratio and total [NAD + + NADH] concentration as a function of cell growth were determined for batch cultures (Figure 4).

Pyruvate-Converting Pathways in L. brevis
In L. brevis, pyruvate is converted to D-and L-lactate by corresponding lactate-dehydrogenases [9] (Table 3).But under certain conditions LAB may use alternative ways of utilizing pyruvate than reduction to lactic acid.Pathways of pyruvate metabolism were investigated in L. brevis ATCC 367 and 8287 strains (Figure 5).Pyruvate oxidase (POX), pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH) and pyruvate formate lyase (PFL) activities were assayed in the cell extracts.The L. brevis ATCC 367 genome was screened for the corresponding genes.Pyruvate oxidase activity was detectable only at the late stationary phase in aerobic conditions being 0.02 and 0.01 U/mg of total cell protein for ATCC 8287 and ATCC 367 strains respectively.The putative pyruvate oxidase-encoding gene was identified in the L. brevis ATCC 367 genome (Table 3).
Although PDH activity was not detected, the genes predictably encoding enzymes of PDH complex were identified in L. brevis ATCC 367 genome (Table 3).No PFL (EC 2.3.1.54)or PDC (EC 4.1.1.1)activities were detected under conditions applied, and the corresponding genes were not identified during genome analysis.Formate produced in the PFL reaction was not found among metabolic end-products.
The genes presumably encoding acetolactate synthase (ALS), alpha-acetolactate decarboxylase (ALDB) and diacetyl acetoin reductase also named butanediol dehydrogenase (BUTB) participating in synthesis of acetolactate, acetoin and 2,3-butenediol from pyruvate, were identified in L. brevis ATCC 367 genome (Table 3).However, these metabolites were not detected in cultural liquid during L. brevis semi-anaerobic or aerobic fermentation in MRS or HM media.
Thus, besides conversion into lactate, pyruvate may be converted into acetyl-phosphate by pyruvate oxidase in strictly aerobic conditions and under glucose limitation.No other overlaps between lactate and ethanol metabolic branches have been detected in L. brevis.
Data of metabolic stoichiometry and enzyme kinetics allowed developing the scheme of carbon and electron flow in L. brevis in anaerobic and aerobic glucose fermentation (Figure 6).

Key Factors Influencing the Direction of Carbon and Electron Flow in L. brevis
L. brevis metabolism is based on carbohydrate fermentation coupled with substrate level phosphorylation.Oxidation of a substrate leads to formation of NADH from NAD + , which has to be regenerated continuously.Stoichiometry analysis of fermentation in various redox conditions (Table 2) proved that L. brevis utilizes sugars through the 6-phosphogluconate (6-PG) pathway.The crucial enzyme of the 6-PG pathway is phosphoketolase, which converts xylulose-5-phosphate (C5) into glyceraldehyde-3-phosphate (C3) and acetyl phosphate (C2) [32] (Figure 6).In semi-anaerobic glucose fermentation this bifurcation leads to formation of lactate (C3) and ethanol (C2) (Table 2, Figure 3(a)) that have a key role in NAD + regeneration.
In comparison with the lactate branch, the NADHoxidizing capacity of the ethanol branch is very low: ALDH activity was about three orders lower than LDH activity (Table 4).It makes ALDH the bottleneck of the L. brevis anaerobic metabolism, despite the highly active ADH following the ALDH in the ethanol-forming pathway.The ethanol branch does not bring additional ATP to the cells and it participates only in the maintenance of red-ox balance that determines the low level of biomass accumulation by L. brevis grown semi-anaerobically in MRS medium (Figure 1(a)).
In aerobic conversion of hexoses, the oxygen acts as the external electron acceptor in a reaction catalyzed by NADH:H 2 O oxidase (NOX) (Figure 6).Due to significantly higher activity and affinity to NADH compared to ALDH (Table 4), the NOX has the strong benefit of NAD + regeneration and thereby prevents ethanol formation.The acetyl-phosphate is completely redirected from acetyl-CoA formation towards the acetate formation (Figure 3(b)) accompanied by synthesis additional ATP in acetate kinase reaction (Figure 6), and hence faster aerobic growth is observed in comparison to semi-anaerobic conditions (Figure 1(a)).
The rapid growth of ATCC 8287 versus ATCC 367 in MRS medium in aerobic conditions (Figure 1(a)) was obviously dependent on higher NOX activity of the ATCC 8287 strain: 1.7 U/mg in ATCC 8287 vs 0.20 U/mg in ATCC 367 (Table 4).
In the recombinant L. brevis strains the native ethanol and recombinant butanol pathways originate from acetyl-CoA [9].3-Hydroxybutyryl-CoA dehydrogenase (HBD) is a first NADH-dependent enzyme of the butanol pathway.In aerobic conditions, the NOX, because of its comparatively high activity and affinity to NADH, efficiently competed for reducing equivalents not only with the ALDH, but with the HBD: the affinity towards NADH of the HBD was about 7 times lower than the NOX affinity (Table 4).For this reason, the redirection of the electron and carbon flow towards butanol synthesis was not achieved aerobically, and the recombinant L. brevis strains produced butanol only in semi-anaerobic fermentation during exponential growth phase [9].
An active role of oxygen and NOX in growth and energy metabolism of various LAB species has been demonstrated in previous studies [35][36][37].For example it was shown that the growth rate and the Y glc of Leuconostoc sp in aerated cultures were higher compared to non-aerated.An NOX-deficient mutant did not shift from ethanol to acetate production, and Y glc was the same aerobically and anaerobically [37].
The ratio of NAD + /NADH and total intracellular [NAD + + NADH] concentration correlated with the sugar consumption and influenced the pathway fluxes at the level of various dehydrogenase reactions.In the loga-rithmic growth phase of a batch culture, the NAD + / NADH ratio increased (Figure 4(a)) while total [NAD + + NADH] concentration decreased (Figure 4(b)).The intracellular NADH concentration dropped from 700 to 50 μM consequently passing through the K m values of LDH, ALDH, ADH, and hence the activity of the corresponding enzymes decreased.In this way the cell automatically regulates carbon fluxes in response to environmental conditions.

Fermentation of Complex Sugar Substrates
Anaerobic heterolactic fermentation of 1 mole glucose through 6-PG pathway gives theoretically 1 mole of lactic acid, ethanol, CO 2 and ATP.Semi-anaerobic incubation of L. brevis in MRS media supplemented with glucose resulted in almost theoretical distribution of lactate and ethanol [9].
A complex fermentation medium changes the balance and the composition of end-products.Lactate, ethanol, acetate and mannitol were produced during semi-anaerobic fermentation of MRS medium containing glucose and fructose (Figure 3(a), Table 2).Mannitol formation indicates that fructose is not only used as a carbon source, but as an additional electron acceptor, and metabolic intensity prevails over efficiency of substrate utilization.Reduction of fructose to mannitol may be catalyzed by NAD + : mannitol dehydrogenase that is presumably encoded in L. brevis ATCC 367 by LVIS 2162 gene.This reaction allows overcoming the low capacity of the ethanol branch for NAD + regeneration arising at the level of aldehyde dehydrogenase [38].Using the mannitol pathway in fructose fermentation was earlier described with Oenococcus oeni [39].
In aerobic conditions NOX prevented not only the ethanol formation, but also the use of fructose as an electron acceptor (Figure 3(b), Table 2).
L. brevis strains can utilize hexoses (glucose, galactose), pentoses (xylose, arabinose) and uronic acids of SEW-hydrolyzate (Figure 2, Table 2).No CO 2 is formed during pentose fermentation in 6-PG pathway and since no dehydrogenation steps are necessary to reach the xylulose-5-phosphate. Consequently the ethanol formation becomes redundant.Instead, acetyl phosphate is used by the acetate kinase yielding acetate and ATP.Fermentation of pentoses thus leads to production of equimolar amounts of lactic and acetic acids [32].Therefore fermentation of HM medium resulted in lower ethanol and higher acetate ratio as well as higher biomass accumulation level compared to MRS medium containing glucose and fructose (Table 2).The uronic acids present in SEWhydrolyzate may be utilized to form lactate and acetate (http://www.genome.jp/kegg-bin/show_pathway?lbr0004 0).

Role of Pyruvate Oxidase in L. brevis Metabolism
Under aerobic conditions and sugar limitation acetate is produced at the expense of lactate as glucose becomes depleted and L. brevis enters the stationary phase of growth (Figures 3(b) and (d)).Various LAB species can use lactate as a carbon source [40,41].The pathway of lactate to acetate conversion could be performed via three enzymatic steps: oxidation of lactate to pyruvate by the NADH-dependent D-and L-LDHs, oxidative decarboxylation of pyruvate to acetyl-phosphate by pyruvate oxidase, and dephosphorylation of acetyl-phosphate to acetate by acetate kinase (Figures 5, 6).ATP formed in ACK reaction provides the cells with energy in the stationary phase.The LDH-POX-ACK pathway is regulated at the level of pyruvate-oxidase activity, which is induced by O 2 and repressed by glucose [41][42][43][44][45].In the L. brevis ATCC 367 genome, the putative pyruvate oxidase-encoding gene was identified.The pyruvate oxidase activity was detected at the late stationary phase of aerobic growth.Lactate to acetate conversion could also be performed aerobically by oxygen-dependent lactate oxidase (lactate 2-mono-oxygenase).However, no similarity with the corresponding genes was found in L. brevis genome analysis.Thereby in aerobic conditions and under glucose limitation L. brevis cells may use pyruvate oxidase as a link between pyruvate and acetyl-phosphate in a pathway from lactate to acetate.Since anaerobic conditions are required for heterologous butanol synthesis, the aerobically active pyruvate oxidase cannot be used for redirecting carbon flux from lactate to butanol synthesis.

Proposed Strategy for Redirection L. brevis Carbon and Electron Flow towards Butanol Synthesis
In Therefore pyruvate redirection may be organized by cloning the pyruvate formate lyase genes (Figure 7).The anaerobically active PFL enzyme system has been shown to be operational in several LAB species [46][47][48].Cloning the formate dehydrogenase (FDH) might help to utilize formate produced in PFL reaction and provide an additional mole of NADH per mol of 1-butanol produced (Figure 7).
Despite the principal possibility to support the butanol synthesis [9], the native L. brevis ALDH, ADH and THL, and recombinant clostridial BCD/EtfAB cannot provide a high capacity pathway for butanol because of 1) low activity of ALDH and THL, 2) low specificity towards C4 substrate of ALDH and ADH [9] and 3) cofactor (FAD and ferredoxin) deficiency for BCD/EtfAB [49].These enzymes should be enhanced or substituted with efficient analogues.

Conclusions
Carbon and electron flow was investigated in two L. bre- vis strains in order to assess metabolic potential of these bacteria for heterologous butanol synthesis.The study approach was based on analysis of fermentation stoichiometry in various redox conditions together with catalytic properties ( , V max ) of the NADH-dependent enzymes from the main metabolic pathways.In general, availability of external electron acceptors (oxygen, fructose) enhances biomass accumulation associated with additional ATP synthesis in acetate kinase reaction, but reduces NADH availability for enzymes of ethanol pathway and, therefore, decreases carbon flux through acetyl-CoA.
Pyruvate metabolism was investigated to find redirection possibilities of competitive carbon fluxes towards butanol synthesis.Pyruvate may be converted into acetyl-phosphate by pyruvate oxidase in strictly aerobic conditions and under glucose limitation.No other overlaps between lactate and ethanol metabolic branches have been detected in L. brevis.
L. brevis strains ferment glucose, galactose, arabinose, and xylose from SEW-hydrolysate of spruce chips, but not utilize mannose.Increased acetate formation accompanied by enhanced cell growth and decreased ethanol synthesis indicated reduced carbon flow though the acetyl-CoA.Thus, SEW-hydrolyzate could be used for initial biomass accumulation of butanol-producing L. brevis strains.Using SEW-hydrolysate for butanol synthesis requires optimization of fermentation media in order to increase NADH availability for enzymes of butanol pathway.
The study provides a basis for rational development of L. brevis strains producing butanol from SEW-hydrolysate and proposes possible engineering ways for rerouting carbon and electron flow towards butanol synthesis.

Acknowledgements
This work has been supported in part by the Ministry of Education and Science of the Russian Federation (EurAsES program, contract № 16.M04.12.0017) and by Academy of Finland (grant № 137469, 15.04.2010 applied by Tom Granström).We are most grateful to Professor Matti Leisola, Aalto University, School of Chemical Technology, for his invaluable advice and helpful discussions.

Figure 2 .
Figure 2. Sugar utilization by L. brevis ATCC 8287 and ATCC 367 strains grown semi-anaerobically on HM medium during 72 h.The mean values of at least three independent experiments are presented.

Figure 6 .
Figure 6.The scheme of carbon and electron flow in L. brevis in anaerobic and aerobic glucose fermentation.The solid arrows indicate native permanently acting reactions.The dotted arrows indicate reactions acting preferably in anaerobic condition.The hollow arrows indicate reactions active aerobically.NOX: NADH oxydase, LDH: lactate dehydrogenase, ADH: alcohol dehydrogenase, ALDH: aldehyde dehydrogenase, PK: phosphoketolase, PTA: phosphotransacetylase, ACK: acetate kinase, POX: pyruvate oxidase.

KK
Conditions necessary for generation of acetyl-CoA and NADH required for butanol biosynthesis have been determined.Key enzymes controlling direction of carbon and electron flow in L. brevis were defined.In anaerobic glucose fermentation, the carbon flow through acetyl-CoA and the slow NAD + regeneration rate are controlled by aldehyde dehydrogenase ALDH ( = 200 µM, V max = 0.03 U/mg).Aerobically, the NOX, due to its comparatively high affinity to NADH and activity ( = 25 µM, V max = 1.7 U/mg), efficiently competes with ALDH for NADH that results in redirecting carbon flow from acetyl-CoA towards acetate formation.In recombinant butanol-producing strains the enzymes of the native ethanol and heterologous butanol pathways compete for acetyl-CoA and NADH.Aerobically, the NOX, because of low , prevents not only the ethanol but also the butanol formation.NADH m K