In Vitro Modification of Bacterial Cyanophycin and Cyanophycin Dipeptides Using Chemical Agents Towards Novel Variants of the Biopolymer
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Variations of the composition of cyanophycin (CGP) have been investigated since the early 2000s. Modifications of the polymer are of academical interest, but also expand the number of putative applications for CGP and its dipeptides in fields like food supplementations, and medical and cosmetic applications. Until recently variations of the composition occurred only in vivo. However, in the last years, two procedures using chemical or enzymatic in vitro modification were successfully applied. Since chemical treatments were more effective and reached higher conversion rates, a search for reagents and their applicability to conduct reactions with CGP was done. The reaction of CGP with methyl isocyanate resulted in the conversion of 50% of the lysine residues, while only 3% of the arginine residues were modified. However, using digested CGP dipeptides, the conversion rates of lysine increased slightly to 72%, while the conversion of arginine reached 96%. Using formaldehyde, CGP could be methylated with a conversion rate of 84% for lysine and 15% for arginine. Acetylation of lysine residues was obtained using acetic anhydride, reaching a conversion rate of 100% for a single acetylation, where 63% of the residues were acetylated twice. Arginine residues could be acetylated at a rate of 89%. Diacetyl could be added to 80% of all arginine residues, while lysine was not targeted by the compound. Other agents were also tested, but showed lesser or no conversion and/or inconclusive results. Overall, the tested reactions confirm the viability of chemical CGP modification for future approaches.
KeywordsCyanophycin Chemical modification Arginine Lysine In vitro
The polyamide cyanophycin (CGP) has been in the focus of various studies over the last few decades and especially since the early 2000s. The main focus of these studies was usually the identification of novel production strains or the optimization of the production of CGP in general. Modifications of the CGP composition were mostly out of the scope of academic interest and were considered as a characteristic of the strain or the CGP-producing cyanophycin synthetase. For example, examining the characteristics of these variations yielded interesting insights in the solubility behavior of the polymer (Wiefel and Steinbüchel 2014). Aside from academic interest, CGP as well as CGP-derived dipeptides also have several putative applications in areas like food supplementations or medical and cosmetic applications were an increased variety in the composition can expand the range of potential uses (Sallam and Steinbüchel 2010). Therefore, actively modifying the composition of CGP has become more relevant in recent years. Usually, variations of the composition occurred only in vivo, either naturally as a characteristic of the producing organism, or intended using selectively chosen and modified strains and/or specifically optimized culture conditions. Strains of E. coli are known to incorporate copious amounts of lysine into the CGP, replacing arginine residues in the side-chains (Kroll et al. 2011, Wiefel et al. 2014), while strains of S. cerevisiae and P. putida incorporate citrulline, also replacing arginine (Steinle et al. 2009; Wiefel et al. 2011).
However, in recent years, two novel procedures using chemical or enzymatic in vitro modification of the CGP were successfully applied. The first chemical modification of CGP was reported by Frommeyer et al. (2014) by the guanidinylation of a lysine-rich CGP with O-methylisourea. This reaction completely converted the lysine residues of CGP to homoarginine (Frommeyer et al. 2014). The feasibility of enzymatic CGP modification was demonstrated by Wiefel and Steinbüchel (2016) using a peptidyl arginine deiminase from Oryctolagus cuniculus, which normally introduces post-translational modifications on several proteins, to catalyze the conversion of arginine residues to citrulline. The conversion rate was, however, only 7.5 mol% of the CGPs amino acids, likely due to product inhibition by the citrulline.
2 Materials and Methods
2.1 Synthesis of CGP and Dipeptides
Escherichia coli HSM174 pCOLADuet::cphA6308 (Kroll et al. 2011) was used for CGP production according to Wiefel and Steinbüchel (2014). Within 26 h, a cell density of 13.3 g/L was obtained, and an overall CGP content of 34.1% of the cell dry mass was measured. 67.7% of the accumulated CGP were insoluble with a lysine content of 15.8 mol%, while the remaining 32.3% of the polymer were soluble with a lysine content of 35.8 mol%.
2.2 Digestion of CGP
CGP was also digested for enzyme tests using CGP dipeptides and for analytical purposes. For that, CGP was digested using a purified CphE (Cyanophycinase, EC 126.96.36.199) from Pseudomonas alcaligenes DIP1 (Sallam et al. 2009) for the generation of a mixture of Asp-Arg and Asp-Lys dipeptides. 10 mg of CGP were mixed with 50 µl of CphE solution (0.8 mg/ml in H2O), adjusted to 1 ml with Tris/HCl-buffer (20 mM, pH 8.0), and incubated at 50 °C for 5 h. Afterwards, the reaction mixture was passed through an ultrafiltration column (10 kDa exclusion size; Vivaspin, Sartorius, Göttingen) to separate the dipeptides from the CphE in the supernatant. A control without CGP was carried along to ensure that all detected signals derived from the digested CGP. CGP modified in reactions was first separated from the reaction mixture by an ultrafiltration column (10 kDa exclusion size) which retained the CGP, before being rebuffered with Tris/HCl (20 mM, pH 8.0), and mixed with CphE. CphE solution was provided by Cysal GmbH, Münster, Germany.
2.3 Reaction with Cyanates
Reactions with cyanates were performed with a modified protocol of Weisgraber et al. (1978). A solution of 1 mg per ml CGP was mixed with 20 mg of cyanate per mg of CGP. Due to the use of mass spectrometric (MS) methods, sodium borate buffer was replaced with H2Odest for GITC and AITC, while the MITC solution also contained 1/4 (v/v) of methanol to solve the cyanate completely. The methanol content was adjusted to the same level after the reaction was completed for analytic purposes. The pH was adjusted by a 2.5% (w/v) ammonia solution. The reaction solution was incubated for 2 h at 30 °C and stopped by cooling on ice, dilution with methanol and water, and lowering the pH to acid levels for MS analysis. The pH dependence of the reaction was studied in a range from pH 8 to pH 10.
2.4 Reaction with Formaldehyde
The reactions with formaldehyde were performed using a modified protocol of Means and Feeney (1968). 100 µl of a CGP or CGP dipeptide solutions (50 mg/ml) was mixed with 900 µl borate buffer (50 mM, pH 8.2). The reaction was started by adding 20 µl of 37% (v/v) formaldehyde, adding additional 20 µl every 10 min over the next 50 min for a total volume of 120 µl. The reaction was performed on ice for 18 h. Afterwards, not digested CGP was digested as previously mentioned, while dipeptides were directly prepared for MS analysis.
2.5 Reaction with Acetic Anhydride
Acetylation with acetic anhydride was performed after a modified protocol of Riordan et al. (1965) using 100 µl of a CGP or CGP dipeptide solutions (50 mg/ml), and mixed with 900 µl NaOAc buffer (40 mM, pH 7.2). The reaction was started by adding 20 µl of acetic anhydride and adding additional 20 µl every 10 min over the next 50 min for a total volume of 120 µl. The reaction was performed on ice for 18 h. Afterwards, undigested CGP was digested as previously mentioned, while dipeptides were directly prepared for MS analysis.
2.6 Reaction with Pyridoxal Phosphate
The reaction protocol for the reaction of amino acids with pyridoxal phosphate was carried out according to the report of Shapiro et al. (1968). Since CGP and amino acids do not have to be buffered, the use of the sodium phosphate was replaced with H2Odest. This also allows the direct measurement of the resulting product in the mass spectrometer. The reaction was set up with 1 mg/ml of CGP and 1 mM pyridoxal phosphate at a pH of 8. The reaction time was 1 h at 30 °C. The stabilization of the resulting Schiff base by sodium borohydride was only performed with not digested CGP to enable the removal of sodium borohydride by ultrafiltration. For this, the reaction with PLP was started as described above. After 30 min, the sodium borohydride was added up to a concentration of 0.6 mg/ml, leading to a discoloring of the yellow pyridoxal phosphate solution.
2.7 Reactions with Diacetyl and Diacetyl trimer
The reactions were performed using 100 µl CGP or CGP dipeptide solutions (50 mg/ml), 900 µl H2O, and 20 µl diacetyl. The reaction was performed on ice for 18 h. In case of the diacetyl trimer, 200 µl were used in the reaction mixture. The diacetyl trimer was prepared by mixing 20 mL borate buffer (50 mM, pH 8.2) with 3.75 mL diacetyl, adjust to pH 9 using NaOH, and adjusting the volume to 25 mL with H2Odest and stirring for 1 h.
2.8 Mass Spectrometry (MS)
Samples were analyzed by MS (LXQ Mass Spectrometer, Finnigan) with LQXTunePlus software (Finnigan). Samples were prepared for analysis by addition of 1/3 of the sample volume of methanol. The production of positive charged ions was supported by adding 0.1% formic acid, yielding a pH value in a slightly acidic range of pH 6–4. The pH value was tested with pH paper (pH 1–11, Macherey–Nagel), and if needed, more formic acid was added until the pH range was reached. The solution was directly injected into the ion source of the MS at a flow rate of 10 µl/min. The basic parameters during measurement used the following values: a capillary temperature of 300 °C, a sheath gas flow rate of 12 Ls/h, an auxiliary gas flow of 6 Ls/h, and a sweep gas flow of 1 L/h. A mass range from m/z = 50 to 1,000 was scanned.
Since both, high lysine content and solubility of the polymer, were desired for the reactions, the soluble lysine-rich CGP produced by E. coli HSM174 pCOLADuet::cphA6308 was chosen for the experiments. This form offered both lysine and arginine residues as targets for chemical modifications.
3.1 Reactions with Cyanates
The reactions of CGP and CGP dipeptides were performed with the cyanates MITC, GITC, and AITC. However, only the reaction with MITC showed a noticeable conversion of the amino acid residues. Both other cyanates gave no noticeable modifications (data not shown).
Conversion rates of cyanophycin (CGP) reacting with methyl isothiocyanate (MITC)
66.5 ± 0.3%
31.0 ± 0.4%
2.5 ± 0.2%
26.5 ± 0.3%
59.0 ± 1.5%
14.5 ± 1.2%
27.8 ± 0.1%
35.6 ± 0.4%
36.6 ± 0.4%
82.4 ± 0.1%
9.4 ± 0.7%
8.2 ± 0.4%
48.5 ± 0.1%
45.5 ± 0.0%
6.0 ± 0.1%
75.6 ± 0.4%
24.4 ± 0.4%
17.1 ± 1.5%
82.9 ± 0.3%
3.5 ± 0.4%
96.5 ± 0.0%
98.2 ± 0.0%
1.8 ± 0.1%
96.8 ± 0.7%
3.2 ± 0.1%
Carrying out the reaction with undigested CGP proved to be problematic, due to solubility issues. The use of 1/3 methanol, which is needed to solve the MITC, in an alkaline pH regime forces the CGP to precipitate. Thus, a chemical contact between the cyanate and the CGP is unlikely, and the subsequent analysis of the reaction products showed only 3.2% of the arginine residues being converted to the expected reaction product. The conversion of lysine at a rate of 50.3%, even though much better compared to arginine, was also noticeably worse to reactions with already digested dipeptides (72.2% conversion). In both cases, the conversion rates were also higher at pH = 10 than under less alkalic conditions. A pH of 9 was not tested.
3.2 Methylation using Formaldehyde
The MS analysis of cyanophycin cleaved by CphE into dipeptides after reaction with formaldehyde exhibited a dominant signal at m/z = 290.17, which corresponds to the unmodified Asp-Arg dipeptide, while a secondary signal at m/z = 262.17 refers to unmodified Asp-Lys. A signal measured at m/z = 276.17 matches with the expected mass of a methylated Asp-Lys dipeptide. The measured conversion rate for unmodified Asp-Lys to methylated Asp-Lys based on the intensities of the two peaks was calculated at 84.4%. There are three very weak additional signals at m/z = 303.17, 312.17, and 332.08 which do not correspond to any predicted product of the reaction. No relevant methylation of the Asp-Arg dipeptide was measured.
3.3 Acetylation with Acetic Anhydride
The calculated conversion rates for the acetylation of CGP dipeptides were > 99% for Asp-Lys; 36.6% of the Asp-Lys was acetylated once, while 63.4% were acetylated twice. 66% of Asp-Arg was acetylated. If the unknown signal at m/z = 314.08 is caused by acetylation of the unknown signal at m/z = 272.2, the conversion rate is 89.2%.
3.4 Reaction with Pyridoxal Phosphate
Performing the same reaction for undigested cyanophycin, allowed for the usage of NaBH4 for stabilization of the resulting Schiff base formed by this reaction, however, there were no significant changes in MS analysis. Only the ratio of the signals at m/z = 493.4–491.4 increased in regard to the already digested cyanophycin. This can be explained by the addition of sodium borohydride which reduces the formed Schiff base from a double bond to a single bond. A conversion rate of about 10% for the lysine dipeptide and 1–2% for the arginine dipeptide was determined, again with no clear correlation regarding the pH.
The MS analysis of cyanophycin incubated with diacetyl exhibits a dominant signal at m/z = 444.08, which corresponds to the reaction of the Asp-Arg dipeptide with two molecules of diacetyl. If water is cleaved of this molecule (m/z − 18), the structure will lead to a signal at approx. m/z = 444.08. Based on this, the conversion rate of Asp-Arg (m/z = 290.17) to the signal at m/z = 444.08 was determined at 79.7%. There was no measurable product of the Asp-Lys dipeptide at m/z = 262.17.
Reactions performed with the generated diacetyl trimer exhibited a large variety of signals, none of which matched with predicted masses and/or signals detected in the reactions with regular diacetyl.
The MS analysis of reaction products of cyanophycin with MITC demonstrates that a modification of CGP is possible and enhanced at high pH values. For the previously digested cyanophycin, a conversion of around 70% for the Asp-Lys dipeptide and around 95% for the Asp-Arg was calculated. Hereby, the Asp-Lys dipeptide shows two possible modifications by single or double addition of MITC, whereas the Asp-Arg dipeptide shows one product. This pattern is explained by the fact that the Asp-Lys dipeptide yielded two amino moieties, the ε-group of the lysine residue and the α-group of the aspartate, which is accessible in digested CGP, contrary to the undigested polymer. Asp-Arg possesses three amino groups, if the guanidine moiety of arginine is also taken into account, which can be modified. Therefore, three possible products should be found. However, due to the mesomeric stabilization of the guanidine residue, this group is not accessible to an addition reaction; therefore, only one product can be detected. This argumentation is supported by control reactions, which show a similar reaction behavior, as well as the reaction with undigested cyanophycin. Despite the solubility problem, a conversion rate of about 50% of Asp-Lys and 3% for Asp-Arg was found. Thus, almost no modified Asp-Arg product was discovered. Also the amount of double modified Asp-Lys dipeptides was significantly decreased. The presence of low amounts of modified Asp-Arg and double modified Asp-Lys dipeptides is explainable by the fact that not all cyanates could be removed after the reaction. The remaining cyanate traces may react with the dipeptides which are resulting from the digestion for analytic purposes. Also, the presence of dipeptides from the cyanophycin isolation or natural degradation process could not be excluded. Since performed control reaction with ornithine was promising, the reaction should also be done with ornithine-rich cyanophycin. By the introduction of an easily modifiable sulfur into cyanophycin upon reaction with MITC, the basis for additional linkage opportunity is built (Baslé et al. 2010). The solubility problem of the undigested CGP may be improved by varying the protocols resulting in a higher yield. The attempts to modify cyanophycin and cyanophycin dipeptides with ATIC and GITC were not successful. The control reaction with free lysine displayed a high conversion rate of approximately 55% (ammonium isothiocyanate) and > 99% (guanidine isothiocyanate); however, the products were not corresponding to the expected masses and could thus not be identified. These other cyanate were used, due to their similar structure to MITC, but more promising may be the reaction with KNCO as described by Weisgraber et al. (1978).
The cleavage products of cyanophycin obtained by incubation with CphE showed nearly no reaction (expected hydroxymethylation) at the Asp-Arg dipeptides, while 84.4% of the Asp-Lys dipeptides were methylated. The reaction setup with dipeptides (Fig. 8) showed completely different results. There was no methylation of Asp-Lys dipeptides measurable based on the done interpretation of the peaks, while 15.4% of the Asp-Arg dipeptides where both methylated and hydroxymethylated and an additional 11.8% was either methylated (5.4%) or hydroxymethylated (6.4%). One explanation for the absence of a methylated Asp-Lys is that a di-methylated Asp-Lys dipeptide may cause a signal at m/z = 290.2, so unmodified Asp-Arg dipeptides and di-methylated Asp-Lys dipeptides may mask each other in the MS analysis. Due to this fact, all done measurements should be interpreted with care, especially the calculated conversion rates. However, the reaction, in general, concerning the conditions and buffers is a viable method.
4.3 Acetic Anhydride
Using undigested CGP as substrate, the acetylated Asp-Lys dipeptides showed a remarkable yield of 78.5% conversion rate. 4.7% of the Asp-Arg dipeptides were acetylated once without any other modification, but there occurred several other signals with substantial intensities which likely refer to acetylated dipeptides potentially with other modifications in common (Fig. 9). There are too many unknown peaks to include them for reasonable calculations of conversion rates. The interpretation of these signals is also difficult. Due to the controls that were done along with the reaction setup, it can be excluded that these signals originate from contaminations with other amino acids in the CGP or from other sources, they have to be modified Arg-Asp or Arg-Lys dipeptides. However, there are a couple of patterns noticeable within the detected signals. There is a recurring difference of m/z = 14, applying to the peaks at m/z = 290.08 and 304.08, to the peaks at m/z = 312.08 and 326.08, to the peaks at m/z = 334.17 and 348.08 and also to the peaks at m/z = 356.08 and 370.08. A difference of 14 m/z might be caused by methylation as seen in the reaction with formaldehyde. A difference of m/z = 44 can be seen between the peaks at m/z = 290.08 and 334.17, the peaks at m/z = 304.08 and 348.08, the peaks at m/z = 312.08 and 356.08, and also to the peaks at m/z = 326.08 and 370.08. Finally, there is also a pattern of a difference of 22 m/z recognizable for the peaks at m/z = 290.08 and 312.08, at m/z = 304.08 and 326.08, at m/z = 312.08 and 334.17, at m/z = 326.08 and 348.08, at m/z = 334.17 and 356.08 and for the peaks at m/z = 356.08 and 370.08. A possible reason for both of these patterns could be the NaOAc buffer used in the reaction. The addition of sodium could result in a shift of m/z = 22, while the acetate could contribute a carboxyl group for a change of m/z = 44. However, this is unlikely since the identically treated dipeptides do not exhibit this change and they did not show up in control reactions with free amino acids. Therefore, further studies and experiments are required to identify the identity of these reaction products.
The measurement of the reactions performed on dipeptides showed notably better results. All Asp-Lys dipeptides where at least acetylated once, 63.4% of them were acetylated twice, 66% of the Asp-Arg dipeptides were acetylated and the structure causing the signal at m/z = 272.2 was acetylated with a remarkable yield of 89.2%. The signal at m/z = 272.2 is puzzling, since the signal was too weak for MS–MS fragmentation to give analyzable results. The difference in mass of m/z = 18 regarding the Asp-Arg dipeptide (m/z = 290.08) might suggest the loss of one molecule of H2O during the reaction or by other means.
4.4 Pyridoxal Phosphate
The achieved result for the reaction of the polymer with pyridoxal phosphate suggests that cyanophycin can be modified by the active form of vitamin B6. The maximal reached conversion rate for the Asp-Lys dipeptide of 10% was observed at a pH value of 9 and the use of sodium borohydride on undigested cyanophycin. It is worth to mention that the use of sodium borohydride increases also the yield, which may be reasoned by the reduction of the Schiff base and thus stabilizing the bond between the amino acid and the pyridoxal phosphate, due to the removal of the double bond. Also experiments studying the dependency of the conversion rate on the sodium borohydride concentration were interesting. The obtained data suggested also that a reaction with arginine is not preferred. Due to the mesomeric stabilization, the guanidine group cannot be attacked by pyridoxal phosphate. Since no significant products of the reaction with arginine were detected, this could indicate the specificity of the reaction towards non-α-amino moieties. This should be substantiated by measurements of the reaction with free lysine in higher m/z ranges, to detect a possible formation of products with two additions of pyridoxal phosphate. Based on the modification of cyanophycin by pyridoxal phosphate, the dephosphorylation of the product by acid phosphatase (Bingham et al. 1976; Masuda et al. 2005) is a promising next step for modifications.
In comparison to the methylation or acetylation, the experiments with diacetyl had totally different preconditions. There is little literature available about this reaction, since it is rarely used. The reaction with diacetyl gave remarkable yields of 79.7% for the peak at m/z = 444 in the reaction setup with CphE cleavage. The reaction with CGP gave no compound with a measurable signal at m/z = 376, while the reaction with dipeptides exhibited a dominant signal. An explanation for this is that the peak at m/z = 376 is caused by a modification which can be done on dipeptides but not on cyanophycin, so probably the amino group of aspartic acid is targeted and not the arginine. Due to these results, the structure causing the peak at m/z = 376 is likely not a modification on arginine as desired. In general, the reactions with diacetyl offer an interesting basis for further experiments, since there is still not much knowledge about these reactions in comparison to established reactions like methylation or acetylation. Reactions performed with the generated diacetyl trimer, while exhibiting various reaction products were difficult to interpret and would require excessive additional experiments, exceeding the aim of this study.
In summary, the tested reactions exhibited significant conversion rates for both arginine and lysine but were often dependent on the enzymatic digestion of the CGP into dipeptides, which except for the methylation of lysine residues, usually gave higher conversion rates than undigested CGP. Some of these modifications might have actual applications, e.g. the aforementioned use of acetylation to increase the permeability of certain drugs through the blood–brain barrier (Pardridge 2012) or conversion of N6-(pyridoxal phosphate)-lysine of the modified Asp-Lys dipeptide into regular pyridoxal (vitamin B6) by dephosphorylation (Masuda et al. 2005). Methylation, in addition to its importance as post-translational modification of arginine and lysine, also has interesting medical applications: Oligo-arginines are studied due to their capability in penetrating cell-walls and as part of herbicides and anti-malaria drugs (Sparr et al. 2013). Grogg et al. (2018) introduced modifications like MeO, Me 2N, or Me3CO to CGP-derived octapeptides, which were used due to their similarity to oligo-arginines and found significant differences in the cell-wall permeability and toxicity of the compounds, depending on the introduced modification. A different set of methylation or other modifications could expand on these results.
Other modifications could be used to further expand the toolbox for introducing novel modifications into the CGP. The introduction of a sulfur group using isothiocyanates opens up a new spectrum of reactions targeting this group instead of the functional groups available on the unmodified arginine or lysine. Acetylation (and in part methylation) can provide protecting groups to block certain groups from modification (Kociensky 2005) which could be desirable for reactions targeting both the arginine and lysine residues. Using undigested CGP acetylation and methylation primarily affected the lysine (78.5% and 84.4%) with only minor changes to the arginine (4.7% and 0.0%).
Overall, the investigated reactions confirmed the viability of chemical CGP modification, especially in comparison to enzymatic modifications.
Compliance with Ethical Standards
Conflict of interest
On behalf of all authors, the corresponding author declares that there is no conflict of interest and no financial and/or personal relationship with a third party.
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