Enabling Selective and Sustainable P450 Oxygenation Technology

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Enabling Selective and Sustainable P450 Oxygenation Technology. Production of 4‑Hydroxy-α-isophorone on Kilogram Scale Iwona Kaluzna,* Thomas Schmitges, Harrie Straatman, Dennis van Tegelen, Monika Müller, Martin Schürmann, and Daniel Mink DSM Ahead R&D BVInnovative Synthesis, P.O. Box 18, 6160 MD Geleen, The Netherlands synthesis of 4-hydroxy-alpah-isophorone on kilogram scale by using P450 oxidation technology.

ABSTRACT: The development of P450 platform technology has enabled the sustainable production of an oxygenated intermediate, 4-hydroxy-α-isophorone, on kilogram scale. Application of a cytochrome P450 enzyme resulted in an unprecedented product concentration of 10 g/L and a space−time yield of 1.5 g/L/h. These findings are highly relevant for the economical evaluation of cytochrome P450 technology and, additionally, provide access to an alternative and cost-effective route toward 4hydroxy-α-isophorone.

2. RESULTS AND DISCUSSION The poor diversity and activity of the P450 enzymes were both tackled by protein engineering as described by Kaluzna et al.15 The combinatorial library was built based on P450-BM3 from Bacillus megaterium (CYP102A1) which was well-expressed in Escherichia coli. The library was designed using a rational approach focusing on creating differently shaped active site pockets. The substrate scope of the library was characterized by screening the entire collection toward 13 structurally different substrates including the precursor of the hydroxylated molecule of interest, α-isophorone. The screening results showed that a number of mutants and the BM3 wild type enzyme were able to selectively hydroxylate α-isophorone to the corresponding 4hydroxy-α-isophorone. Testing the library on microtiter plate (MTP) scale in the presence of glucose and glucose dehydrogenase (as the regeneration system for NADPH) for the selective hydroxylation of α-isophorone (Figure 1) led to

1. INTRODUCTION The increasing demand for oxygenated molecules in food, feed, flavor and fragrance, cosmetic, pharma, and materials applications has revealed an unmet need for cost-effective and sustainable routes toward the aforementioned targets.1−4 One of the widely acknowledged approaches for the oxidation of C− H bonds is the application of highly versatile catalysts such as cytochrome P450 enzymes (CYPs or P450s).5−7 The extraordinary ability of these enzymes to catalyze a diverse variety of oxidation reactions in a chemo-, regio-, and stereoselective mode under mild conditions8 makes them highly desirable catalysts for the synthesis of fine chemicals, active pharmaceutical ingredients (APIs), and nutritional intermediates.9 It is noteworthy that the potential of P450 enzymes has not been fully exploited since there are a number of limiting factors which hinder the wider implementation of these catalysts for commercial purposes.10−12 The main restrictions of these enzymes have been recognized as limited diversity and selectivity, poor expression and activity, and, most importantly, unsatisfactory performance under industrially relevant conditions.13,14 In order to address these limitations, and hereby be able to synthsize new molecules, DSM has successfully applied protein engineering.15 In addition, the application of reaction engineering principles for enzymatic systems allowed us to accomplish successful scale up from the laboratory to our pilot plant facility. This newly developed technology was applied on α-isophorone. It is worth pointing out that the stereoselective oxidation of α-isophorone to 4hydroxy-α-isophorone by chemical approaches is challenging.16 However, the use of P450 technology enabled the production of this valuable intermediate to be accomplished on kilogram scale with relatively high product concentrations and volumetric productivities. The process which has been developed is selective and environmentally friendly and represents efficient route toward © XXXX American Chemical Society

Figure 1. Bio-oxidation/hydroxylation reaction of α-isophorone to 4hydroxy-α-isophorone.

the identification of many variants affording the desired product. However, considering the existing broad knowledge about the growth and expression of the BM3 WT,17,18 it was decided to perform further optimization studies with this wild type enzyme. The outcome of the initial screening results obtained for the BM3-WT with α-isophorone in the presence of continuously regenerated NADPH on the microtiter scale is summarized in Table 1. Received: September 9, 2015

A

DOI: 10.1021/acs.oprd.5b00282 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Table 1. Performance of the BM3 Wild Type on a 2 mL Scale

a

enzyme

α-isophorone (mM)

4-hydroxy-α-isophorone (mM)

keto-isophoronea (mM)

regioisomerb (mM)

regio-selectivity (%)

conversion (%)

wild type BM3

12.5

8.1

0.13

0.38

96

69

Overoxidized product, 4-keto-isophorone. bRegio-isomer, 3-hydroxymethyl-α-isophorone.

air with a flow of 0.11 L/h on 20 mL scale and a flow of 0.42 L/ h (0.42 vvm) on 1 L scale. However, the use of air caused protein denaturation and foaming of the reaction mixture. Slowing down the aeration only helped reducing foam formation to a small extent and, additionally, led to the reaction suffering from oxygen limitation and consequently extended reaction times compared to the 20 mL scale reactions shown in (Figure 2).

Several options for identifying the most desired formulation for process optimization work with the wild type P450 enzymes have been previously explored in our laboratories. These include fermentation broth as such, the use of whole cell pastes, and the use of isolated biocatalysts such as (cell-free) extract or (partially) purified liquid enzyme formulations. As described in excellent reviews earlier,19,20 each approach has its advantages and disadvantages. For the particular example described in this article we chose to work with permeabilized whole cell formulation (whole cells disrupted by a freezing/thawing step) since this overcomes mass transfer limitations and to a certain extent increases the coupling efficiency of the system (often a large proportion of the electrons required for product formation are lost due to side product formation, mainly hydrogen peroxide and/or other active oxygen species), and this also partially solves the stability problem of the enzyme.21 Furthermore, whole cell formulations can utilize intracellular cofactors for the desired biocatalytic reaction. To avoid addition of the required cofactor, heterologous coexpression of a glucose dehydrogenase was used to improve the cofactor regeneration.22 Overall, we used a permeabilized whole cell formulation consisting of the BM3 wild type coexpressed with an NAD(P)H-dependent glucose dehydrogenase in E. coli. The concentration of properly folded P450 was determined by CO assay. The specific activity of glucose dehydrogenase was determined spectrophotometrically (Table 2).

Figure 2. Progress of the P450 hydroxylation of α-isophorone to 4hydroxy-α-isophorone on 1 L scale with aeration at 20 mL/min.

The application of pure oxygen solved the foam formation problem and, moreover, the P450 reaction was no longer limited by oxygen (Figure 3). For safety reasons, the oxygen

Table 2. Cytochrome P450 Concentration and GDH Specific Activity of the Wild Type BM3 (BM3-WT) exp no.

clone

fermentation volume

P450a (nmol/g)

P450b (nmol/L)

GDH (U/mg)

A B

BM3WT/GDH BM3WT/GDH

10 L 1000 L

1625 1875

52 000 30 000

7 5

a

Nanomoles of P450 per gram of total protein: measurement of the BM3 expression with a properly inserted heme group. bTotal P450 produced per liter: measurement of the volumetric productivity of fermentative BM3 production.

Systematic optimization was performed (using cells obtained from a 10 L fermentation (Exp A)) on 20 mL scale in order to identify the most suitable conditions. The optimum conditions turned out to be pH 7.5, a temperature of 28 °C, and a substrate load of 2% (w/v, 130 mM), leading to conversions of up to 78% (102 mM of 4-hydroxy-α-isophorone). Consequently, further optimization was done on 1 L scale in a downscaled reactor which mimicked the 200 L miniplant reactor that was to be used for the production of 4-hydroxy-αisophorone on kilogram scale. The systematic optimization studies were done with cells derived from a 10 L fermentation (Table 2, A). Cytochrome P450 reactions are complex systems demanding various components, with oxygen being one of the key parameters. It has a dual role, with one oxygen atom being inserted in to the substrate and a second oxygen atom acting as acceptor of the electrons supplied by the nicotinamide cofactor NADPH.23 In the first experiments oxygen was delivered using

Figure 3. Application of pure oxygen for the P450 hydroxylation of αisophorone to 4-hydroxy-α-isophorone on 1 L scale.

concentration in the reaction mixture and in the headspace was continuously monitored. For safe operation, the set point of the maximum oxygen concentration in the headspace was 20%, and the set-point for oxygen in the reaction mixture was 5 ppm. The oxygen intake was adjusted to keep the concentration below these set points, resulting in an oxygen flow of 0.6 L/h (0.6 vvm). It is important to mention that the solubility of oxygen in water at 30 °C and atmospheric pressure is comparatively low (0.24 mmol/L).24 Therefore, the development of an efficient cytochrome P450 system requires that the oxygen transfer rate (OTR) of the setup must be faster than the oxygen conditions during reaction. The maximum amount of oxygen transferred under limited oxygen conditions has been calculated and was found to be 0.6 mmol/min·L for the 1 L B



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experiment. As shown in Figure 3, using pure oxygen increased the reaction rate by a factor of 4. However, only a small portion of the oxygen supplied was actually used for formation of the desired product; a large proportion was either utilized for the metabolism of partially living cells (i.e., of the permeabilized whole cell formulation) or was consumed unproductively without product formation (low coupling efficiency). This unproductive oxygen consumption led to the formation of active oxygen species which had a negative impact on the P450 catalyst stability (hereby lowering the total turnover number (TTN). This phenomenon is very often associated with the P450 class of enzymes.25 Additionally, important factors such as stirring speed/power input were optimized in order to achieve efficient stirring without affecting the stability of the enzymes. Based on these optimization studies, the following parameters were applied on 1 L scale, 400 rpm (tip speed 1.76 m/s), and thus, a power input of 0.53 W/m3. Another essential parameter which required attention was the continuous regeneration of the cofactor NADPH, in our case enabled by a coexpressed glucose dehydrogenase (GDH) in the presence of glucose. It is noteworthy that, dependent on the nature of the substrate, the provided cofactor electrons are only partly used by the desired reaction. Often a large proportion of these electrons are lost due to side product formation, mainly hydrogen peroxide and/or other active oxygen species as mentioned earlier (low coupling efficiency). Other challenges to overcome in order to develop an industrially viable process include (i) the fast acidification of the reaction mixture due to the formation of byproducts such as gluconic acid (which comes from the GDH/glucose regeneration system) and acetate (which is produced by living cells) and (ii) the oxygen mass transfer rate at ambient pressure and temperature as mentioned earlier (e.g., at 28 °C). To ensure that the regeneration system is not rate-limiting, each component of the regeneration system (NADP, GDH, and glucose) was added in excess and evaluated individually (data not shown). An excess of glucose was required due to uncoupling, and an additional amount of external NADP was found to be needed. Due to the utilization of a permeabilized whole cell formulation, the transfer of NADP through the cell membranes was not limiting. To summarize, the reaction was completed within 6 h with a substrate load of 10 g/L and afforded a conversion of 61% with a productivity of 1.5 g/L·h (Table 4). The total turnover number of the P450 biocatalyst under optimized reaction conditions on a 1 L scale was 13 200 molisophor/molP450. Overall, despite many challenges we have shown that the systematic investigation of the enzymatic and cosubstrate limitations (using modeling studies and reactor engineering principle) enabled process conditions to be identified, and the enzymatic oxidation could be applied on a multi kilogram scale (vide infra). The enzymatic hydroxylation/oxidation reaction was performed on 100 L scale in a temperature, pH, and oxygen controlled stirred tank reactor (200 L total volume). Due to the aforementioned acidification of the reaction (caused by use of the GDH/glucose cofactor regeneration system), the pH of the reaction was kept constant by automated dosing of 5 M NaOH. The titration profile during the course of the reaction is shown in Figure 4. To ensure a sufficient supply of electrons during the reaction, a 10-fold excess of glucose was used to compensate for the aforementioned uncoupling problem. The

Figure 4. Progress of the P450 catalyzed hydroxylation of αisophorone to 4-hydroxy-α-isophorone on 100 L scale in two independent batches.

substrate α-isophorone was added at a concentration of 1 wt %. The biocatalyst, P450/GDH coexpressed in E. coli, was added as a whole cell permeabilized slurry (slurry derived from fermentation on 1000 L scale, Table 2B) in a ratio of 7:1 on weight basis with respect to the substrate. Modeling studies allowed us to identify the most optimal stirrer as well as the optimum position for the oxygen sparger in the reactor. In silico studies had indicated that the positioning of the sparger plays a key role and that the ideal location would be at the bottom of the reactor below the stirrer. This position would ensure a good dispersion of the small oxygen bubbles generated by the sintered metal frit of the sparger. However, the actual geometry of the 200 L glass lined reactor and its Pfaudler stirrer (a three-blade, retreat curve impeller) meant that only a semioptimal oxygen transfer rate (OTR) of 0.4 mmol/min/L could be reached in practice. The reaction was performed with an oxygen flow of 72 L/h (0.72 vvm) and a stirrer speed of 140 rpm (tip speed 3.08 m/s). This required a power input of 0.71 W/m3. In order to run the process on this scale with the required safety standards, the headspace of the reactor was flushed continuously with nitrogen in order to keep the O 2 concentration below 20%. An O2 electrode was also placed in the reaction mixture to monitor the dissolved oxygen. Despite having optimized all of the parameters in the downscaled 1 L reactor, the first 2 h of reaction time in the 100 L reactor were characterized by slow product formation and buildup of unconsumed oxygen in the headspace. Acceleration of the reaction only occurred when the dissolved oxygen concentration increased and the concentration of oxygen in the head space decreased. This clearly indicated the importance of oxygen in the reaction. Correspondingly, the oxygen mass balance showed a significant amount of unconsumed oxygen within the first 2 h of the reaction. As aforementioned, an efficient oxygen supply is important parameter for P450 process development work in order to ensure enhanced reaction rates. Two consecutive batches were executed on 100 L. The reactions were completed within 10 h and afforded substrate conversions of 80% and 82%, respectively (Figure 4, Table 3). The hydroxylated product was obtained with a high enantiomeric excess (>99%), a high purity (>98% HPLC, GC, NMR), and with isolated yields of 51% and 61%, for batch 1 and batch 2, respectively. Overall, the two batches delivered 1 kg of 4-hydroxy-α-isophorone. We also have observed that one of the most critical parameters when using cytochrome P450 catalysts, their limited C



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Table 3. Comparison of the Results and Reaction Conditions on 1 and 100 L Scale exp. no.

U (μmol/min)

U/μmolP450

U/g cells

conv. (%)

TTN (molisophor/molP450)

productivity (g/L·h)

substrate load (g/L)

OTR (mmol/min·L)

1L 100 L

219 19 173

50 42

2.8 1.3

61 80−82

13 200 18 000

1.5 1.0

10 6

0.6 0.4

Figure 5. HPLC spectrum of concentrated oil.

Figure 6. HPLC spectrum of recrystallized product.

total turnover number (TTN), was scale-independent. This remains, therefore, one of the most desired parameters to be improved. The TTN for the P450 reaction in the 200 L reactor, 18 000 molisophor/molP450, was slightly higher than that achieved on 1 L scale, 13 200 molisophor/molP450, but this is still an order of magnitude lower than typical TTN’s reported for enzymatic processes performed by alcohol dehydrogenases.26

Table 4. Description of HPLC Purity of the Product

3. CONCLUSION Kilogram quantities of hydroxylated product were obtained using P450 technology. High product concentrations (of more than 6 g/L) and volumetric productivities (of more than 1.0 g/ L·h) were achieved on 100 L scale by using a P450 produced in a recombinant microorganism. The demonstrated P450 process was still restricted by the oxygen transfer rate (OTR) and by self-deactivation of the catalyst (resulting in a limited total turnover number). Despite these limitations, we were still able to launch a novel P450 technology which broadens the industrial applicability of this class of enzymes. Moreover, this offers alternative oxidation and hydroxylation solutions for numerous challenging molecules in many different application fields within the life sciences arena.

Table 5. Analysis Parameters

4. EXPERIMENTAL SECTION Standard Procedure Used in Batch 1 and Batch 2. To a 200 L stirred reactor, 30 kg of water, 5 kg of 100 mM potassium

phosphate (KPi) buffer pH 7.5, and 18.7 kg of D-(+)-glucose monohydrate (94 mol) were added, and the reaction mixture was stirred for 60 min. The headspace of the reactor was

compound

oil area %

crystal area %

4-hydroxy-α-isophorone 3-hydroxymethyl-α-isophorone (regio-isomer) keto-isophorone α-isophorone

94.45 2.71 0.92 0.64

99.22 0.14 0.06 0.10

column eluent temperature flow gradient

stoptime detection injection volume

D

symmetry shield, C18 150 mm × 4.6 mm, ID 3.5 μm A: water B: acetonitrile 40 °C 1.5 mL/min time (min) %B 0.00 5.0 15.00 35.0 18.00 35.0 18.10 5.0 25.0 min UV 240 nm 1 μL



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Figure 7. Spectra of 600 MHz proton NMR.

flushed with nitrogen (oxygen concentration below 10%), and during the whole reaction a nitrogen flow of 100 L/h was maintained into the headspace. The reactor outlet was equipped with a 4 °C reflux condenser. Subsequently 70 g of NADP (97 mmol) and 897 g of α-isophorone (6.3 mol) were added to the mixture. The temperature was adjusted to 28 °C and the pH to 7.5 with 5 M NaOH (final amount 3.45 L). The reaction was started by addition of 30.5 kg cell paste to the reactor. All containers were rinsed with 14.8 kg water, which was added to the reactor. The total starting reaction weight was 100 kg. After adding the biocatalyst, the oxygen sparging as well as the auto titration to pH 7.5 by 5 M NaOH was started. The oxygen concentration in the reaction mixture and in the headspace was continuously monitored. The set point for the oxygen concentration in the headspace was 20%, and the set point for oxygen in the reaction mixture was 5 ppm. The oxygen intake was adjusted to keep the concentration below the set points, resulting in an oxygen flow of 72 L/h (0.72 vvm). The reaction progress was followed by time samples which were analyzed by HPLC.

After completion of the reaction (10 h), 50 L of methanol was added, and the temperature was adjusted to 50 °C. The reaction mixture was stirred overnight. Dicalite (filter aid) was added (8 kg), and the reaction mixture was filtered over a Nutsche filter. The filter cake was washed with methanol (20 L). The filtrate was concentrated under vacuum to remove methanol. The residue was extracted three times with isopropyl acetate (25 L each). The combined extracts were washed with brine and concentrated to 0.95 kg of orange residue which crystallized upon standing. HPLC Purity. The concentrated oil as well as the crystals were analyzed by HPLC. For both the chromatograms are shown below (Figures 5 and 6) as well as the area % of both the samples (Table 4). The HPLC samples were prepared by mixing 50 mg of reaction sample with 600 μL of acetonitrile and centrifuged, and the supernatant was injected. Analysis. The parameters for analysis are shown in Table 5. NMR Details. The crystals were analyzed by 600 MHz proton NMR. As solvent, deuterated dimethyl sulfoxide was used and para-nitro-toluene as the internal standard. The spectra are given in Figure 7. The purity of 4-hydroxy-αisophorone was determined to be >95%. E



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(21) Brummund, J.; Müller, M.; Sonke, T. Org. Process Res. Dev. 2015, 19, 1590−1595. (22) Schewe, H.; Kaup, B. A.; Schrader, J. Appl. Microbiol. Biotechnol. 2008, 78, 55−65. (23) Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev. 2004, 104, 3947−3980. (24) Hass, V. C.; Pörtner, R. Praxis der Bioprozesstechnik; Spektrum: Heidelberg, 2011; p 180. (25) Shakunthala, N. Expert Opin. Drug Metab. Toxicol. 2010, 6, 1− 15. (26) Wolberg, M.; Filho, M. V.; Bode, S.; Geilenkirchen, P.; Feldmann, R.; Liese, A.; Hummel, W.; Muller, M. Bioprocess Biosyst. Eng. 2008, 31, 183−191. (27) Hennig, M.; Püntener, K.; Scalone, M. Tetrahedron: Asymmetry 2000, 11, 1849−1858.

Melting Point. The melting point of 4-hydroxy-αisophorone was measured by Mettler FP62 and has been determined as 71.4 °C. Optical Purity. The recrystallized 4-hydroxy-α-isophorone which based on NMR showed purity >99% were analyzed. The obtained optical rotation value, [α]21 D of +106.7 (c = 1.00, methanol) indicated that produced 4-hydroxy-α-isophorone had enantiomeric purity of >99% by comparison to the literature reference (pure (R)-enantiomer [α]22 D of +105.9 (c = 1.00, methanol)).27

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Address: DSM Ahead R&D BVInnovative Synthesis, Urmonderbaan 22, NL-6167 RD Geleen, The Netherlands. Present Address

D.v.T.: Pathonostics, Randwycksingel 45, 6229 EG Maastricht, The Netherlands. Notes

The authors declare no competing financial interest.

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REFERENCES

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Enabling Selective and Sustainable P450 Oxygenation Technology

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