Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

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Redox self-sufficient whole cell biotransformation for amination of alcohols Stephanie Klatte, Volker F. Wendisch ⇑ Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitaetsstr. 25, 33615 Bielefeld, Germany

a r t i c l e

i n f o

Article history: Received 24 February 2014 Revised 2 May 2014 Accepted 9 May 2014 Available online xxxx Keywords: Redox self-sufficient amination Whole cell biotransformation Escherichia coli Alcohol dehydrogenase Transaminase Alanine dehydrogenase Cofactor recycling Diamines

a b s t r a c t Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/ pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an L-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with L-alanine dependent transamination and L-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor L-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD+ was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The demand of sustainable and green production of synthetics and fine chemicals from renewable resources increases continuously in order to replace common chemical routes which are dependent on restricted fossil feedstocks. Among others, amine functionalized organic compounds such as polyamines are of special interest as they serve as building blocks for polyamides mainly used in coatings. Polyamines are aliphatic, saturated organic compounds with two terminal primary amino groups and, optionally, varying numbers of secondary amino groups.1 They may be used to produce dyes, agrochemicals, and pharmaceuticals or they can be applied as tensides. Common chemical routes to produce amines are, for example, alkylation or arylation of ammonium applying metal- or organocatalysts.2 In nature, amines, diamines and polyamines can be found in many living organisms and are involved in the transcription and translation mechanism,3 acid resistance4 and protection from oxygen toxicity.5 The Gram-negative bacterium Escherichia coli produces polyamines such as 1,4diaminobutane (putrescine), 1,5-diaminopentane (cadaverine) or spermidine naturally that derive from amino acids like L-lysine, L-ornithine, L-arginine or L-aspartic acid via decarboxylation. ⇑ Corresponding author. Tel.: +49 521 106 5611; fax: +49 521 106 5626. E-mail address: [email protected] (V.F. Wendisch).

Recombinant E. coli or Corynebacterium glutamicum strains have been constructed for fermentative production of, for example, 1,4-diaminobutane1,6,7 and 1,5-diaminopentane.8,9 In biocatalysis, stereoselective enzymes, for example, x-transaminases which transfer amino groups from an aminated co-substrate to an aldehyde, are mainly employed for the amine functionalization of chemicals. The selection of transaminases for efficient amination is challenging as they do not only exhibit specificity to amino group acceptors but also to amino group donors.10 Furthermore, the reaction equilibrium must be shifted to drive the reaction towards product formation, for example, by co-product removal.11 Biocatalytic amination usually starts from aldehyde precursors but requires reduction equivalents. Cascading initial alcohol oxidation by alcohol dehydrogenases with subsequent reductive amination provides a means of redox cofactor recycling.12 Alternative redox cofactor recycling systems including, for example, formate dehydrogenase, glucose dehydrogenase or glutamate dehydrogenases may also be employed.13,14 In whole cell biotransformation, the hosts’ metabolism may be used for redox cofactor regeneration, for example, by catabolism of carbon sources like glucose. In whole-cell biotransformation, redox cofactor supply can be improved further by genetic engineering focusing on NADH or NADPH regenerating pathways.15–17 Direct reductive amination of ketoacids is catalyzed, for example, by amino acid dehydrogenase such as L-alanine dehydrogenase which reductively aminates

http://dx.doi.org/10.1016/j.bmc.2014.05.012 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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pyruvate to L-alanine using ammonium in an NADH-dependent manner. Typically, these amino acid dehydrogenases only act on a narrow spectrum of a-keto acids, but coupling to aminotransferase offers the access to a wider substrate spectrum. Coupling of L-alanine dehydrogenase of Bacillus subtilis to branched-chain amino acid transaminase AvtA from C. glutamicum led to reductive amination of 2-keto-3-methylvalerate to L-isoleucine by an engineered E. coli strain when coupled to glucose catabolism for redox cofactor recycling.10 Coupling of L-alanine dehydrogenase of Bacillus subtilis to an x-transaminase and cascading with an alcohol dehydrogenase in vitro was followed for redox self-sufficient amination of

several primary alcohols in cell-free biocatalysis.12,15 The cascading of multiple enzymes in biocatalysis requires moderate conditions for every enzyme to gain high product yield. Additionally, a broad substrate spectrum might be versatile for amination of various substrates. In the present study, this concept was extended for whole cell biotransformation in E. coli. A three-enzyme-cascade employing alcohol dehydrogenase from Bacillus stearothermophilus, x-transaminase from Vibrio fluvialis and L-alanine dehydrogenase from Bacillus subtilis was encoded in an artificial operon and used for redox self-sufficient amination of alcohols. In a first step, the alcohol is oxidized to the aldehyde intermediate which is further aminated by an L-alanine dependent transaminase (Fig. 1). The resulting pyruvate and NADH are recycled by L-alanine dehydrogenase during reductive amination of pyruvate to L-alanine. Using several primary and secondary linear mono- and dialcohols as well as cyclic and aromatic monoalcohols the capacity of the cascade could be demonstrated. 2. Material and method 2.1. Bacterial strains, plasmids and oligonucleotides

Figure 1. Scheme of the redox self-sufficient amination of alcohols by employing an alcohol dehydrogenase for substrate oxidation, a transaminase for aldehydeintermediate amination and an alanine dehydrogenase to recycle the cofactor and amino group donor in one step. Substrates for amination: Primary and secondary aliphatic mono- and dialcohols (6-hexanol, 8-hexanol, 10-hexanol, 12-hexanol, 2hexanol, cyclohexanol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol); Primary aromatic monoalcohol (benzylalcohol).

The E. coli strains, plasmids and oligonucleotides used in this study are listed in Table 1. Competent cells and vector cloning was performed according to standard DNA work procedure.18 In this study three E. coli expression vectors based on IPTG-inducible pTrc99A were constructed. Therefore, PCR-derived gene product adhB.stearothermophilus19 (ADH_KpnI_RBS_for; ADH_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-adh. To construct pTrc99A-ald-adh-ta and pTrc99A-ta-ald-adh the artificial operon was built by

Table 1 Strains, plasmids and oligonucleotides Name

Relevant characteristics

Reference

Strains E. coli DH5a E. coli W3110 W3110/pTrc99A W3110/pTrc99A-ald-adh-ta W3110/pTrc99A-ta-ald-adh

Fthi-1 endA1 hsdr17(r, m) supE44 DlacU169 (/80lacZDM15) recA1 gyrA96 relA1 F k INV(rrnD–rrnE)1 E. coli W3110 harboring pTrc99A E. coli W3110 harboring pTrc99A-ald-adh-ta E. coli W3110 harboring pTrc99A-ta-ald-adh

38

Plasmids pTrc99A pTrc99A-adh pTrc99A-ald-adh-ta

pTrc99A-ta-ald-adh

38

This study This study This study 39

Ptrc, pBR322ori, rrnB T1, rrnB T2, lacIq pTrc99A carrying adh from B. stearothermophilus pTrc99A carrying ald-adh-ta synthetic operon ald from B. subtilis 168 ta from V. fluvialis adh from B. stearothermophilus pTrc99A carrying ta-ald-adh synthetic operon genes’ origin see pTrc99A-ald-adh-ta

This study This study

This study

Name

Sequence 50 ? 30

Cut site

Use in this study

Oligonucleotides ADH_KpnI_RBS_for ADH_BamHI_rev AlaDH_KpnIRBSfor

caaGGTACCcaggaaacagaccATGAAAGCAGCAGTTGTGGAAC gttggatccTTATTTATCTTCCAGGGTCAG

KpnI BamHI KpnI

pTrc99A-adh pTrc99A-adh pTrc99A-ald-adh-ta

— —

pTrc99A-ald-adh-ta pTrc99A-ald-adh-ta

— —

pTrc99A-ald-adh-ta pTrc99A-ald-adh-ta

BamHI KpnI

pTrc99A-ald-adh-ta pTrc99A-ta-ald-adh

— — — — BamHI

pTrc99A-ta-ald-adh pTrc99A-ta-ald-adh pTrc99A-ta-ald-adh pTrc99A-ta-ald-adh pTrc99A-ta-ald-adh

AlaDH_RBS99A_rev ADH_RBS99A_for ADH_RBS99A_rev

xTA_RBS99A_for xTA_BamHI_rev xTA_KpnI_RBS_for xTA_RBS99A_rev AlaDH_RBS99A_for AlaDH_RBS99A_rev ADH_RBS99A_for ADH_BamHI_rev

caaGGTACCcaggaaacagaccATGATCATAGGGGTTCCTAAAG ggtctgtttcctgTTAAGCACCCGCCACAGATG caggaaacagaccATGAAAGCAGCAGTTGTGGAAC ggtctgtttcctgTTATTTATCTTCCAGGGTCAGAAC caggaaacagaccATGAACAAACCGCAGAGCTGG gttGGATCCTTACGCAACTTCCGCGAAAAC caaGGTACCcaggaaacagaccATGAACAAACCGCAGAGCTG ggtctgtttcctgTTACGCAACTTCCGCGAAAAC caggaaacagaccATGATCATAGGGGTTCCTAAAG ggtctgtttcctgTTAAGCACCCGCCACAGATG caggaaacagaccATGAAAGCAGCAGTTGTGG gttGGATCCTTATTTATCTTCCAGGGTCAG

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overlap-extension-PCR before treatment with KpnI and BamHI and ligation with KpnI/BamHI treated pTrc99A. For the ald-adh-ta operon oligonucleotides AlaDH_KpnIRBSfor/AlaDH_RBS99A_rev, ADH_RBS99A_for/ADH_RBS99A_rev and xTA_RBS99A_for/ xTA_BamHI_rev, were used to amplify ald15 [NCBI GI: 936557], adh19 [NCBI GI: 440137] and ta20 [NCBI GI: 327207066], respectively. The construction of pTrc99A-ta-ald-adh was performed similarly but xTA_KpnI_RBS_for/xTA_RBS99A_rev, AlaDH_RBS99A_ for/AlaDH_RBS99A_rev and ADH_RBS99A_for/ADH_BamHI_rev oligonucleotides were used for single gene amplification and xTA_KpnI_RBS_for and ADH_BamHI_rev to build the synthetic operon ta-ald-adh. KpnI/BamHI treated synthetic operon and pTrc99A were then ligated and E. coli DH5a was transformed with the ligation products. CaCl2-competent E. coli DH5a were heatshocked for the uptake of ligation products. Newly derived expression vectors were proven by sequencing and E. coli W3110 was transformed with correct plasmids pTrc99A-ald-adh-ta, pTrc99Ata-ald-adh and the empty vector pTrc99A for production experiments and pTrc99A-adh for investigation of substrate spectrum in vitro. 2.2. 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 lg/mL ampicillin was supplemented to the medium. 2.3. Toxicity tests E. coli W3110 was grown in LB-medium overnight and cells for the final optical density at 600 nm OD600 = 10 in 20 mL final volume were harvested ðvolumeharvest ¼ OD600final  OD600culture =volumefinal Þ and washed once with one volume of sterile 50 mM Hepes buffer pH 6. The cell pellet was resuspended and filled up to 20 mL with sterile buffer in a 100 mL Schottbottle. Testing aliphatic monoalcohols and monoamines 10 mM of the compound was added while for aliphatic dialcohols and diamines only 5 mM was tested due to low solubility of C10 and C12 substrates. Test reactors containing cells and the compound of interest were incubated at 37 °C and 200 rpm for 10 h. To test the cell viability cells were diluted in sterile water to a maximum of 107 after 0 and 10 h and 100 ll was spread on LB-plates to count the colony forming (CFU) units per mL the next day and calculate the relative CFU per mL in respect to the initial colony forming units. 2.4. Preparation of cell free extract and enzyme assay The E. coli W3110 derivatives were grown in LB + 100 lg/mL ampicillin until an optical density at 600 nm of 0.6–0.8, induced with 1 mM isopropyl-b-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 min (cycle 0.5; amplitude 55%). Cell cebris was centrifuged at 10.000g at 4 °C for 1 h and clear cell extract was used for the measurement of the enzyme activity. 2.4.1. 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.

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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 min. The assay was performed in triplicates and one enzyme unit was calculated to be the amount of enzyme that catalyzes the conversion of 1 lmol substrate in 1 min. 2.4.2. 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 min and one enzyme unit was calculated to be the amount of enzyme that catalyzes the conversion of 1 lmol 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. 2.4.3. 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)-a-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 ll 16% perchloracetic acid. The samples where neutralized by the addition of 40 ll 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 lmol product in 1 min. 2.5. Whole cell biotransformation with resting cells E. coli W3110/pTrc99A and its derivatives W3110/pTrc99A-aldadh-ta and W3110/pTrc99A-ta-ald-adh were inoculated to an initial OD600 = 0.1 in LB-medium plus 20 mM Mops and 100 lg/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. After 20–22 h cells were harvested for a final OD600 = 10 in 20 mL final volume, once washed with 50 mM Hepes buffer pH 6 and prepared for whole cell biotransformation in a resting buffer system with the mentioned buffer. Supplements like NH4Cl, L-alanine, pyridoxal-5-phosphate (PLP) and nicotinamide adenine dinucleotide (NAD+) 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 and 200 rpm and samples for HPLC-analytics were taken continuously throughout the production. 2.6. HPLC-analysis Extracellular (di)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 10.000g for 5 min 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-5l, 40  4.6 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and the main column (LiChrospher 100

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RP8 EC-5l, 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 (di)amine detection. Here, through a precolumn (LiChrospher 100 RP 18–5 EC; 40  4 mm) and the main column (LiChrospher 100 RP18 EC-5l; 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. 3. Results 3.1. Influence of primary linear mono- and dialcohols on E. coli W3110 viability Substrate and product toxicity might limit biocatalysis by the production host. To assess potential toxicity of the alcohol substrate and amine products, primary linear mono- and dialcohols as well as primary linear mono- and diamines with chain lengths ranging from 6 to 12 carbon atoms were added to resting E. coli W3110 wild type cells at an optical density at 600 nm of 10 and cell viability was determined by plating. Colony forming units (CFU) on LB agar were determined after exposure of up to 10 h, a time period typical for whole cell biotransformations (Fig. 2). Long chain dialcohols and diamines were tested at 5 mM whereas 10 mM was used for monoalcohols and monoamines. Cell viability in the mock treatment, that is, no addition to 50 mM Hepes pH 6 was hardly affected and decreased by only 27% after 10 h at 37 °C. Toxicity by aliphatic alcohols and amines varied with chain length and (di)amine or (di)alcohol functional groups. Monoamines were shown to be the most toxic compounds as cell viability

Figure 2. Toxicity of primary linear mono-/dialcohols and mono-/diamines to E. coli W3110. Resting cells were treated with primary linear monoalcohols/-amines (10 mM) and dialcohol/-amines (5 mM) for 10 h ranging from C6 to C12 chain length. Counting colony forming units on LB-Agar the rel. CFU (%) was calculated by considering the initial CFUs. The H2O-control was performed to visualize overall cell death in the buffer over time and became reference to plot cell survival after treatment with the test compounds.

was reduced 2000-fold by treatment with hexylamine and more than 107 fold after treatment with octyl-, decyl- or dodecylamine. By contrast, among the diamines only diaminododecane reduced cell viability drastically. The dialcohols hardly affected cell survival and only 1,10-decanediol reduced CFU counts 20-fold. The monoalcohol 1-octanol was toxic while the shorter 1-hexanol and the longer 1-decanol and 1-dodecanol reduced viability less than 5fold. This analysis revealed that E. coli is suitable as host for whole cell amination of some, but not all (di)alcohols. 3.2. Heterologous expression of genes for alanine dehydrogenase, alcohol dehydrogenase and transaminase in the E. coli host strain W3110 For overexpression of ald, adh and ta these genes were cloned into the IPTG-inducible E. coli expression vector pTrc99A as an artificial operon with the pTrc promoter upstream of ald and ribosome binding sites upstream of each gene. Two expression vectors with different gene orders were constructed and used to transform E. coli W3110 to yield strains W3110/pTrc99A-ald-adh-ta and W3110/pTrc99A-ta-ald-adh, respectively. Enzyme activity assays with crude cell extracts of these strains revealed that the three genes could be functionally expressed in the heterologous host. The activities varied depending on the gene position in the artificial operon. For the alanine dehydrogenase an activity 8.5 U/mg was determined when it was encoded in the first position of the artificial operon and 7.9 U/mg when encoded in the second position. Crude extracts of the strain carrying pTrc99A-ald-adh-ta displayed an alcohol dehydrogenase activity of 0.54 U/mg and 0.36 U/mg for transaminase activity. The strain carrying pTrc99A-ta-ald-adh showed lower activities of both, alcohol dehydrogenase and transaminase, that is, 0.36 U/mg and 0.12 U/mg, respectively. However, assaying the crude extract of W3110/pTrc99A-ald-adh-ta all three enzymes encoded in the artificial operon showed highest enzyme activities and that is why this strain was used for testing the redox self- sufficient amination of alcohols in vivo. 3.3. Amination of aliphatic and aromatic alcohols by whole cell biotransformation 3.3.1. Linear mono- and dialcohols Hydroxyl functionalized substrates like primary and secondary linear as well as cyclic and aromatic alcohols were chosen to determine the substrate spectrum for the three enzyme-coupled cascade in vivo. The enzyme equipment in W3110/pTrc99A-aldadh-ta has displayed higher enzyme activities for all three enzymes compared to W3110/pTrc99A-ta-ald-adh and was used for further studies. To start out first (di)amine production, W3110/pTrc99Aald-adh-ta was cultivated in LB-medium plus 20 mM Mops and 1 mM IPTG to overexpress the genes ald, adh and ta and, subsequently, resting cells in Hepes buffer were incubated with 10 mM linear mono-and dialcohols with chain lengths ranging from 6 to 12 C-atoms for amine functionalization. The resting cells were supplemented with 250 mM alanine, 0.35 mM PLP, 0.75 mM NAD+ and 275 mM NH4Cl as it was described for enzyme catalysis12 and samples were taken regularly within 12 h. Efficient amination was dependent on chain length, mono- or dialcohol function and primary or secondary hydroxyl group but maximum yield was achieved after six to eight hours except for the H2O-control where no product formation has been detected (Fig. 3). Whereas primary linear monoalcohols with short chain length such as 1-hexanol were excellent substrates the product yield was reduced for monoalcohols with extended chain length ranging from C8, C10 to C12 (100%, 80%, 0.2% 0.4%). Contrarily, the incubation with short linear dialcohols such as 1,6-hexanediol showed no product formation, but 87% conversion was observed for

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S. Klatte, V. F. Wendisch / Bioorg. Med. Chem. xxx (2014) xxx–xxx Table 2 In vitro oxidation of mono- and dialcohols by ADH Substrate

km [mM]

Vmax [U/mg]

V5mM [U/mg]

Vmax/Km

1-Hexanol 1-Octanol 1,6-Hexanediol 1,8-Octanediol Cyclohexanol Benzylalcohol 2-Hexanol

6.6 0.98 2.7 3.7 — 7.9 5.2

0.24 0.067 0.4 0.47 — 0.32 1

0.1 0.04 0.26 0.27 0 0.14 0.5

0.036 0.068 0.15 0.13 — 0.045 0.19

3.5. Proof of principle of the redox self-sufficient cascade

Figure 3. Maximum (di)amine production by W3110/pTrc99A-ald-adh-ta under specified conditions. The substrate specifity of production strain W3110/pTrc99Aald-adh-ta was visualized by plotting the maximal (di)amine production against the substrate chain length. The H2O-control was used as reference as no corresponding (di)amine production was detected here. Reaction conditions: 10 mM substrate, 250 mM L-alanine 0.35 mM PLP, 0.75 mM NAD+, 275 mM NH4Cl.13.

1,8-octanediol and 100% conversion was achieved with 1,10decanediol. Increasing the chain length to C12 decreased conversion to 60%. Unlike the primary 1-hexanol, the secondary 2-hexanol was poorly aminated since only 0.3 mM 2-hexylamine accumulated after 24 h. 3.3.2. Primary cyclic and aromatic substrates Since 1-hexanol was converted well, also cyclohexanol and benzylalcohol were used for the amine functionalization in vivo to test if cyclic and aromatic monoalcohols are also amenable by the described whole cell biotransformation system. However, cyclohexanol was poorly aminated to cyclohexylamine as only 0.3 mM were detected after 24 h. The aromatic benzylalcohol was aminated quite efficiently and full conversion was detected after 6 h.

3.5.1. Rotation of ald, adh and ta in the artificial operon Further investigation on the improvement of the three-enzymecascade was done with the substrate 1,10-decanediol as complete conversion was achieved by W3110/pTrc99A-ald-adh-ta and monitoring the intermediate 1-amino-10-decanol may be helpful to guide cascade improvement. The permutation of the sequence of the genes ald, adh and ta in the artificial operon to ta, ald and adh in the strain W3110/pTrc99A-ta-ald-adh might influence the production rate as the gene encoding the transaminase is supposed to be higher expressed in the first position. In vivo amination of 1,10-decanediol by E. coli W3110/pTrc99A-ald-adh-ta was complete after eight hours and little of the intermediate 1-amino-10decanol accumulated intermittently (Fig. 4). Using E. coli W3110/ pTrc99A-ta-ald-adh only 68% conversion was observed after 24 h. The intermediate 1-amino-10-decanol accumulated in the first eight hours and was slowly aminated subsequently. The negative control E. coli W3110/pTrc99A did not produce any 1,10-diaminodecane (data not shown). 3.5.2. Redox cofactor recycling The three-enzyme-cascade allows redox self-sufficient amination of alcohols in principle. To test if this cascade functions under redox self-sufficient conditions in the whole cell biotransformation setup, conversion and specific production rates were determined in the presence or absence of various supplements with 0.5 g cell dry weight per L of E. coli W3110/pTrc99A-ald-adh-ta and 1.7 g/L 1,10dodecanol (Fig. 5, Table 3). The addition of 250 mM L-alanine,

3.4. The role of the employed alcohol dehydrogenase for substrate selectivity of the whole cell biotransformation The whole cell biotransformation described here showed substrate preference of certain alcohols, in particular 1-hexanol, 1,10-decanediol and benzylalcohol. Besides constraints in substrate uptake, toxicity of intermediates or export of products, the substrate spectrum of the enzymes in the cascade might impair conversion. To determine if the substrate spectrum of the first enzyme of the cascade, the alcohol dehydrogenase, limits the substrate range in the whole cascade, cell extracts of IPTG-induced E. coli W3110/pTrc99A-adh cells were assayed for alcohol oxidation using various alcohols as substrates. Due to low solubility of C10 and C12 linear (di)alcohols only C6 and C8 mono- and dialcohols were tested and Km and Vmax values were estimated and catalytic efficiency was calculated as Vmax/Km. Oxidation of cyclohexanol was not detected (Table 2), which indicates that cyclohexanol is only a very poor substrate of the employed alcohol dehydrogenase and may explain why no amination of cyclohexanol in the whole cell biotransformation was observed. On the other side, the employed alcohol dehydrogenase was not the bottleneck explaining poor amination of 1,6-hexanediol of 2-hexanol in the whole cell biotransformation since both substrates were oxidized using crude extracts of E. coli W3110/pTrc99A-adh (Table 2).

Figure 4. Influence of the position of ald, adh and ta in the artifical operon on the 1,10-diaminodecane production. The positions of ald, adh and ta were rotated in W3110/pTrc99A-ta-ald-adh compared to W3110/pTrc99A-ald-adh-ta and cells which have overexpressed the genes were incubated with 10 mM 1,10-decanediol in a resting buffer system. Conditions: 50 mM Hepes pH 6, 0.35 mM PLP, 0.75 mM NAD+, 275 mM NH4Cl; solid lines, 1,10-diaminodecane; dashed lines, 1-amino-10decanol; closed circles, W3110/pTrc99A-ald-adh-ta; closed triangles, W3110/ pTrc99A-ta-ald-adh.

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0.75 mM NAD+, 0.35 mM PLP and 275 mM NH4Cl resulted in 100% conversion after 12 h corresponding to the highest production rate (0.06 g/g⁄cdwh) (Table 3, No. 1). Neither addition of NAD+ nor of pyridoxal-5-phosphate was required, but their combined lack reduced the production rate (Table 3, No. 2–4). Thus, efficient redox selfsufficient amination was achieved in the whole cell biotransformation setup. 3.5.3. Nitrogen source for the amination cascade L-Alanine is the amino donor of x-transaminase and might be recycled to some extent by L-alanine dehydrogenase using ammonium chloride for reductive amination of pyruvate. Running the cascade only with 250 mM NH4Cl supply did not yield diamine. The absence of ammonium chloride reduced 1,10-diaminodecane formation to 60% conversion and about 40% remained as intermediate 1-amino-10-decanol while it accumulated only transiently under the other conditions (Table 3, No. 5–6, Fig. 5). Thus, while amination did not occur in the absence of L-alanine, both ammonium chloride and L-alanine contributed to amination by the whole cell system. 3.5.4. Optimization of reaction conditions To determine if the concentrations of L-alanine and NH4Cl as nitrogen donors can be reduced, various combinations of L-alanine and ammonium chloride were tested. When various ammonium chloride concentrations were added to 250 mM L-alanine, complete conversion was achieved with 50 to 200 mM ammonium chloride, but the conversion rate dropped to 0.02 g/g⁄cdwh (Table 3, Nos. 7–9 as compared to No. 4). Since increasing the ammonium concentration to 400 mM reduced conversion to 69% (Table 3, No.10), 100 mM ammonium chloride in combination with 0– 400 mM L-alanine were tested (Table 3, Nos. 11–14). Near-complete conversion (96%) was observed for 100 mM L-alanine and 100 mM NH4Cl but 400 mM L-alanine and 100 mM NH4Cl let to highest maximal production rate (0.05 g/g⁄cdwh). With 100 mM L-alanine the NH4Cl concentration could be reduced 50 mM while maintaining complete conversion. However, 250 mM L-alanine and 275 NH4Cl are favored since complete conversion was reached faster.

Figure 5. Proof of principle of the three enzyme cascade and the cofactor recycling. Different conditions were chosen to proof the redox self-sufficient amination of 10 mM 1,10-decanediol by W3110/pTrc99A-ald-adh-ta in a resting buffer system consisting of 50 mM Hepes pH 6. Quantification of 1,10-diaminodecane (solid lines) and 1-amino-10-decanol (dashed lines); circles: 250 mM alanine, 0.75 mM NAD+, 0.35 mM PLP, 275 mM NH4Cl; triangle upside down: 250 mM alanine, 0.35 mM PLP, 275 mM NH4Cl; squares: 250 mM alanine, 0.75 mM NAD+, 275 mM NH4Cl; diamonds: 250 mM alanine, 275 mM NH4Cl; triangles: 250 mM alanine; cross: 275 mM NH4Cl.

4. Discussion 4.1. Cascading enzymes in whole cell biotransformation In whole cell biotransformation the use of several enzymes for complex substrate substitution is challenging because of the enzymes´ demand of specific reaction conditions like pH and temperature. However, efficient in vivo and in vitro cascading enzyme has been demonstrated and conditions to ensure that all enzymes are active for product formation were found for a number of multienzyme cascades.15,21 In this study, cascading of an alcohol dehydrogenase, a transaminase and an alanine dehydrogenase was shown to sustain redox-self sufficient amination of mono- and dialcohols in vivo even though pH and temperature optima varied among the employed enzymes.22,23 In whole cell biotransformation homeostasis of the host’s intracellular pH determines that the biotransformation occurs at about pH-neutral conditions and requires enzymes to be active at the host’s intracellular pH. To adjust the interplay between the cascaded enzymes their synthesis may be balanced, for example, to overcome cofactor limitations in redox biocatalysis.24 When two artificial operons with different gene order of ald, adh and ta in the two strains W3110/ pTrc99A-ald-adh-ta and W3110/pTrc99A-ta-ald-adh only W3110/ pTrc99A-ald-adh-ta led to 100% conversion of 1,10-decanediol to 1,10-diaminodecane within eight hours (Fig. 4). Based on the enzyme activity assays of crude extracts of these strains, AlaDH activity was comparable, but ADH and TA activities were about ten-fold and about three fold, respectively, higher in W3110/ pTrc99A-ald-adh-ta than in W3110/pTrc99A-ta-ald-adh. Commensurate with higher AlaDH and TA enzyme activities transient accumulation of the intermediate 1-amino-10-decanol was less pronounced using strain W3110/pTrc99A-ald-adh-ta (Fig. 4). Substrate specificities of cascaded enzymes pose a challenge to develop broad substrate-spectrum biotransformation systems, but may also help to avoid side products in very specific conversions. As described here, the W3110/pTrc99A-ald-adh-ta-based whole cell biotransformation of 1,6-hexanediol and 2-hexanol did not yield 1,6-diaminohexane and very low concentration of 2-hexylamine, respectively, although the alcohol dehydrogenase from B. stearothermophilus used here exhibited the highest catalytic efficiency (expressed as Vmax/Km-value and measured in crude extracts) for both substrates among the tested alcohols (Table 2). By contrast, complete biotransformation of 1-hexanol to 1-hexylamine was possible although the catalytic efficiency of alcohol dehydrogenase for 1-hexanol was even two-fold lower than that for 1,6-hexanediol. Thus, alcohol dehydrogenase activity did not limit 1,6-hexanediol and 2-hexanol biotransformation which suggests that transaminase specificity might limit amine formation in this cascade. The opposite case was shown for the amination of cyclohexanol to cyclohexylamine where no product formation could be detected by resting W3110/pTrc99A-ald-adh-ta but xtransaminase of V. fluvialis is proposed to aminate cyclohexanone to cyclohexylamine.25 Here, the alcohol dehydrogenase from B. stearothermophilus might have not oxidized cyclohexanol (Table 2) as already demonstrated elsewhere.23 Comparing the in vivo and in vitro data it can be concluded that substrate specificities of the cascaded enzymes is responsible for product formation in vivo rather than substrate or product toxicity to the whole cell biocatalyst (Fig. 2). 4.2. Employment of transaminases in biocatalysis Among the transaminases x-transaminases are known to have a broader substrate spectrum than a-transaminases.10,26 Thus, xtransaminases are generally preferred for broad substrate-spectrum biotransformations, but their wide usage in biocatalysis faces

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S. Klatte, V. F. Wendisch / Bioorg. Med. Chem. xxx (2014) xxx–xxx Table 3 Conversion and maximal specific production rate of 1,10-diaminodecane production by W3110/pTrc99A-ald-adh-ta under varying conditions Nr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Biotransformation condition +

250 mM alanine, 0.35 mM PLP, 0.75 mM NAD , 275 mM NH4Cl 250 mM alanine, 0.35 mM PLP, 275 mM NH4Cl 250 mM alanine, 0.75 mM NAD+, 275 mM NH4Cl 250 mM alanine, 275 mM NH4Cl 0 mM alanine, 275 mM NH4Cl 250 mM alanine 250 mM alanine, 50 mM NH4Cl 250 mM alanine, 100 mM NH4Cl 250 mM alanine, 200 mM NH4Cl 250 mM alanine, 400 mM NH4Cl 0 mM alanine, 100 mM NH4Cl 50 mM alanine, 100 mM NH4Cl 100 mM alanine, 100 mM NH4Cl 400 mM alanine, 100 mM NH4Cl 100 mM alanine, 50 mM NH4Cl 100 mM alanine, 200 mM NH4Cl 100 mM alanine, 400 mM NH4Cl

Conversion [%]

Maximal spec. production rate [g/gcdw h]

100a 100a 90a 100a 0 60a 100b 100b 100b 69b 0 75a 96b 92a 100b 100b 70b

0.06 ± 0.003 0.06 ± 0.002 0.05 ± 0.002 0.05 ± 0.003 0 0.03 ± 0.002 0.02 ± 0.001 0.02 ± 0.001 0.02 ± 0.002 0.02 ± 0.008 0 0.03 ± 0.009 0.02 ± 0.001 0.05 ± 0.0004 0.02 ± 0.0004 0.02 ± 0.002 0.02 ± 0.008

Maximum 1,10-diaminodecane production after 12 ha, 30 hb.

some challenges. Equilibria of x-transaminase reactions are not well understood but can influence product formation significantly.26,27 For the amination of acetophenone to a-methylbenzylamine by x-transaminase from Bacillus thuringiensis with alanine as amino donor an equilibrium constant K of 0.00088 was calculated.28 By contrast, a K value of around 26 was calculated the transamination of 4-phenyl-2-butylamine with cyclohexanone to 4-phenyl-2-butanone and cyclohexylamine by a x-transaminase ATA041 from cLecta (Leipzig, Germany).25 Different strategies e.g. co-product removal may help to overcome the equilibrium which was demonstrated for amination of acetophenone to a-methylbenzylamine catalyzed by the x-transaminase of V. fluvialis.27 Additionally, it was demonstrated that pyruvate inhibits the employed transaminase from V. fluvialis and its removal improved product formation.20,22,29,30 In the study described here, reductive amination of pyruvate by L-alanine dehydrogenase also brought about co-product removal and might have promoted product formation. As alternative to co-product removal enzymes more resistant to inhibition may be selected by directed evolution.31 In contrast to pyruvate high concentrations of the amino group donor L-alanine were necessary for complete conversion to 1,10diaminodecane by resting W3110/pTrc99A-ald-adh-ta (Table 3) and apparently did not inhibit transaminase activity. With L-alanine as substrate the x-transaminase from Chromobacterium violaceum showed 16.5-fold higher intial rates than the x-transaminase from V. fluvialis chosen here.10 In this study, 100% conversion was possible when reducing the L-alanine concentration to 100 mM, but to further reduce the concentration of this supplement employment of a different x-transaminase with higher affinity for L-alanine is desirable.

have been successfully applied by cascading with recycling enzymes.36,37 In this study, the interplay of two redox reactions which connects initial substrate oxidation and the recycling of the cofactor NAD+ were used in a whole cell biotransformation process as an extension of cell-free biocatalysis.12 Mainly, cofactor regeneration, stable environment for the enzymes and the lack of the cofactors PLP and NAD+ were beneficial in the whole cell system. It could be shown that only L-alanine and NH4Cl were sufficient to keep the conversion of 1,10-diaminodecane to 100%. High activity of L-alanine dehydrogenase was required since less conversion was observed with E. coli W3110/pTrc99A-ta-ald-adh than with W3110/pTrc99A-ald-adh-ta and the intermediate 1amino-10-decanol accumulated (Fig. 4).

4.3. Cofactor recycling for the redox self-sufficient amination of alcohols

We would like to acknowledge Drs. Philip Engels, Jan Pfeffer and Thomas Haas (Evonik Industries AG) for provision of strains and their collaboration within the BMBF-cofunded BioIndustrie 2021 Project, Biooxidations- und Aminierungstechnologie als Plattform für funktionelle Amine als Monomerbausteine’.

The regeneration of cofactors for redox-reactions avoids their costly addition in redox biocatalysis. The employment of dehydrogenases for the recycling of redox-equivalents like NAD+ and NADP+ was introduced successfully in whole cell biotransformation and cell-free biocatalysis.12,14,32 In addition, in whole cell biotransformation the host´s metabolism may be used for cofactor regeneration by catabolism of e.g. glucose and cofactor supply can be further increased by genetic engineering of the biocatalyst.33–35 Different recycling strategies like the oxidation of a second substrate or the reduction of an oxidized intermediate to recycle the desired reduced or oxidized state of the cofactor

5. Conclusions The constructed whole cell biocatalyst E. coli W3110/pTrc99Aald-adh-ta allowed amine functionalization of primary aliphatic mono- and di-alcohols and aromatic alcohols. Best results have been obtained for 6-hexanol, 1,10-decanediol and benzylalcohol but also 8-octanol, 1,8-octanediol, 1,12-dodecanediol were aminated. During amination of dialcohols, accumulation of only low aminoalcohol concentrations indicated efficient NAD+ recycling and, most probably, removal of inhibitory pyruvate by the alanine dehydrogenase. In comparison to cell-free biocatalysis cofactors NAD+ and PLP were not required for complete conversion. Moreover, the biocatalyst can easily be prepared by growing E. coli W3110/pTrc99A-ald-adh-ta and by inducing enzyme production. Acknowledgments

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