Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli
- Thomas G Palmen†1,
- Jens Nieveler†1,
- Bettina Frölich2,
- Wiltrud Treffenfeldt3,
- Martina Pohl2, 4 and
- Jochen Büchs1Email author
© Palmen et al; licensee BioMed Central Ltd. 2010
Received: 5 July 2010
Accepted: 19 October 2010
Published: 19 October 2010
The benzoylformate decarboxylase (BFD) from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR) as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS).
Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro) had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala) showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln) behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences in metabolic activity of their respective host strain.
The BFD-variants with high cofactor binding affinity (wild type, His281Ala, Ser181Thr) obviously extract thiamine from the medium and bind it tightly to the enzyme. This might explain the hampered growth of these clones. In contrast, growth of clones expressing variants with low cofactor binding affinity (Leu476His, Leu476Pro, Leu476Pro-Ser181Thr) is not impaired. Leu476Gln has an intermediate cofactor binding strength, thus, growth of its host strain depends on the specific cultivation conditions. This paper shows that slight differences of the amino acid composition can affect protein expression and cultivation and might require an adaptation of media components. Effects such as the observed are hardly foreseeable and difficult to detect in conventional screening processes. Via small scale experiments with on-line measurements in shake flasks such effects influencing the cultivation and product formation can be detected and avoided.
Biocatalysts, i. e. whole cell systems or enzymes have found increased use in industrial biotechnology. To further increase the number of industrial biotechnological processes, new biocatalysts are needed. By utilizing the existing biodiversity , enzymes from microorganisms found in nature are screened to find suitable biocatalysts for such processes. Besides these naturally occurring sources of potential enzymes, techniques such as directed evolution  are applied. With this technique, mutations in selected genes can be generated. If applied to enzymes, single or few amino acids in these enzymes can be exchanged, resulting in different enzyme variants. In this way, clone libraries of microorganisms harbouring the genes for different variants are created which are screened for desired attributes, e. g. improved enzyme activity, thermostability or stereoselectivity.
One enzyme with an already high stereoselectivity  is the benzoylformate decarboxylase from Pseudomonas putida. It is named BFDC in recent publications. In this work, we stick to the older abbreviation BFD to be consistent with our previous publications [4–6]. While its main reaction, the non-oxidative decarboxylation of benzoylformate is part of the mandelate biosynthetic pathway [7–9], the physiological function of the biotechnologically interesting side reaction, the carboligation  is unknown. In this side reaction, BFD catalyses the formation of chiral 2-hydroxy ketones from benzaldehyde or benzaldehyde derivates as acyl donors and acetaldehyde as the acyl acceptor . 2-Hydroxy ketones are important structural subunits in many biologically active natural products and are also important building blocks for stereoselective syntheses , e. g. for the synthesis of ephedrine or bupropion . Depending on the acyl donor, the enantiomeric excess of the product synthesised by BFD catalysis varies as well as the amount of converted substrate in a given time [3, 11]. To improve the carboligase activity of BFD, a combination of directed evolution and site-directed mutagenesis has been applied . Upon induction of protein expression, these respective recombinant E. coli clones expressed different BFD-variants. Other attempts aimed to alter the substrate specificity of BFD [5, 13] and to improve the stereoselectivity of BFD for different substrates .
Kensy et al.  showed that the induction of cultures can affect the growth of the applied microorganisms. Therefore, expressing different BFD-variants might result in different kinetics of the metabolic activity of the applied strains. Hence, in screening processes, the growth of the cultures has to be monitored. Without appropriate measurement systems, endpoint measurements or costly sampling during cultivation have to be conducted in screening processes. Evaluation of the cultivation performance and product formation on the basis of these few data is problematic. In this study, the RAMOS (Respiratory Activity MOnitoring System) was applied to measure the oxygen transfer rate (OTR) on-line as a parameter for the growth and metabolism of the investigated organisms. In this work, the thiamine auxotroph strain E. coli SG13009 pKK233-2 was applied to express different BFD-variants. Thiamine auxotroph strains, such as E. coli DH5α, E. coli JM109 or E. coli M15 are routinely utilized in laboratories all over the world. They are also used to express thiamine dependent enzymes [16–19].
The aim of this study is to show the physiological relation between the respiration activity and the heterologous expression of selected BFD-variants under different culture conditions. This example should increase the awareness for effects that can occur during cultivation and that may influence the expression of the product and the cultivation itself.
Applied clones and abbreviations
Escherichia coli SG13009:pKK233-2-BFD-wt-His6
Escherichia coli SG13009:pKK233-2-BFD-His281Ala-His6
Escherichia coli SG13009:pKK233-2-BFD-Leu476Gln-His6
Escherichia coli SG13009:pKK233-2-BFD-Leu476His-His6
Escherichia coli SG13009:pKK233-2-BFD-Leu476Pro-His6
Escherichia coli SG13009:pKK233-2-BFD-Leu476Pro-Ser181Thr-His6
Escherichia coli SG13009:pKK233-2-BFD-Ser181Thr-His6
To prepare stock cultures, precultures of E. coli SG13009 pKK233-2 clones in LB medium were made. After reaching an optical density (OD) of 3, glycerol solution was added as a cryoprotective, resulting in a final glycerol concentration of 300 g/L. The cultures were stored at -20°C in 1 mL aliquots in cryo-vials.
E. coli SG13009 pKK233-2 was cultivated in buffered LB medium with 10 g/L glycerol. It consists of 5 g/L yeast extract, 10 g/L tryptone, 12.54 g/L K2HPO4 and 2.31 g/L KH2PO4. Another complex medium, TB medium, was also applied for the cultivations. It consists of 24 g/L yeast extract, 12 g/L tryptone, 12.54 g/L K2HPO4, 2.31 g/L KH2PO4 and 5 g/L glycerol. Furthermore, modified PanG mineral medium  was used for the cultivations. It consists of 1.6 g/L NaH2PO4*H2O, 3.2 g/L KH2PO4, 2.6 g/L K2HPO4, 0.2 g/L NH4Cl, 2.0 g/L (NH4)2SO4, 0.6 g/L MgSO4, 0.2 g/L CaCl2*H2O, 5 g/L glycerol and 1 mL/L trace element solution. The trace element solution consists of 5 mL/L H2SO4 (conc.), 6 g/L CuSO4*5H2O, 0.08 g/L KI, 3 g/L MnSO4*H2O, 0.3 g/L Na2MoO4, 0.02 g/L H3BO3, 0.5 g/L CoCl2, 20 g/L ZnCl2 and 65 g/L FeSO4*7H2O. The pH of all media was adjusted to 7.0. Furthermore, 0.1 g/L ampicillin was added to each medium. As the applied strain has a neomycin/kanamycin selection marker, additionally 0.05 g/L neomycin was added to each medium. Although E. coli SG13009 pKK233-2 is a thiamine auxotroph strain, no additional thiamine was added to the complex LB and TB medium, according to the literature [21–23]. For the cultivations with additional thiamine, sterile filtrated thiamine hydrochloride stock solution (1 g/L) was added. Final thiamine concentrations were 0.002 mg/L, 0.02 mg/L, 0.1 mg/L, 0.2 mg/L, 2 mg/L, 10 mg/L and 20 mg/L.
For all cultivations, precultures were made. The precultures were inoculated with a stock culture of the given E. coli SG13009 pKK233-2 clone and cultivated in the same cultivation vessel under the same cultivation conditions as the given main culture. Main cultures were inoculated with an inoculation volume of 1% (v/v) of the main culture volume.
To determine the BFD portion of total protein, the carboligation activity in crude extract and the optical density cultivations were conducted in 250 mL Erlenmeyer flasks filled with 25 mL buffered LB medium with 10 g/L glycerol. The cultures were grown at 37°C, a shaking diameter of 50 mm and a shaking frequency of 150 rpm.
The oxygen transfer rate (OTR) in shake flasks was measured online with a self made RAMOS (Respiratory Activity MOnitoring System) device, as described by Anderlei et al. . The RAMOS cultivations were performed in modified 250 mL Erlenmeyer flasks (RAMOS flasks) as described by Anderlei and Büchs . The applied RAMOS device allows to run up to 8 modified Erlenmeyer flasks in parallel. The cultures were grown in 10 or 25 mL medium (buffered LB medium with 10 g/L glycerol, TB medium or modified PanG mineral medium) at 37°C, a shaking diameter of 50 mm and a shaking frequency of 150, 320 or 400 rpm. No antifoaming agents were added to the medium during the cultivations with high shaking frequencies.
BFD expression was induced by adding IPTG stock solution (100 mM) resulting in a final IPTG concentration of 1 mM.
Cell disruption was applied as described by Losen et al. . After centrifuging 3 mL culture medium for 15 min at 600 g, the cell pellets were frozen at -20°C and subsequently resuspended in extraction buffer (50 mM K3PO4, 5 mM MgSO4, 0.5 mM thiamine diphosphate (ThDP), pH 7). These samples were centrifuged for 10 min at 10000 g and the pellet was resuspended in extraction buffer with 1 mg/mL lysozyme. After incubation for 1.5 h at 30°C the samples were ultrasonicated for 5 min and centrifuged for 15 min at 10000 g. The supernatants were used to determine the BFD portion of total protein, the protein determination and the volumetric carboligation activity in crude extract.
BFD portion of total protein
Specific decarboxylase activity of selected BFD-variants
Specific decarboxylation activity [U/mg BFD]
Volumetric carboligation activity in crude extract
To determine the carboligation activity of the wild type BFD and the BFD-variants, cell extract was diluted 1/20 in reaction buffer (1.5 M ethanol, 50 mM K3PO4, 2.5 mM MgSO4, 0.1 mM ThDP). An equal volume of substrate solution (1 M acetaldehyde, 80 mM benzaldehyde) was added. After incubation for 30 min at 30°C, the reaction was stopped by heating for 2 min at 95°C. The amount of formed 2-hydroxy-1-phenyl-propanone (2-HPP) was measured using a HPLC system. Separation was performed on a RP8-column (Macherey & Nagel, Düren, Germany) using 0.5%/20% acetic acid/acetonitrile (v/v) as eluent. The flow rate was 1.1 mL/min.
To determine the cell density, the optical density was measured with a spectrophotometer (Uvikon 922 A, Kontron Instruments, Milano, Italy) at a wavelength of 600 nm. 10 mm cuvettes were applied. To keep the measurements in the linear range between 0.03 and 0.3, the samples were diluted with NaCl solution (9 g/L). Cuvettes containing only NaCl solution (9 g/L) were used as blanks. Furthermore, the optical density of sterile medium was subtracted from the measured optical density.
Purification of selected BFD-variants
Purification of the wild type BFD and BFD-variants was performed as described for pyruvate decarboxylase  using potassium phosphate buffer (50 mM, pH 7.0) for Ni-NTA chromatography and potassium phosphate buffer (50 mM, pH 6.0) containing ThDP (0.5 mM) and MgSO4 (2.5 mM) as elution buffer for the subsequent gel chromatography. Lyophilised BFD-variants were stored at -20°C.
Enzymatic synthesis in buffer
The initial carboligase activities were measured as described by Lingen et al. . 10-50 μg purified wild type BFD or BFD-variant were incubated in 0.5 mL of 50 mM KPi, pH 7.0, containing 0.5 mM ThDP, 2.5 mM MgSO4 in the presence of 20, 40 or 60 mM benzaldehyde and 500 mM acetaldehyde for 30 min at 30°C. The enzymes were heat inactivated and the resulting 2-HPP formed was measured using an analytical HPLC system (Gynkotek, Germering, Germany) with an ultraviolet monitor (263 nm). Separation was performed on a 318-Hypersil column (C&S, Langerwehe, Germany) using 0.5%/20% acetic acid/acetonitrile (v/v) as eluent. The flow rate was 1.1 mL/min. The retention time of 2-HPP was 12.8 min.
Stability of cofactor binding
The stability of cofactor binding was tested as described by Lingen et al. . 0.1 mg/mL of the BFD-variants were incubated in potassium phosphate buffer without the cofactors ThDP and MgSO4 for 24 h. Then, 50 μL samples were removed and decarboxylase activity was determined. For this purpose, a coupled enzymatic test as described by Iding et al. was conducted . An assay mixture was applied consisting of 100 μL benzoylformate solution (50 mM, pH 6.0), 100 μL NADH (3.5 mM), 50 μL horse liver alcohol dehydrogenase (HLADH) (10 U) and 700 μL potassium phosphate buffer (50 mM, pH 6.0). After mixing and incubating at 30°C, 50 μL BFD solution was added to initiate the reaction. The descending curve was examined at 340 nm and the linear slope was calculated from 0 to 90 s. The activity of the different BFD-variants was compared to the activity of the given BFD-variant under the same conditions but with 700 μL potassium phosphate buffer (50 mM, pH 6.0) additionally containing 0.5 mM thiamine diphosphate and 2.5 mM MgSO4. One unit is defined as the amount of enzyme that catalyses the decarboxylation of 1 μmol benzoylformate per minute at pH 6.0 and 30°C.
Results and Discussion
Cultivation of the E. coli SG13009 pKK233-2 clones under the same conditions in TB medium results obviously in two different types of OTR curves (Figure 2B). Again, Leu476Pro-Ser181Thr and Leu476Pro reach a plateau of 0.013 mol/L/h after an exponential increase. This plateau is again due to an oxygen limitation. Its level is slightly lower than for LB medium, as TB is a richer medium resulting in lower oxygen solubility and diffusivity . After 24 h, the OTRs drop. The OTR of the second type (wild type and Ser181Thr) also increase exponentially to 0.013 mol/L/h. However, they decline already after 18 h to about 0.06 mol/L/h. Further respiration activity at this lower level ensues before a second decline follows after ca. 25 h. In contrast to the first cultivation shown in Figure 2A, here, Leu476Gln and His281Ala behave like the first type and the second type, respectively.
To surely avoid an oxygen limitation, another experiment with a lower filling volume (10 mL) and higher shaking frequency (400 rpm) was conducted (Figure 2C). No plateau of the OTR was observed. Again, different types of OTR curves depending on the clones occurred. Up to about 7 h, all OTRs increase exponentially to 0.065 mol/L/h, yet the following respiration behaviours differ. The OTRs of the first type (Leu476Pro-Ser181Thr, Leu476Gln and Leu476Pro) slightly drop, before another increase until ca. 8.5 h follows. All three curves drop after 11 h. The curves of the second type (the clone expressing the wild type BFD, Ser181Thr and His281Ala), however, drop to ca. 0.025 mol/L/h after 8 h. The OTR of His281Ala remains at this level, while the curves of the clone expressing the wild type BFD and Ser181Thr recover to over 0.03 mol/L/h after 9.5 h. Subsequently, all three curves decline steadily.
From Figure 2A-C, three different groups of clones can be distinguished on basis of the OTR curves. The first group consists of Leu476Pro-Ser181Thr and Leu476Pro, which show typical OTR curves under oxygen limited and not oxygen limited conditions. In contrast, the OTR of the second type shows an earlier drop of OTR which is followed by further respiration activity on a lower level in TB medium. The third type (Leu476Gln and His281Ala) shows a variable behaviour, depending on the cultivation conditions. All these clones, having only very small differences in their genetic construction, behave surprisingly different in their metabolic activity, proving the differences that were already found in their expression properties shown in Figure 1.
The addition of thiamine seems to have no effect on the growth of Leu476Pro. This clone shows no limitation in TB medium without additional thiamine and the OTR curves of Leu476Pro in TB medium with and without additional thiamine hardly differ. For the induced clone expressing the wild type BFD, the addition of thiamine to the medium leads to an OTR curve without early decrease. Apart from a slower increase due to the metabolic burden, the curve of the induced culture with additional thiamine is similar to the curves of the non-induced cultures. These results support the assumption that the early drop of OTR during the cultivation of the clone expressing the wild type BFD in TB medium without additional thiamine was caused by the level of thiamine concentration.
The thiamine concentration was varied between 0.002 mg/L and 20 mg/L (Figure 4B). The highest maximum OTR of 0.05 mol/L/h is obtained by the culture with 2 mg/L thiamine. Up to this concentration, increasing the thiamine concentration results in an increased maximum OTR. However, the addition of 20 mg/L thiamine leads to a lower maximum OTR of about 0.04 mol/L/h. While a concentration of 0.02 mg/L thiamine is sufficient to allow the growth of the applied clone, a concentration of 0.05 mg/L thiamine was selected for further cultivations to ensure a sufficient thiamine concentration during further cultivations with induction.
The OTR curves of the clones expressing Leu476His, Leu476Gln and Ser181Thr without induction are also similar (Figure 5B). They show a maximum OTR of about 0.04 mol/L/h after ca. 18 h. For Leu476His, the OTR of the induced culture does not change in comparison to the not induced culture, but has a reduced maximum OTR of 0.03 mol/L/h and a decreased slope upon induction, which is again caused by the metabolic burden. For the clones Ser181Thr and Leu476Gln, however, the curves of the induced cultures vary strongly compared to the curves of the not induced cultures. With a maximum OTR of 0.01 mol/L/h, the OTR of the induced Ser181Thr culture is similar to the OTR of the clone expressing the wild type BFD shown in Figure 5A. After induction at 16 h, the OTR of Leu476Gln increases slowly to 0.015 mol/L/h after 24 h.
Regarding the induced cultures in Figure 5A and 5B, three different types of OTR curves can be distinguished. The first type (Leu476Pro-Ser181Thr and Leu476His) has a similar respiration activity as the non-induced cultures with a slightly reduced maximum OTR and a reduced initial increase of OTR upon induction due to the metabolic burden. In contrast to the OTR in complex medium (Figure 2C and 3B), the OTR of the second type (the clone expressing the wild type BFD and Ser181Thr) in mineral medium does not drop after a certain time, but shows a strongly decreased maximum OTR. Upon induction, the slope of the OTR curve of the third type (Leu476Gln) is drastically reduced, resulting in a maximum OTR of only 0.02 mol/L/h after 24 h.
According to Lingen et al. , Leu476 is located near, but not in the active centre of the enzyme and is involved in contacts between two of the four monomers the enzyme consists of . The authors suppose it to have an influence on the cofactor binding strength, thus, by exchanging the amino acid at this position the cofactor binding strength of the enzyme can be altered. While the exchange of leucine by histidine or proline leads to a lower cofactor binding strength, exchanging it for glutamine also reduces the cofactor binding strength, but to a lesser degree. Contrarily, the replacement at the positions 181 and 281 does not reduce the cofactor binding strength.
These different cofactor binding strengths might explain the different OTR curves of the clones. Thiamine is a cofactor of BFD as well as of enzymes of the central carbon metabolism of E. coli, such as the pyruvate dehydrogenase complex (PDHc) . The PDHc catalyses the formation of CO2, Acetyl-CoA (which is further utilised in the citric acid cycle) and NADH2+ (which is used in the electron transport chain), from pyruvate, coenzyme A and NAD+. Here, thiamine pyrophosphate (ThDP) is a cofactor of the subunit pyruvate dehydrogenase that deacetylates pyruvate under CO2-formation. ThDP is supposed to actively participate in the reaction of pyruvate dehydrogenase [34–37]. Thus, if thiamine is bound by other enzymes such as BFD and, in consequence, is not available for the PDHc, the reaction of the PDHc might be hampered, resulting in different OTR curves.
Besides its role as cofactor for several enzymes, thiamine in its phosphorylated form ThDP is also involved in the regulation of genes of the thiamine biosynthesis. In E. coli, the operons thiCEFSGH, thiMD and tbpAthiPQ, which code for enzymes of the thiamine biosynthesis and thiamine transporters, are regulated by ThDP . ThDP binds at a conserved, untranslated RNA structure that is called thi box without involvement of protein cofactors. When thiamine is bound at this thi box riboswitch, the structure of the mRNA changes, masking the Shine-Dalgarno box and, thus, hindering the initiation of translation . In consequence, the gene expression of the thi box riboswitch regulated genes is reduced  if thiamine is present in the medium, whereas in case of a lack of thiamine, the Shine-Dalgarno box is not masked and the gene expression is, thus, not hampered.
As shown in this study, addition of thiamine to the medium can prevent the observed differing growth kinetics and is, thus, strongly recommended. While thiamine was identified as the cause for the differing growth kinetics in these experiments, other cultivations might be influenced by different effects. Unexpected phenomena such as the observed are hardly detectable in conventional screening processes. This might lead to wrong selection of enzyme variants, wrong assumptions about the optimal point of harvest and, ultimately, to wasted resources. To realise such hardly foreseeable effects influencing cultivation and expression of products, the application of on-line monitoring systems is, therefore, advised in screening processes.
While this study was conducted with the thiamine auxotroph strain E. coli SG13009, further studies should focus on prototrophic strains, such as E. coli BL21, to investigate, if the different binding strengths of the BFD-variants effect the OTR of these strains, too.
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