Role of L-alanine for redox self-sufficient amination of alcohols
© Klatte and Wendisch; licensee BioMed Central. 2015
Received: 5 September 2014
Accepted: 30 December 2014
Published: 23 January 2015
In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578–5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed.
The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst.
The replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions.
KeywordsRedox self-sufficient amination Whole cell biotransformation Escherichia coli Transaminase Chromobacterium violaceum Energy maintenance Acetate formation Pyruvate oxidase Phosphate acetyltransferase Acetate kinase
White biotechnology is the key technology for alternative and sustainable production of e.g. fine chemicals. Its application in biocatalysis is considered a branch of Green Chemistry which can replace or complement routes of chemical modification and functionalization. Enzymes catalyze reactions under mild conditions contrarily to chemical catalysts which often demands high pressure and temperature as well as toxic solvents. Among others, amine functionalization of chemical compounds is an important approach in biocatalysis to produce (poly)amines which are components of for example synthetics and coatings. This can be performed by amino acid dehydrogenases catalyzing NADH-dependent reductive amination of oxo-acids with ammonium or by transaminases transferring an amino group from a donor amine to a carbonyl compound. The cofactor pyridoxal-phosphate is covalently bound to the catalytic center of ω-transaminases to transfer the amino group to the acceptor molecule [1,2].
Construction of the whole cell biocatalyst W3110/pTrc99A-ald-adh-ta Cv and its comparison to W3110/pTrc99A-ald-adh-ta Vf in vitro and in vivo
The whole cell biocatalyst W3110/pTrc99A-ald-adh-ta Vf was previously shown to enable redox self-sufficient amination of a variety of alcohols  and involved thermo-sensitive transaminase from Vibrio fluvialis. Due to the thermostable alcohol dehydrogenase of B. stearothermophilus an increased reaction temperature for redox self-sufficient amination was considered to improve the production rate. Therefore, the gene for the transaminase of V. fluvialis was replaced by the gene for the transaminase of C. violaceum in the IPTG-inducible vector pTrc99A-ald-adh-ta Vf. The vector was used to transform E. coli W3110 to yield the whole cell biocatalyst W3110/pTrc99A-ald-adh-ta Cv. Enzyme activity assays of the newly constructed whole cell biocatalyst revealed that all three genes were functionally expressed. The crude extracts displayed enzyme activities of 9.8 ± 1.1 and 0.55 ± 0.03 U/mg for the alanine dehydrogenase and the alcohol dehydrogenase, respectively, which was similar to the activities in W3110/pTrc99a-ald-adh-ta Vf . However, with (S)-(−)α-methylbenzylamine as substrate, the specific activities of transaminase TaCv of 0.62 ± 0.01 in W3110/pTrc99a-ald-adh-ta Cv were two-fold higher than that of TaVf in W3110/pTrc99a-ald-adh-ta Vf.
In vitro estimation of the catalytic efficiencies of transaminases Ta Vf and Ta Cv with L-alanine and hexanal as substrates
Origin of the transaminase
K m for L-alanine [mM]
V max [U/mg]
V max /K m
20.00 ± 1.10
0.30 ± 0.01
35.00 ± 2.20
2.00 ± 0.07
Comparison of the redox self-sufficent amination of 1,10-decanediol by W3110/pTrc99a- ald-adh-ta Vf and W3110/pTrc99a- ald-adh-ta Cv with varying L-alanine concentrations
Reaction temperature [°C]
Max. conversion [%]
Alanine consumption [mM]
W3110/pTrc99a- ald-adh-ta Cv
100 mM alanine, 100 mM NH4Cl
50 mM alanine, 100 mM NH4Cl
20 mM alanine, 100 mM NH4Cl
W3110/pTrc99a- ald-adh-ta Vf
100 mM alanine, 100 mM NH4Cl
50 mM alanine, 100 mM NH4Cl
20 mM alanine, 100 mM NH4Cl
Influence of the reaction temperature on amination of 1,10-decanediol to 1,10-diaminodecane by W3110/pTrc99a-ald-adh-ta Vf and W3110/pTrc99a-ald-adh-ta Cv
The role of L-alanine for the redox self-sufficient amination of alcohols in a whole cell process
Whole cell biotransformation with W3110/pTrc99a- ald - adh - ta Cv at 42°C with 100 mM NH 4 Cl and various L-alanine concentrations
0 mM +20 mM pyruvate
L-alanine added was utilized completely (at 5 and 10 mM) or partially (at 20 and 50 mM) during the whole cell biotransformation approach. Under certain conditions, acetate accumulated as by-product. When L-alanine was present at the same or lower concentrations as the substrate 1,10-decanediol, acetate accumulation was not observed, however, at higher L-alanine excess increasing acetate concentrations could be observed (Table 3). L-alanine (20 mM) was catabolized entirely in the absence of the substrate 1,10-decanediol. Pyruvate only partially replaced L-alanine since only 70% product formation were detected with 20 mM pyruvate. Taken together, these results indicate L-alanine consumption and acetate formation by the host’s central carbon metabolism.
Redox self-sufficient amination of alcohols by whole cell biotransformation benefitted from replacing the transaminase from V. fluvialis used previously  by transaminase TaCv from C. violaceum  since it showed higher activity at 42°C. Moreover, it showed higher catalytic efficiency with L-alanine as substrate (Table 1). The low activities of transaminases at 42°C appeared to be limiting the efficiency of the whole cell biocatalyst since alcohol dehydrogenase of B. stearothermophilus and L-alanine dehydrogenase are rather thermostable (Figure 2) [14,15]. Indeed, TaCv, which showed higher activity at 42°C than TaVf, allowed for efficient conversion of 1,10-decanediol to 1,10-diaminodecane at 42°C. Shifting the biotransformation temperature from 37°C to 42°C led to about three-fold faster conversion employing W3110/pTrc99a-ald-adh-ta Cv (Figure 3). Since at 37°C both W3110/pTrc99a-ald-adh-ta Cv and W3110/pTrc99a-ald-adh-ta Vf showed comparable production rates, the lower catalytic efficiency of TaVf did not limit product formation under these conditions.
Addition of L-alanine was required for full and fast conversion of 1,10-decanediol to 1,10-diaminodecane using the whole cell biocatalyst for alcohol amination. The consumption of L-alanine over time suggested insufficient L-alanine recycling and loss of pyruvate via the cellular catabolism (Figure 1B; Table 3). This was less pronounced at higher 42°C possibly because under these conditions activities of both L-alanine dehydrogenase and alcohol dehydrogenase were increased allowing for more efficient redox cofactor recycling.
L-alanine served two functions in the biotransformation: As substrate in the transaminase reaction and to provide energy and reduction equivalents to the whole cell biocatalyst by catabolism of pyruvate, the co-product of L-alanine-dependent transamination (Figure 1). Under the non-growth conditions of whole cell biotransformation up to 20 mM of alanine were consumed (Table 3) with a rate of about 0.04 g/g*h, which is in the same order of magnitude as non-growth maintenance energy (0.055 to 0.07 g of glucose/g*h). Pyruvate addition only partially replaced L-alanine addition since product formation in the presence of 20 mM pyruvate was incomplete (70%; Table 3). In part, L-alanine was catabolized to acetate. If pyruvate is oxidatively decarboxylated to acetate by pyruvate oxidase PoxB, a reduction equivalent (ubiquinol) is formed which may be used (indirectly) for reductive amination by L-alanine dehydrogenase in the cascade. E. coli is known to produce acetate as overflow metabolite even under fully aerobic conditions, e.g. with excess glucose  when 10% - 30% of carbon flux is directed to acetate formation . Acetate may be formed under aerobic growth conditions by the combined activities of pyruvate dehydrogenase complex PDHC, phosphotransacetylase Pta and acetate kinase AckA (Figure 1B). Besides the reduction equivalent NADH, this pathway yields ATP. A third pathway may be active as a mutant devoid of poxB, pta and ackA still produced acetate . Fast catabolism of glucose to acetate followed by its reuse via acetyl-CoA synthetase (Figure 1B) may be advantageous in comparison to other microorganisms present in its natural habitat that slowly convert the limiting carbon source glucose . In the absence of pyruvate oxidase PoxB conversion of 1,10-decanediol to 1,10-diaminodecane was reduced in about the same way as when PoxB and all other known enzymes for pyruvate degradation were missing, thus, indicating that PoxB is the major enzyme for pyruvate degradation under the chosen biotransformation conditions. In the biotransformation described here, the whole cell biocatalysts were harvested in the stationary phase when PoxB dominates.
The newly derived whole cell biocatalyst W3110/pTrc99a-ald-adh-ta Cv allowed for the improvement of redox self-sufficient amination of alcohols displayed by an increase in production rate. This was achieved by replacing the transaminase of V. fluvialis by the transaminase of C. violaceum, which showed higher activity at 42°C. The whole cell biocatalyst for redox self-sufficient amination of alcohols required L-alanine in concentrations equimolar to the dialcohol substrate for complete conversion to the diamine. L-alanine catabolism occurred primarily via pyruvate oxidase PoxB under the biotranformation conditions.
Materials and method
Bacterial strains, plasmids and oligonucleotides
Strains, plasmids and oligonucleotides used in this study
E. coli DH5α
F− thi-1 endA1 hsdr17(r−, m−) supE44 ΔlacU169
(ɸ80lacZΔM15) recA1 gyrA96 relA1
E. coli W3110
F− λ− INV(rrnD – rrnE)1
E. coli MG1655
F− λ− ilvG- rfb-50 rph-1
E. coli YYC202
ΔaceEF pfl1 poxB1 pps4 rpsL zbi::Tn10
E. coli BW25113
lacI q rrnB T14 lacZ WJ16 hsdR514 araBA-D AH33 rhaBAD LD78
E. coli W3110 harboring pTrc99A-ald-adh-ta with the transaminase of Vibrio fluvialis
E. coli W3110 harboring pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
E. coli MG1655 harboring pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
E. coli YCC202 harboring pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
E. coli BW25113 harboring pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
JW0855-1/pTrc99A-ald-adh-ta Cv (BW25113ΔpoxB::kan)
F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ − , ΔpoxB772::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514; harboring the plasmid pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
/Transformation in this study
JW2293-1/pTrc99A-ald-adh-ta Cv (BW25113ΔackA::kan)
F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ − , ΔackA778::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514; harboring the plasmid pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
/Transformation in this study
JW2294-1/pTrc99A-ald-adh-ta Cv (BW25113Δpta::kan)
F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ − , Δpta779::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514; harboring the plasmid pTrc99A-ald-adh-ta with the transaminase of Chromobacterium violaceum
/Transformation in this study
pTrc99A carrying ta of Vibrio fluvialis
pTrc99A carrying ta of Chromobacterium fluvialis
pTrc99A carrying ald-adh-ta Vf synthetic operon
ald from B. subtilis 168
adh from B. stearothermophilus
ta from V. fluvialis
pTrc99A-ald-adh-ta Vf with BamHI cut site upstream of ta Vf
pTrc99A carrying ald-adh-ta Cv synthetic operon
ald from B. subtilis 168
adh from B. stearothermophilus
ta from C. violaceum
Sequence 5′→ 3′
Competent cells and vector cloning was performed according to standard DNA work procedure . In this study two different cloning strategies for E. coli expression vectors based on IPTG-inducible pTrc99a were used. Firstly, cut sites were used for inserting a gene into a vector. Therefore, PCR-derived gene product ta of Chromobacterium violaceum [GI: 34105712; codon-optimized] (taCv_KpnIRBS_for; taCv_BamHI_rev) amplified by KOD Hot Start Polymerase Kit (Novagen) was cut with KpnI and BamHI and used for ligation with also KpnI and BamHI treated pTrc99A to generate pTrc99a-ta Cv. To construct pTrc99a-ald-adh-ta Cv the BamHI cut site was inserted upstream of ta Vf within the artificial operon ald-adh-ta Vf of pTrc99a- ald-adh-ta Vf by site directed mutagenesis using the oligonucleotides pTrc99a-ald-adh-taVf_mut_for . The newly derived vector pTrc99a-ald-adh-taVf_mut was then cut by BamHI and ligated with BamHI cut ta of Chromobacterium violaceum that was amplified by KOD Hot Start Polymerase Kit (Novagen, pTrc99a-ald-adh-taCv _for, pTrc99a-ald-adh-taCv_rev). Secondly, to construct pTrc99A-ta Vf the gene ta of Vibrio fluvialis was amplified with the oligonucleotides taVf_RBS_for and taVf_rev and assembled with EcoICRI restricted pTrc99a using Gibson assembly method . Then, E. coli DH5α was transformed with the ligation products. CaCl2-competent E. coli DH5α were heat-shocked for the uptake of ligation products. Newly derived vectors were proven by sequencing and E. coli W3110 was transformed with correct plasmids pTrc99a-ta Vf, pTrc99a-ta Cv and pTrc99A-ald-adh-ta Cv and E. coli MG1655 as well as E. coli YCC202 with pTrc99A-ald-adh-ta Cv.
Cultivation conditions and media
Standard cultivation of E. coli was performed in Luria-Bertani medium (LB-medium: 10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract) at 37°C and 200 rpm in baffled flasks or plated on LB-Agar as it is not declared otherwise. When strains harboring plasmid pTrc99A and its derivatives, 100 μg/mL ampicillin was supplemented to the medium. Strain YYC202 and its derivatives were supplemented with 25 μg/ml streptomycin and 10 μg/ml tetracycline, additionally.
Preparation of cell free extract and enzyme assay
The E. coli derivatives were grown in LB + 100 μg/mL ampicillin until an optical density at 600 nm of 0.6 – 0.8, induced with 1 mM isopropyl-β-D-thiogalactopyranosid (IPTG) and harvested in the exponential phase at OD600 = 3.5. 10 mL of the cell culture was harvested and always kept on ice. The cells were once washed with buffer for the enzyme assay, resuspended in 1 mL of the same buffer and then lysed by sonication (Ultrasonic processor UP200S, Hielscher Ultrasound Technology, Teltow, Germany) for 2 minutes (cycle 0.5; amplitude 55%). Cell cebris was centrifuged at 10.000 x g at 4°C for 1 hour and clear cell extract was used for the measurement of the enzyme activity.
Measurement of alanine dehydrogenase activity
50 mM Na2CO3 pH 10 was used for cell washing. For measuring the activity of alanine dehydrogenase pyruvate was converted to L-alanine by NADH consumption spectrophotometrically at 340 nm. Therefore, 50 mM Na2CO3 pH 8.5, 50 mM NH4Cl, 10 mM pyruvate and 0.25 mM NADH where mixed in a cuvette, filled up to 1 mL ddH2O and upon the addition of crude extract the reductive amination was initiated and measured for 3 minutes. The assay was performed in triplicates and one enzyme unit was calculated to be the amount of enzyme that catalyzes the conversion of 1 μmol substrate in 1 min.
Measurement of alcohol dehydrogenase activity
25 mM Sodium phosphate buffer pH 8 was used for cell washing. For measuring the alcohol dehydrogenase activity 1,4-butanediol was oxidized to hydroxybutyraldehyde and NADH formation followed spectrophotometrically at 340 nm. Therefore, 25 mM Na-P-buffer pH 8, 18 mM 1,4 butanediol and 10 mM NAD+ were mixed in a cuvette, filled up to 1 mL ddH2O and reaction was initiated upon the addition of crude extract (triplicates). The NADH formation was followed over 3 minutes and one enzyme unit was calculated to be the amount of enzyme that catalyzes the conversion of 1 μmol substrate in 1 min. To analyze substrate specificity to 1-hexanol, 1-octanol, 1,6-hexanediol, 1,8-hexanediol, cyclohexanol, benzylalcohol and 2-hexanol same conditions were used but different substrate concentration were added to estimate Km and Vmax-values via Lineweaver-Burk Plot.
Measurement of transaminase activity
100 mM Potassium-phosphate buffer pH 7.4 was used for cell washing. Reaction conditions were: 100 mM K-P-buffer pH 7.4, 50 mM (S)-α-MBA and 10 mM pyruvate. The transamination was initiated upon the addition of crude extract and samples were taken continuously. The reaction was stopped with 75 μl 16% perchloracetic acid. The samples where neutralized by the addition of 40 μl buffer containing 20 mM Tris/HCl pH 8 and 23 mM K2CO3. L-alanine formation was measured via HPLC and one enzyme unit was calculated to be the amount of enzyme to catalyze the formation of 1 μmol product in 1 min.
The experimental procedure for the estimation of catalytic efficiency was equal but hexanal was used as substrate instead of (S)-α-MBA. The Reaction conditions were: 100 mM K-P-buffer pH 7.4, 10 mM hexanal and varying concentrations of L-alanine. Hexylamine formation was measured via HPLC and one enzyme unit was calculated to be the amount of enzyme to catalyze the formation of 1 μmol product in 1 min.
Whole cell biotransformation with resting cells
E. coli W3110/pTrc99A and its derivatives W3110/pTrc99A-ald-adh-ta and W3110/pTrc99A-ta-ald-adh were inoculated to an initial OD600 = 0.1 in LB-medium plus 20 mM Mops and 100 μg/mL ampicillin and incubated at 37°C and 200 rpm. At an OD600 = 0.6-0.8 1 mM IPTG was added to the expression culture to induce the cells and cultivation was continued as described above. 15 hours cells were harvested for a final OD600 = 10 in 20 mL final volume, once washed with 50 mM Hepes buffer pH 7 and prepared for whole cell biotransformation in a resting buffer system with the mentioned buffer. NH4Cl and L-alanine were added to the system when necessary and concentrations are given in the text. The test reaction containers (100 mL Schottbottle) where incubated at 37°C or 42°C and 200 rpm and samples for HPLC-analytics were taken in intervals throughout the production.
Extracellular amines and 1-amino-10-decanol were analyzed by high-pressure liquid chromatography (HPLC, 1200 series, Agilent Technologies Deutschland GmbH, Böblingen, Germany). Samples were centrifuged at 10.000 × g for 5 minutes and the clear supernatant was taken for HPLC-measurement. For the detection samples were derivatized with ortho-phthaldialdehyde (OPA) automatically before entering the precolumn (LiChrospher 100 RP8 EC-5 μ, 40 × 4.6 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and the main column (LiChrospher 100 RP8 EC-5 μ, 125 × 4.6 mm, Langerwehe, Germany) for separation. The used mobile phase was made of A: 0.25% (v/v) Na-acetate buffer pH 6 and B: Methanol; 0 min 30% B, 1 min 30% B, 8 min 70% B, 13 min 90% B, 16 min 70% B, 18 min 30% B. 1,7-diaminoheptane was used as internal standard.
The detection of amino acids were performed with a quicker HPLC-method but derivatization with OPA was used equally to amine detection. Here, through a precolumn (LiChrospher 100 RP 18–5 EC; 40 × 4 mm) and the main column (LiChrospher 100 RP18 EC-5 μ; 125 × 4.6 mm; CS-Chromatographie Service GmbH, Langerwehe, Germany) amino acids were separated and detected by a FLD-detector. As an internal standard L-asparagine was used and the gradient for improved separation was made of A: 100 mM Sodiumacetate pH 7.2 and B: Methanol; 0 min 25% B, 0.5 min 45% B, 4 min 65% B, 7 min 70% B, 7.2 min 80% B, 7.4 min 85% B, 8 min 20% B, 10.6 min 20% B.
Overflow metabolites were separated by the Organic Acid Resin column (800 × 8 mm) from CS-Chromatographie Service GmbH (Langerwehe, Germany) and detected with DAD-detector. An isocratic elution with 5 mM H2SO4 and a flow rate of 0.7 ml/min for the separation of the samples.
We would like to acknowledge Drs. Philip Engels, Jan Pfeffer and Thomas Haas (Evonik Industries AG) and Prof. Dr. Kroutil (University Graz) for provision of strains and plasmids and their collaboration within the BMBF-cofunded BioIndustrie 2021 project “Biooxidations- und Aminierungstechnologie als Plattform für funktionelle Amine als Monomerbausteine”. We acknowledge support of the publication fee by Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University.
- Liu W, Peterson PE, Carter RJ, Zhou X, Langston JA, Fisher AJ, et al. Crystal structures of unbound and aminooxyacetate-bound Escherichia coli gamma-aminobutyrate aminotransferase. Biochemistry-Us. 2004;43:10896–905.View ArticleGoogle Scholar
- Lyskowski A, Gruber C, Steinkellner G, Schurmann M, Schwab H, Gruber K, et al. Crystal structure of an (R)-selective omega-transaminase from Aspergillus terreus. PLoS One. 2014;9(1):e87350. doi:10.1371/journal.pone.0087350.View ArticleGoogle Scholar
- Wichmann R, Vasic-Racki D. Cofactor regeneration at the lab scale. Adv Biochem Eng Biotechnol. 2005;92:225–60.Google Scholar
- Lorenz E, Klatte S, Wendisch VF. Reductive amination by recombinant Escherichia coli: whole cell biotransformation of 2-keto-3-methylvalerate to L-isoleucine. J Biotechnol. 2013;168:289–94.View ArticleGoogle Scholar
- Sattler JH, Fuchs M, Tauber K, Mutti FG, Faber K, Pfeffer J, et al. Redox self-sufficient biocatalyst network for the amination of primary alcohols. Angew Chem Int Edit. 2012;51:9156–9.View ArticleGoogle Scholar
- Klatte S, Wendisch VF. Redox self-sufficient whole cell biotransformation for amination of alcohols. Bioorg Med Chem. 2014;22:5578–85.View ArticleGoogle Scholar
- TZB Gerharz, Takors R, Bott M. Produktion von Pyruvat aus Glucose mit Escherichia coli. Biospektrum. 2001:29–33Google Scholar
- Chang YY, Wang AY, Cronan JE. Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma-factor encoded by the rpoS(katf) gene. Mol Microbiol. 1994;11:1019–28.View ArticleGoogle Scholar
- Abdel-Hamid AM, Attwood MM, Guest JR. Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli. Microbiol-Sgm. 2001;147:1483–98.Google Scholar
- Weber H, Polen T, Heuveling J, Wendisch VF, Hengge R. Genome-wide analysis of the general stress response network in Escherichia coli: sigma(S)-dependent genes, promoters, and sigma factor selectivity. J Bacteriol. 2005;187:1591–603.View ArticleGoogle Scholar
- Kuhn D, Fritzsch FSO, Zhang XM, Wendisch VF, Blank LM, Buhler B, et al. Subtoxic product levels limit the epoxidation capacity of recombinant E. coli by increasing microbial energy demands. J Biotechnol. 2013;163:194–203.View ArticleGoogle Scholar
- Vijayendran C, Polen T, Wendisch VF, Friehs K, Niehaus K, Flaschel E. The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12. J Biotechnol. 2007;128:747–61.View ArticleGoogle Scholar
- Kaulmann U, Smithies K, Smith MEB, HaileS HC, Ward JM. Substrate spectrum of omega-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis. Enzyme Microb Tech. 2007;41:628–37.View ArticleGoogle Scholar
- Fiorentino G, Cannio R, Rossi M, Bartolucci S. Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements. Protein Eng. 1998;11:925–30.View ArticleGoogle Scholar
- Shin JS, Yun H, Jang JW, Park I, Kim BG. Purification, characterization, and molecular cloning of a novel amine : pyruvate transaminase from Vibrio fluvialis JS17. Appl Microbiol Biot. 2003;61:463–71.View ArticleGoogle Scholar
- Veit A, Polen T, Wendisch VF. Global gene expression analysis of glucose overflow metabolism in Escherichia coli and reduction of aerobic acetate formation. Appl Microbiol Biotechnol. 2007;74:406–21.View ArticleGoogle Scholar
- Farmer WR, Liao JC. Reduction of aerobic acetate production by Escherichia coli. Appl Environ Microb. 1997;63:3205–10.Google Scholar
- Phue JN, Lee SJ, Kaufman JB, Negrete A, Shiloach J. Acetate accumulation through alternative metabolic pathways in ackA(−) pta(−) poxB(−) triple mutant in E. coli B (BL21). Biotechnol Lett. 2010;32:1897–903.View ArticleGoogle Scholar
- Sambrook J, Russell D. Molecular Cloning. A Laboratory Manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratoy Press; 2001.Google Scholar
- Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006 0008.View ArticleGoogle Scholar
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983;166:557–80.View ArticleGoogle Scholar
- Liu HT, Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology. 2008;8:91. doi:10.1186/1472-6750-8-91.View ArticleGoogle Scholar
- Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6:343–1.View ArticleGoogle Scholar
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