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Bio-derived Production of via a Three Step Biocatalytic Cascade and Metabolic Engineering

DOI: 10.1039/C7GC03325G

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Citation for published version (APA): Klumbys, E., Zebec, Z., Weise, N., Turner, N., & Scrutton, N. (2018). Bio-derived Production of Cinnamyl Alcohol via a Three Step Biocatalytic Cascade and Metabolic Engineering. Green Chemistry. https://doi.org/10.1039/C7GC03325G Published in: Green Chemistry

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Bio-derived Production of Cinnamyl Alcohol via a Three Step Biocatalytic Cascade and Metabolic Engineering

6Received 00th July 20xx, a a a a ,a Accepted 00th July 20xx Evaldas Klumbys, Ziga Zebec, Nicholas J. Weise, Nicholas J. Turner, Nigel S. Scrutton*

DOI: 10.1039/x0xx00000x www.rsc.org/

The construction of biocatalytic cascades for the production of media, sourcing of catalysts from renewable feedstocks and chemical precursors is fast becoming one of the most efficient chemo-, regio- and enantioselectivity. These features often approaches to multi-step synthesis in modern chemistry. enable the efficient combination of multiple enzymes within a However, despite the use of low solvent systems and renewably- single reaction vessel under common conditions without resourced catalysts in reported examples, many cascades are still unwanted cross-reactivity or the need for complex and dependent on petrochemical starting materials, which as of yet wasteful purification of intermediate compounds.3-6 Despite cannot be accessed in a sustainable fashion. Herein we report the this, a large number of biocatalytic routes make use of starting production of the versatile chemical building block cinnamyl materials obtained from traditional sources, such as alcohol from the primary metabolite and fermentation product L- petroleum. As such, there is a drive to find methods employing phenylalanine. Through the combination of three biocatalyst biocatalysts for the conversion of sustainably-resourced classes (phenylalanine ammonia lyase, carboxylic acid reductase substrates to products associated with multiple and / or and ) the target compound could be industrially-important syntheses. reached in high purity, demonstrable at 100 mg scale achieving 53 Cinnamyl alcohol is a simple versatile chemical implicated in % yield using ambient temperature and pressure in aqueous the production of various compounds of applied and solution. This system represents a synthetic strategy in which all commercial interest. Simple esterification of this alcohol can components present at time zero are biogenic and thus afford a variety of cinnamyl esters, which find use in the minimising damage to the environment. Further we extend this flavour and fragrance industries,7, 8 as well as being used as biocatalytic cascade by its inclusion in a L-phenylalanine precursors for the production of smart polymer materials.9 overproducing strain of Escherichia coli. This metabolically Examples of these include cinnamyl acetate, which is used to engineered strain produces cinnamyl alcohol in mineral media confer spicy and floral aromas, and cinnamyl methacrylate - a using a glycerol and glucose as carbon source. This study monomer which can be polymerised by both radical and demonstrates the potential to establish green routes to the photochemical means to create a dual crosslinked product. synthesis of cinnamyl alcohol from a waste stream such as glycerol The amination of cinnamyl alcohol has also been reported, derived, for example, from lipase treated biodiesel . allowing the facile production of various cinnamyl amines.10-12 Notable examples of these include the clinically approved In an effort to widen the remit of green chemistry in the drugs flunarizine and naftifine (used for the treatment of production of materials, additives and pharmaceuticals, fungal infections and peripheral vascular conditions, several avenues are being explored to reduce the long-term respectively). As well as one-step conversions yielding environmental impact often incurred in such processes. One compounds of interest, cinnamyl alcohol is also reported as a strategy that has seen sustained attention is recent years is starting material for the multistep synthesis of the drug biocatalysis: the use of enzymes to complement and replace dapoxetine and a widely used cancer treatment drug taxol 1, 2 chemical routes to various target molecules. The advantages (Figure 1).13, 14 of using enzymes as opposed to organic synthetic methods The current provision for bio-derived production of cinnamyl include mild reaction conditions, compatibility with aqueous alcohol is poor, due to the low levels in which it occurs naturally as a metabolite. As such, synthesis of this compound usually involves the chemoselective reduction of the corresponding aldehyde using chemical reducing agents, finite and expensive metals or complex catalyst formulations, such as nanotubes or nanoparticles.15-18 Chemical reduction of allyl

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COMMUNICATION Journal Name alcohols often requires fine control of reaction conditions, due Results and discussion to the possible non-selective reduction of both the hydroxyl 19 As an initial test of the feasibility of the three enzyme cascade and ene functionalities. Whilst there are examples of alcohol proposed in Figure 2, separate biotransformations were set up dehydrogenase (ADH) enzymes used to perform this same 20, 21 to represent the first two steps. To this end the PAL from the reaction in a more mild and chemoselective fashion, cyanobacterium Anabaena variabilis was chosen due to its access to is not as easy as various primary Table 1. Effect of catalyst loading on the conversion of L-phenylalanine (1) to trans- (2) by PAL[a]

Catalyst Loading / mg % composition in aqueous phase[b] mL-1

1 2

0 >99 <1

1 44 56

2 36 74

3 18 82

4 13 87

5 10 90 [a]1–5 mg of lyophilised E. coli cells producing AvPAL, 100 mM potassium phosphate buffer (pH 7.5), 10 mM L-Phe, final volume 1 ml, 30 °C, 250 rpm vertical shaking, 24 h.[b]determined via reverse-phase HPLC analysis on a non-chiral phase.

extensive use in biocatalytic literature.24-26 Previous studies report the use of this enzyme within lyophilised whole cells. As such a pET-16b plasmid containing the codon-optimised avpal gene was used to transform E. coli BL21(DE3) cells and subsequently produce the dry whole cell formulation as

previously reported.26 Reactions with varying quantities of Figure 1.Examples of industrially-relevant chemicals which can be synthesized from AvPAL incubted for 24 hr at 30oC with 10mM L-phenylalanine cinnamyl alcohol. Cinnamyl amines including naftifine and flunarizine,10-12 cinnamyl 7, 8 showed increasing conversions up to 90% as seen by reverse- esters including various fragrances and photocrosslinking monomers, multi-step synthesis of compounds such as dapoxetine and taxol.13, 14 phase HPLC (Table 1). Next a CAR from Mycobacterium marinum (MCAR) was metabolites, widely available as fermentation products. To this produced in its active form via co-transformation of the end, we envisaged the addition of well-documented carboxylic encoding gene from pET-16a vector with that for a acid reductase (CAR)22, 23 and phenylalanine ammonia lyase phosphopantetheinyl transferase from Bacillus subtilis in (PAL) biocatalysts to allow production directly from the pCDF-1b. This system was deemed suitable for cascade proteinogenic amino acid L-phenylalanine (L-Phe) (Figure 2). construction, due to its successful incorporation into a reported method for the multi-step enzymatic production of chiral piperidines.27 Initial studies of the whole lyophilised cells with trans-cinnamic 2 acid gave only 10 % conversion to the corresponding aldehyde. Indeed the major products formed gave a mass spectrum consistent with cinnamyl alcohol 4 and 3-phenylpropanol 6. It could be envisaged that these products were formed via the action of CAR and a host cell ene- reductase in either order, followed by host cell aldehyde reduction. It has been demonstrated that E. coli ADHs have a broad specificity28 and reduce cinnamaldehyde as defence Figure 2.A biocatalytic route to cinnamyl alcohol (4) from bio-derived L-phenylalanine mechanism.29 In addition various ene-reductases are known (1) using a combination of phenylalanine ammonia lyase (PAL), carboxylic acid for their high affinity towards cinnamaldehyde.30 This situation reductase (CAR) and alcohol dehydrogenase (AHD) enzymes. Deamination reaction does not require any additional cofactors however the following both reductions need was found to be remedied (in part) by the use of purified NADPH and ATP for CAR enzyme activity and either NADPH or NADH for ADH. MCAR, with 84 % conversion of 2 seen, giving a product ratio of ~20:1 (3:4) with steady state kinetic parameters exhibited in

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Journal Name COMMUNICATION table 2. This was possibly due to the presence of co-purified E. The presence of the unwanted alcohol 6 and higher coli ADH enzymes and / or use of a glucose dehydrogenase percentage of 2 in the cascade reaction were presumably due (GDH) with low activity for the aldehyde. Evidence of the to the increased overall cell loading (with associated ene- action of endogenous ADH enzymes in both the whole cell reductase activity) and lower stability of CAR and/or ADH systems prompted investigation of PAL-CAR combinations to enzymes ascertain whether cinnamyl alcohol could be produced Table 2. Conversion of 1, 2 and 3 by combinations of PAL, CAR and GDH without the addition of a third biocatalyst. The use of both biocatalysts.[a] AvPAL and MCAR in whole cell formulations was indeed found to yield both the unsaturated alcohol 4 and saturated by- product 6 at a ratio of ~1:2.4 within the extracted organic layer, with the rest predominantly staying as the acid 2. A more favourable ratio of ~4:1 (4:6) could be achieved through the use of purified MCAR in the presence of an additional GDH-based cofactor recycling system (Table 2). However, the conversion was found to be lower when using PAL enzyme in lyophilised and MCAR in purified enzyme form compared to both enzymes in lyophilised cells. The apparent reduction of Biocatalyst(s) Substrate % composition in extracted phase[b] ene-reductase activity within the system was expected due to 2 3 4 5 6 the lower cell mass used for this biotransformation. Further [c] CAR 2 <1 11 33 <1 56 investigation revealed the low impact of cinnamaldehyde [d] reduction by the GDH recycling system (~1 % conversion of the CAR 2 17 79 4 <1 <1 substrate). PAL/CAR[e] 1 18 1 57 <1 24 In order to ensure selective recovery of cinnamyl alcohol from [f] the reaction mixture, the conversion of trans-cinnamic acid to PAL/CAR 1 45 <1 44 <1 11 the alcohol product was studied further with a combination of GDH[g] 3 - 99 1 <1 <1 MCAR and a commercially-available ADH from Saccharomyces Steady state kinetic parameters for conversion of 2 to 3 using MCAR.[h] cerevisiae. It was reasoned that addition of this enzyme to -1 -1 -1 kcat (min ) KM (mM) KI (mM) kcat/KM (min mM ) biotransformations would act to remove cinnamaldehyde 184.54 ± 8.9 0.424 ± 0.059 24.2 ± 5.3 435.24 produced by CAR at a higher rate, pushing the overall [a]general reaction conditions containing PAL: 100 mM potassium phosphate conversion higher and minimising the effect of ene-reductase buffer (pH 7.5), 5 mM substrate, 30 °C, 250 rpm vertical shaking. For any other - enzymes, where whole cells were used. The use of 0.25 mg mL reactions containing CAR enzymes additionally or GDH 10 mM MgCl2, 15 mM D- 1 -1 ScADH in conjunction with 1 mg mL whole cells harbouring glucose, 10 mM ATP, 10 U GDH, 500 µM NADP+ were added. [b] determined via MCAR was found to give increasingly high consumption of GC-MS on a non-chiral phase. [c] CAR biocatalyst used as a whole lyophilised cell -1 cinnamate (>90% after 2 hours) to the desired alcohol, with a formulation (1 mg mL ) [d] CAR biocatalyst used as an isolated enzyme formulation (2 µM) [e] CAR and PAL biocatalyst used as a whole lyophilised cell small amount of cinnamaldehyde also being detected after 8 formulation (1 and 3 mg mL-1 respectively) [f] CAR biocatalyst used as an isolated and 24 hour time points. Increasing the MCAR whole cell enzyme formulation and PAL as whole lyophilised cell formulation (2 µM and 3 -1 biocatalyst component to 2.5 mg mL was found to give mg mL-1) [g] GDH biocatalyst used in recycling reaction quantities. The almost complete conversion after 2 hours with only traces of conversions were calculated according to peak area on GC device. The cinnamate or cinnamaldehyde. Longer reaction times (4, 8 and conversions of PAL are not accounted in this table and supposed to be around ~70 % known from Table 1. [h] MCAR kinetic parameters were recorded by 24 hours) resulted in accumulation first of cinnamaldehyde monitoring the rate of NADPH oxidation at 340 nm. Reaction conditions: 100 and then 3-phenylpropanol contaminants (Figure 3). These mM KH2PO4/K2HPO4, pH 7.5, 10 mM MgCl2, 1 mM ATP, 200 µM NADPH, 0.3 µM results indicate the importance of time course assays in the MCAR enzyme, 30 °C. development an optimised cascade procedure and possibly highlight issues with the reversibility of ScADH in the 100% mitigation of side product formation. 90% Having demonstrated the possibility of high conversion with 80% minimal side reactions, the system was extended to include 70% 4 AvPAL starting from L-phenylalanine. The effect of varying the 60% 6 50% AvPAL whole cell loading on the overall composition of the 3 40% 2

volatile reaction intermediates / products could be easily Composition 30% tested by extraction of these from any remaining 20% phenylalanine in organic solvent. The full three enzyme 10% reaction was performed both as a one-pot cascade and by 0% implementation of the PAL and CAR-ADH portions under 2.5 1 2.5 1 2.5 1 2.5 1 2.5 1 2.5 1 temporal separation (figure S1). Overall the partition reaction 30 60 120 240 480 1440 gave better results with higher percentage compositions of 4, CAR loading / mg no by-product 6 and only small amounts of 2 or 3 detected. Time / min

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Figure 3.The effect of catalyst loading on composition of CAR-ADH cascade reactions 10 U GDH, 0.05 mg/ml ADH, 500 µM NADP+, 500 µM NAD+ final volume 1 ml at 30 °C, over time. Reaction performed in 100 mM potassium phosphate buffer (pH 7.5) 5 mM 250 rpm. trans-cinnamic acid, 10 mM MgCl2, MCAR dried cells, 30 mM D-glucose, 10 mM ATP,

100

90

80

70

60 1 50 2 40 Percentage% / 30 4

20

10

0 0 30 60 120 240 420 480 1320 1330 1350 1380 1500 1650 1680 Time / min Figure 4.Composition profile for the conversion of 1 to 4 via addition of PAL (t=0)performed in 100 mM potassium phosphate buffer (pH 7.5), 3 mg/ml of AvPAL dried cells and 10 -1 mM L-Phe (107 mg) at 30 °C, 250 rpm 65 ml final volume. (t=1320) reaction mixture was spun down and supernatant supplemented with 10 mM MgCl2, 1 mg mL MCAR dried cells, 30 mM D-glucose, 10 mM ATP, 10 U GDH, 0.05 mg/ml ADH, 500 µM NADP+, 500 µM NAD+ final volume 130 ml at 30 °C, 250 rpm.

A) B)

Cinnamyl-alcohol Cinnamaldehyde Cinnamic-acid 24h 48h 72h 400 400 350 350 300 300 -1 250 -1 250 mg L 200 mg L 200 150 150 100 100 50 50 0 0 TB-24h TB-48h TB-72h TB M9 M9-Gly M9-Glu

Figure 5. In-vivo production of cinnamyl alcohol in E. coli NST using the plasmid pZZ-Eva2, measured by GC after 24h, 48h AND 72h. (A) Production in TB media, and (B) production of cinnamyl alcohol comparing different carbon sources in M9 media. Error bars represent standard deviation (SD) of three biological replicates.

compared to AvPAL. In an attempt to increase flux through the was investigated, demonstrating that a molar ratio of 1:2 second part of the partitioned reaction, loading of ScADH was (substrate to ATP) was sufficient for conversion of the trans varied between 0.025 to 0.25 mg mL-1 with either a single or cinnamic acid to cinnamyl alcohol. Lower molar ratios of double batch addition at t=0 min and t=300 min (Figure S2). substrate to ATP result in insufficient conversion (Figure S3), These experiments revealed a siphoning effect resulting in which might be related to the substrate stability or to lower increased purity of 6 for higher ADH concentrations, with catalytic activity of enzymes. batch addition also giving lower percentage compositions of 2 Furthermore a full time course experiment was performed for and 3. Additionally, the effect of altering ATP concentration the two biocatalytic systems using a combination of reverse

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36 phase HPLC (to monitor the initial PAL reaction) and GC (to reaction components, might present a future green and follow product formation with the CAR-ADH cascade).The economically viable solution. reaction was performed with 107 mg (in 65 ml) as starting material around 85% being converted to 2 after a 22 hour PAL We have also demonstrated that metabolic engineering in E. coli reaction before complete double reduction (volume increased NST can be applied to produce cinnamyl alcohol from analytical 33 to 130 ml after PAL separation) after a further 5.5 hours grade glucose and / or glycerol. Crude glycerol is a by-product of (Figure 4). The reaction was performed in sequential manner biodiesel production and is therefore a potential attractive carbon separating PAL and CAR with ADH reactions. 6 hours after source for E. coli37 in metabolic engineering programmes that aim addition of the second and third biocatalysts, the product to establish green routes to the production of valuable chemicals. could be easily extracted and purified using flash Most crude glycerol is derived from alkali treatment of biodiesel. chromatography from the remaining L-phenylalanine, with an Crude glycerol however contains different contaminants that might isolated yield of 43.5 mg (molar yield 53 %). The cinnamyl interfere with fermentation. However, lipase treatment of 1 13 alcohol was confirmed by H and C NMR as well as GC-MS biodiesel38 yields a multitude of different plant derived oils and has chromatogram and ionisation spectrum. The high resolution 39 reached industrial production levels. Glycerol as a by-product of MS however did not show any ionisation pattern (Figure S4). 40 enzymatic (lipase) biodiesel production, is easily removable, In order to convert the L-phenylalanine to cinnamyl alcohol in an contains reduced levels of contaminants,41 and should therefore be economical and environmentally friendly way, the enzymatic directly suitable as a green carbon source for the production of reactions were transferred from an in vitro setup into an in vivo cinnamyl alcohol. system. The substrate, L-phenylalanine was produced by E. coli NST strain31 using glycerol / glucose mixture as carbon source (1 g L-1).32

Additionally, E. coli NST was transformed with the vector pZZ-Eva2 (Fig. S6) enabling the new strain to produce cinnamyl alcohol in TB media. Production was measured by the extraction of cinnamyl Conclusions alcohol from the culture medium using , further The chemical building block cinnamyl alcohol has many uses in derivatization and final quantification performed by GC, following the literature as a precursor to various fragrance compounds, 24h, 48h and 72h of cell growth (Figure 5a). A maximum of 300 mg smart materials and commercially-available pharmaceuticals. cinnamyl alcohol per litre of culture was produced after 24h of Through use of a three enzyme cascade, we have 32 incubation, with an estimated 30 % conversion of L-phenylalanine demonstrated the simple production of this compound in good to cinnamyl alcohol. Cinnamyl aldehyde production was constant at yield and high purity using biocatalytic functional group -1 75mg L over the entire 72h, while the production of trans- interconversion. The optimised method has several attractive cinnamic acid increased over the course of the experiment to a features including a bio-derived starting material (L- maximum of 65 mg L-1. Alternatively we tested the production in phenylalnine), renewably resourced catalysts (phenylalanine mineral M9 media, supplemented with the same glycerol / glucose ammonia lyase, carboxylic acid reductase and alcohol mix and with either glycerol or glucose as carbon source (Figure 5b dehydrogenase enzymes), ambient, low energy reaction The production of cinnamyl alcohol in M9 media was the highest conditions and facile, inexpensive extraction of the final with glucose as carbon source (80 mg L-1), followed by production product. This method opens up routes to the conversion of with the glycerol/glucose mix (40mg L-1), and glycerol as the sole biomass to useful products in an industrial setting as well as carbon source yielded (20mg L-1). Regardless of the relatively low synthetic biology approaches to create designer organisms for cinnamyl alcohol titres, the major advantage of using mineral M9 the direct fermentative production of cinnamyl alcohol from primary metabolic processes. media is that no identifiable side product formation was observed in any of the biological replicas over the whole course of the experiment. Acknowledgements

Here we demonstrate cinnamyl alcohol production via a three step biocatalytic cascade and by metabolic engineering in E. coli NST. This work was funded by the Biotechnology and Biological The production of cinnamyl alcohol using the biocatalytic cascade Sciences Research Council (BBSRC) and Glaxo-SmithKline (GSK) might be advantageous as a result of the high loading capacity of under the Strategic Longer and Larger (sLoLa) grant initiative ref. BB/K00199X/1. NSS is an Engineering and Physical Sciences enzymes, which is consistent with their use with higher Research Council (EPSRC) Established Career Fellow. NJT concentrations of substrate / product. However, such an approach acknowledges the ERC for an Advanced Grant. is potentially disadvantageous regarding the additional cost and reduced simplicity in an industrial setting (e.g. attributed to biocatalyst preparation and cofactor supplementation). Notes and references Nevertheless recent improvements in the field of biocatalysis,34 1. R. Regil and G. Sandoval, Biomolecules, 2013, 3, 812-847. 35 particularly regarding coimmobilization of enzymes and cofactors 2. U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore and and / or using coimmobilization to enable reuse of different K. Robins, Nature, 2012, 485, 185-194.

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3. S. P. France, L. J. Hepworth, N. J. Turner and S. L. Flitsch, ACS Catalysis, 2016, acscatal.6b02979-acscatal.02976b02979. 4. S. Schoffelen and J. C. M. Van Hest, Current Opinion in Structural Biology, 2013, 23, 613-621. 5. E. Ricca, B. Brucher and J. H. Schrittwieser, Advanced Synthesis and Catalysis, 2011, 353, 2239-2262. 6. F. Lopez-Gallego and C. Schmidt-Dannert, Current Opinion in Chemical Biology, 2010, 14, 174-183. 7. D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim, J. M. Hanifin, A. E. Rogers, J. H. Saurat, I. G. Sipes and H. Tagami, Food and Chemical Toxicology, 2007, 45. 8. G. Reuss, W. Disteldorf, A. O. Gamer and A. Hilt, Ulmann's Encyclopedia of iIndustrial Chemistry, 2012, 15, 735-768. 9. D. Lerari, Macromolecular Research, 2015, 18, 1008-1014. 10. B. Emayavaramban, M. Roy and B. Sundararaju, Chemistry (Weinheim an der Bergstrasse, Germany), 2016, 22, 3952-3955. 11. H. Kanno and R. J. K. Taylor, Tetrahedron Letters, 2002, 43, 7337-7340. 12. T. Ohshima, Y. Miyamoto, J. Ipposhi, Y. Nakahara, M. Utsunomiya and K. Mashima, Journal of the American Chemical Society, 2009, 131, 14317- 14328. 13. C. Bonini and G. Righi, J. Chem. Soc., Chem. Commun., 1994, 2767-2768. 14. K. Venkatesan and K. V. Srinivasan, ARKIVOC, 2008, 2008, 302-310. 15. E. Castillejos, P. J. Deboutti??re, L. Roiban, A. Solhy, V. Martinez, Y. Kihn, O. Ersen, K. Philippot, B. Chaudret and P. Serp, Angewandte Chemie - International Edition, 2009, 48, 2529-2533. 16. H. Li, X. Chen, M. Wang and Y. Xu, Applied Catalysis A: General, 2002, 225, 117-130. 17. H. Ma, L. Wang, L. Chen, C. Dong, W. Yu, T. Huang and Y. Qian, Catalysis Communications, 2007, 8, 452-456. 18. T. Mitsudome, Y. Mikami, H. Funai, T. Mizugaki, K. Jitsukawa and K. Kaneda, Angewandte Chemie - International Edition, 2008, 47, 138-141. 19. P. Gallezot and D. Richard, Selective Hydrogenation of α,β-Unsaturated Aldehydes, 1998. 20. F. Chamouleau, C. Hagedron, O. May and H. Groger, Flavour and Fragrance Journal, 2007, 22, 169-172. 21. P. Zucca, M. Littarru, A. Rescigno and E. Sanjust, Bioscience, biotechnology, and biochemistry, 2009, 73, 1224-1226. 22. M. K. Akhtar, N. J. Turner and P. R. Jones, Proceedings of the National Academy of Sciences, 2013, 110, 87-92. 23. D. Gahloth, M. S. Dunstan, D. Quaglia, E. Klumbys, M. P. Lockhart-Cairns, A. M. Hill, S. R. Derrington, N. S. Scrutton, N. J. Turner and D. Leys, Nature chemical biology, 2017, 13, 975-981. 24. M. C. Moffitt, G. V. Louie, M. E. Bowman, J. Pence, P. Joseph and B. S. Moore, Biochemistry, 2008, 46, 1004-1012. 25. F. Parmeggiani, S. L. Lovelock, N. J. Weise, S. T. Ahmed and N. J. Turner, Angewandte Chemie (International ed. in English), 2015, 54, 4608-4611. 26. N. J. Weise, S. T. Ahmed, F. Parmeggiani, E. Siirola, A. Pushpanath, U. Schell and N. J. Turner, Catalysis Science & Technology, 2016, 6, 4086-4089. 27. S. P. France, S. Hussain, A. M. Hill, L. J. Hepworth, R. M. Howard, K. R. Mulholland, S. L. Flitsch and N. J. Turner, ACS Catalysis, 2016, 6, 3753- 3759. 28. S. Atsumi, T.-y. Wu, E.-m. Eckl, S. D. Hawkins, T. Buelter and J. C. Liao, 2010, 651-657. 29. J. Visvalingam, J. D. Hernandez-doria and A. Holley, 2013, 79, 942-950. 30. H. S. Toogood, D. Mansell, J. M. Gardiner and N. S. Scrutton, Elsevier Science, 1 edn., 2012, pp. 216-260. 31. D. E. Tribe, Google Patents, 1987. 32. N. Yakandawala, T. Romeo, A. D. Friesen and S. Madhyastha, Applied microbiology and biotechnology, 2008, 78, 283-291. 33. S. S. Konstantinović, B. R. Danilović, J. T. Ćirić, S. B. Ilić, D. S. Savić and V. B. Veljković, Chemical Industry and Chemical Engineering Quarterly, 2016, 19- 19. 34. M. Bilal, H. M. Iqbal, G. Shuqi, H. Hu, W. Wang and X. Zhang, International Journal of Biological Macromolecules, 2017. 35. C. Schmidt-Dannert and F. Lopez-Gallego, Microbial biotechnology, 2016, 9, 601-609. 36. S. Peirce, J. J. Virgen-Ortíz, V. G. Tacias-Pascacio, N. Rueda, R. Bartolome- Cabrero, L. Fernandez-Lopez, M. E. Russo, A. Marzocchella and R. Fernandez-Lafuente, RSC Advances, 2016, 6, 61707-61715. 37. P. Srinophakun, S. Reakasame, M. Khamduang, K. Packdibamrung and A. Thanapi, Chiang Mai J Science, 2012, 39, 59-68. 38. S. Hama, H. Noda and A. Kondo, Current Opinion in Biotechnology, 2018, 50, 57-64. 39. J. Price, M. Nordblad, H. H. Martel, B. Chrabas, H. Wang, P. M. Nielsen and J. M. Woodley, Biotechnology and bioengineering, 2016, 113, 1719-1728. 40. G. Vicente, A. Coteron, M. Martinez and J. Aracil, Industrial crops and products, 1998, 8, 29-35. 41. E. Garcia, M. Laca, E. Pérez, A. Garrido and J. Peinado, Energy & fuels, 2008, 22, 4274-4280.

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Bio-derived Production of Cinnamyl Alcohol via a Three Step Biocatalytic Cascade and Metabolic Engineering

Evaldas Klumbys, Ziga Zebec, Nicholas J. Weise, Nicholas J. Turner, and Nigel S. Scrutton

BBSRC/EPSRC Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology & School of Chemistry, University of Manchester, 131 Princess Street, M1 7DN, Manchester, UK

Table of contents Page

General methods ...... S2 Experimental procedures ...... S3 In-vivo production of Cinnamyl-alcohol in E. coli NST……………………………………………………………………………………………. S4 Analytical methods ...... S5 Supplementary data figures ...... S6 Characterisation data of 4 from preparative scale biotransformation ...... S15 References ...... S16

S1

General methods

Analytical grade reagents and solvents were obtained from Sigma-Aldrich, AlfaAesar or Fisher Scientific and used without further purification, unless stated otherwise. NADH and NADPH were acquired from Melford. ATP, L- Phenylalanine, trans-Cinnamic and trans-Cinnamaldehyde were purchased from Sigma-Aldrich. Restriction enzyme kits, expression vectors and laboratory strain E. coli were purchased from New England Biolabs (NEB). The L- phenylalanine over production strain, E. coli NST (ATCC 31882) was purchased from ATCC®. Microbiological media ingredients were obtained from ForMediumTM (LB, TB and M9) and prepared according to the recommended protocols provided. GDH (CDX-901) was kindly supplied by Codexis alcohol dehydrogenase from Saccharomyces cerevisiae (ScADH) (≥300 units/mg protein) was obtained from Sigma. Mycobacterium marinum carboxylic acid reductase (MCAR) and Bacillus subtilis phosphopantetheine transferase (Sfp) were provided in pET-21a and pCDF- 1b expression vectors respectively. Anabaena variabilis phenylalanine ammonia lyase (AvPAL) was provided in a pET-16b plasmid.

Experimental Procedures

Preparation of the lyophilised biocatalyst

A pET-16b expression plasmid containing the His6-tagged open reading frame for AvPAL was used as for previous studies1 and transformed into E. coli BL21(DE3) protein production strain (New England Biolabs) according to the supplier’s protocol. Expression of the gene encoding AvPAL was conducted according to previously reported methods.1 LB medium (5 mL, supplemented with kanamycin or ampicillin) was inoculated with a single colony of E. coli BL21(DE3) containing the suitable plasmid and grown for 16 h at 37°C and 250 rpm. This starter culture was then used to inoculate LB-based auto-induction medium2 (800 mL, supplemented with 50 µg mL-1 kanamycin), which was incubated at 18 °C and 250 rpm for 4 days. The cells were pelleted by centrifugation (4000 rpm, 12 min) and separated from the supernatant for storage of the wet cell mass at -20°C until further use. In the case of AvPAL a lyophilised dry cell powder formulation was used as reported previously.3 The isolated cell mass was flash frozen in liquid nitrogen and freeze dried using a Heto Power Dry LL1500 Freeze Dryer for 16-24 h. The dry cell mass was then ground into a fine powder and stored at –20°C until required.

MCAR Whole Cell Biocatalyst Preparation For pre-culture a single colony was grown in 100 ml either Terrific Broth (TB) or Lysogeny Broth (LB) containing 100 μg/ml ampicillin and 50 μg/ml streptomycin over night at 37 °C and 190 rpm. MCAR expression was followed as mentioned next. 5 ml of pre-culture was transferred to 500 ml TB containing the same concentration of antibiotics and grown at 37 °C and 180 rpm shaking. At exponential growth phase (OD600=0.6-0.8) cells were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside and incubated overnight (20h) at 20 °C and 180 rpm. Cells were pelleted (6,000 rpm, 8 min, 4°C) and washed with 0.5 % sodium chloride solution. The cells were then pelleted again (4,000 rpm, 8 min, 4 °C) and lyophilized using a Heto Power Dry LL1500 Freeze Dryer for 16-24 h. The dry cell mass was then ground into a fine powder and stored at –20°C until required.

Purification of MCAR E. coli cells with overexpressed MCAR PPant were lysed in 100 ml 50 mM potassium phosphate buffer pH 8.0, 500 mM NaCl and 10 mM imidazole buffer. Cells were disrupted by the French Press at 1500 psi and cell debris removed by centrifugation at ~48,000 x g, 4 °C for 1 h. Cell lysate was filtrated using 0.45 μm Minisart NML syringe filters (surfactant free cellulose acetate membrane) and loaded on Ni-IDA resin (Generon). Stepwise elution performed with 50 ml buffer at 4°C. The sample after Ni-IDA was concentrated by the combined use of Amicon stirred cell concentrator (76 mm, 100,000 MWCO polyethersulfone membrane Discs from generon) and Vivaspin

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20 (100,000 MWCO PES membrane GE Healthcare) at 2,800 x g up to 6 ml at 4 °C. 3 ml was loaded onto the HiLoad 16/60 Superdex 200 with 5 ml loop and isocratically eluted in 50 mM potassium phosphate buffer pH 8.0, 150 mM

NaCl at 1 ml/min flow rate at 4 °C. Eluent with high A280 absorbance was collected and appropriate fractions selected according to SDS-PAGE analyses. These were then concentrated using a Vivaspin 20 centrifugation tube (100,000 MWCO PES membranes from GE Healthcare) at 2,800 x g (4°C). The retained enzyme isolate was then frozen as several protein ball stocks by dropping protein solution slowly in liquid nitrogen for storage at -80 °C.

Reaction with AvPAL AvPAL reactions were performed in 1 ml volume containing 10 mM L-Phe, 100 mM potassium phosphate buffer (pH 7.5), 1 – 5 mg of AvPAL dried cells at 30 °C, 250 rpm vertical shaking and 24 h. 400 µl of sample was spun down and filtered using standard filter vial 0.45 µm PVDF (Thomson instrument company) for HPLC analysis.

CAR and CAR-ADH Biotransformations

Reactions were performed in 100 mM potassium phosphate buffer (pH 7.5) containing 10 mM MgCl2, 1 mg/ml MCAR dried cells, 15 mM D-glucose, 10 mM ATP, 10 U GDH, 500 µM NADP+ and 5 mM trans-cinnamic acid in 1ml volume. In addition, 0.05 mg/ml ADH, 500 µM NAD+ and 15 mM D-glucose were supplied to test the double reduction cascade. Biotransformations were incubated at 30 °C, 250 rpm vertical shaking. Following an appropriate reaction time, the mixture was acidified with 100 µl concentrated HCl and extracted using 2 x 400 µl ethyl acetate before drying with anhydrous magnesium sulphate. The sample was then derivatised using 200 µl methanol and 10 µl 2.0 M (Trimethylsilyl) diazomethane solution in hexane (28 °C, 60 min, 250rpm). Derivatization was terminated by addition of 2 µl glacial acetic acid before incubation for a further 20 min. Product and starting material were identified using GC and GC-MS to assign peaks, with conversions determined via comparison of integrated peak areas.

Preparative Scale Synthesis of Cinnamyl Alcohol The reaction was performed in 100 mM potassium phosphate buffer (pH 7.5) with 3 mg/ml of AvPAL dried cells and 10 mM L-Phe (107 mg, 65 ml final volume) in a round bottom flask (30 °C, 250 rpm). After sufficient conversion to cinnamic acid the reaction mixture was centrifuged to remove lyophilized AvPAL-containing cells and the supernatant separated and supplemented with 10 mM MgCl2, 1 mg/ml MCAR dried cells, 30 mM D-glucose, 10 mM ATP, 10 U GDH, 0.05 mg/ml ADH, 500 µM NADP+, 500 µM NAD+ (130 ml final volume). The new reaction mixture was then incubated at 30 °C with 250 rpm agitation until full conversion was reached. Monitoring of the AvPAL and CAR-ADH reactions was performed via sampling and HPLC / GC analyses as previously described. Following the complete biotransformation, the reaction mixture was centrifuged down at 4,000 rpm for 20 min and products extracted 3 times with ethyl acetate (1:1/v:v). The organic phase was dried with anhydrous magnesium sulphate, filtered and concentrated using rotary evaporator to obtain crude yellowish oil which solidified upon cooling to room temperature. The cinnamyl alcohol product was then purified by flash chromatography using a silica gel column (pore si e 0 Å, 220-440 mesh particle size, 35-75 µm particle size) with isocratic elution (dichlomethane:methanol - 98:2). This yielded colourless oil which solidified upon cooling. A small 1 13 sample of the isolated product was dissolved in 800 µl CDCl3 for H- and C-NMR analyses. These were recorded using Bruker Biospin instrument operating at 400 MHz.

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In-vivo production of cinnamyl-alcohol in E. coli NST

Molecular cloning

In order to convert L-phenylalanine to cinnamyl alcohol, we constructed the plasmid pZZ-Eva2 (Figure S6) that contains the most efficient genes/parts (srPAL, ADH/KRED, CAR11, Sfp and P-Rham) determined by biocatalysis. First, each part was separately amplified from the corresponding expression vector by polymerase chain reaction (PCR) using the Phusion-polymerase (NEB). The parts were gel purified (Macherey-nagel) and DpnI digested (NEB), always following the manufacturers protocol. Next, we fused srPAL-ADH/KRED and P-Rham-Sfp using overlap extension PCR (OE-PCR) to form a single part,4 resulting in a total of three parts (srPAL-ADH/KRED =2704bp, CAR= 5 3636bp and P-Rham-Sfp =881bp). Finally, inverse PCR was employed to linearize the plasmid backbone pBbE8kRFP. All three parts and the backbone were fused together to form a single vector (Figure S6) in a molar ratio 2:2:2:1 using the In-Fusion®-Kit (Takara). The plasmid’s insert region, which represents the bioengineered pathway, was fully sequenced by Sanger sequencing. All details regarding oligonucleotides, templates and PCR product-sizes can be found in Table S1&S2. The srPAL-ADH/KRED-CAR11 operon is under the control of an L-arabinose inducible promoter, and Sfp is under the control of an L-rhamnose inducible promoter. The pZZ-Eva2 encodes kanamycin resistance gene and a colE1 origin of replication with 20-30 copies per cell.

Culturing conditions, cinnamyl alcohol extraction and quantification

We obtained the L-phenylalanine overproduction strain E. coli NST (ATCC 31882)6 in order to have a stable intracellular supply of substrate for in-vivo production of cinnamyl alcohol. The E. coli NST strain can produce up to 1mg/L of L-phenylalanine in 24h,7 using glycerol (1.5% v/v) and glucose (0.25%) as carbon sources that enter the cell glycolysis pathway for overproduction of L-phenylalanine via the shikimate pathway. Our production media was TB or M9 with glycerol (1.5% v/v) and glucose (0.25%) as carbon sources, while M9-Gly had only glycerol (1.75% v/v) and M9-Glu had only glucose (1.75%) (Figure 5 and S5). Since E. coli NST is auxotrophic for tyrosine and tryptophan, the production media were supplemented with 30 mg/L of each of the two amino acids.

E. coli NST was grown in LB medium until optical density (OD) of 0.2, and then made electro-competent for transformation with pZZ-Eva2. In order to prepare the inoculum for the in-vivo production experiment, three single colonies of E. coli NST transformed with pZZ-Eva2 were picked, transferred into 1.5ml tubes and grown in 300 µl of LB media supplemented with 50 mg/L kanamycin for 3h at 30°C while shaking at 1000rpm (Eppendorf, Thermo- shaker). 5ml of each production media variant (TB, M9, M9-Gly, M9-Glu) was inoculated with 50µl of the starting inoculum of each starting colony and incubated at 30°C for 3h at 200rpm (New Brunswick Scientific). All cultures were induced with 50mM arabinose and 0.01% L-rhamnose and harvested 24h, 48h and 72h after induction to test cinnamyl alcohol production and measure OD. To extract cinnamyl alcohol from the culture, 1ml of culture was transferred to a 2ml tube, and 100 µl 1M HCl was added with 500µl ethyl acetate and vortexed for 10 s. The phases were separated by centrifugation for 5 min at 13000 rpm, and the organic phase was collected in a fresh

1.5ml tube containing anhydrous MgSO4 to remove any residual aqueous phase. This process of organic solvent extraction was repeated twice (2 x 500µl), while collected in the same tube filed with anhydrous MgSO4 for drying, briefly vortexed and centrifuged for 3min at 13000rpm. Derivatization was performed in a 2ml tube mixing 600µl of the dried ethyl acetate, 200 µl methanol and 20 µl 2.0 M trimethylsilyldiazomethane, incubated for 1h at 28 °C, 600 rpm (thermo-block) in a fume hood. To stop the reaction 4 µl glacial acetic acid (2 µl/10 µl derivatization agent) was added to the mixture, followed by 20 min incubation at 28 °C, 600 rpm. Finally, 600µl of ethyl acetate containing the internal standard secondary benzene (0.1%) was added to the mix in the 2ml tube, mixing the ethyl acetate extract from the culture and the ethyl acetate with the internal standard in a 1:1 ratio. This final solution was transferred to a GC vial, processed on GC (see below) and quantified by GC area using standards (Sigma).

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Analytical Methods

HPLC analysis Reverse phase HPLC was performed on an Agilent 1200 Series LC system equipped with a G1379A degasser, a G1312A binary pump, a G1329 autosampler unit, a G1316A temperature controlled column compartment and a G1315B diode array detector. Conversion for the PAL-catalysed reaction was calculated from reverse phase liquid chromatography performed using a ZORBAX Extend-C18 column (50 mm × 4. mm × 3.5 μm, Agilent). Mobile -1 phase: NH4OH buffer (0.35% w/v, pH 10.0) / MeOH (in a ratio of 90:10). Flow rate: 1 mL min . Temperature: 40°C. Detection wavelength: 210 nm. Peaks were assigned via comparison with commercially available standards. Starting material and product distributions were derived from integrations of peak areas with using a response factor of 2.3 to account for the higher UV absorbance of 2. Retention times for compounds 1 and 2 were 2.3 and 5.4 minutes respectively.

GC Analysis Volatile extracts (1 μL) from the CAR, CAR-ADH and in vivo cultures were analysed by gas chromatography on an Agilent Technologies 7890A GC system equipped with an FID detector and a 7693 autosampler. A DB-WAX column (30 m; 0.32 mm; 0.25 μm film thickness; JW Scientific) was used to separate the compounds. The injector temperature was set at 220°C with a split ratio of 10:1 (1 μL injection). The carrier gas was helium with a flow rate of 1.5 mL/min and a pressure of 9.2 psi. The following oven program was used: 100°C (0 min hold), ramp to 200°C at 4°C/min (0 min hold), and ramp to 240°C at 20 °C/min (1 min hold). The FID detector was maintained at a temperature of 250°C with a flow of hydrogen at 30mL/min.

GC-MS Analysis Reaction products were primarily analysed by GCMS using an Agilent Technologies 7890B GC equipped with an Agilent Technologies 5977A MSD. The products were separated on a DB-WAX column (30 m x 0.32 mm i.d., 0.25 μM film thickness, Agilent Technologies). The injector temperature was set at 240 °C with a split ratio of 20:1 (1 μL injection). The carrier gas was helium with a flow rate of 2 mL/min and a pressure of 4.6 psi. The following oven program was used: 100°C (0 min hold), ramp to 20°C at 4 °C/min (0 min hold), and ramp to 240 °C at 20 °C/min (1 min hold). The ion source temperature of the mass spectrometer (MS) was set to 230 °C and spectra were recorded from m/z 50 to m/z 250. Compound identification was carried out using authentic standards and comparison to reference spectra in the NIST library of MS spectra and fragmentation patterns.

HRMS of compound 4 HRMS analyses were performed using an Agilent 6510 Q-TOF mass spectrometer connected to an Agilent 1200 Series LC system.

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Supplementary Data Figures

100 90 80

70

60 2 50 3 40 6 Composition(%) 30 4 20 10 0 10 5 2.5 1 10 5 2.5 1 Concentration AvPAL cells (mg/ml)

Figure S1. Comparison of one-pot triple enzyme cascade (left) and partition process (right) involving addition of CAR and ADH after completion of the PAL reaction. Partition reaction was performed in 100 mM potassium phosphate buffer (pH 7.5), 3 mg of AvPAL dried cells and 10 mM L-Phe at 30 °C, 250 rpm 1 ml final volume. After 20 h the reaction mixture was spun down and 500 µl supernatant supplemented with 10 mM MgCl2, 1 mg/ml MCAR dried cells, 30 mM D-glucose, 10 mM ATP, 10 U GDH, 0.05 mg/ml ADH, 500 µM NADP+, 500 µM NAD+ up to final volume of 1 ml. Incubated at 30 °C, 250 rpm 20h. One-pot reaction had the same conditions but starting concentration of substrate was 5 mM instead of 10 mM and reaction was stopped after 20 h.

100 90 80

70 60 2 50 3 40

Percentage(%) 30 6 20 4 10 0 0.25 0.25 0.15 0.15 0.1 0.1 0.05 0.05 0.025 0.025 (2x) (2x) (2x) (2x) (2x) ADH amount (mg/ml)

Figure S2. Effect of ScADH loading and batch addition on conversion of 2 in combination with MCAR, following initial production by AvPAL.

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100 90 80 70 60 2 50 3 40 6

Percentage (%) Percentage 30 20 4 10 0 1 2 3 4 6 10 ATP concentration (mM)

Figure S3. Effect of ATP loading on conversion of 2 by MCAR and ScADH following initial production by AvPAL.

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Characterisation data of 4 from preparative scale biotransformation

1H and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer (400.1 MHz) without additional internal standard. Chemical shifts are reported as δ in parts per million (ppm), calibrated against residual solvent signal.

(R)-3-amino-3-(3-fluorophenyl)propanoic acid (1c)

1 H NMR (CDCl3): δ 7.1 -7.31 (m, 5H, ArH), 6.50-6.54 (d, 1H, J = 16 Hz, C=CH), 6.23-6.30 (dt, 1H, J = 16, 8 Hz, 13 C=CHCH2 ), 4.21-4.23 (dd, 2H, J = 8, 4 Hz, C=CHCH2); C NMR (CDCl3): 136.70, 131.09, 128.60, 128.54, 127.69, 126.48, 63.65.

Figure S4. Top graph shows chromatogram from GC-MS after flash purification. Bottom graph shows the ionization spectrum and prediction patterns of the main peak from GC-MS chromatogram.

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48h 72h 24h 90

80

70

60

50

40 mg/L/OD 30

20

10

0 TB M9 M9-Gly M9-Glu

Figure S5. Quantification of in-vivo Cinnamyl alcohol produced 24h, 48 and 72h after induction, normalized by cell density (OD). Error bars present the standard deviation (SD) of three biological replicates.

Figure S6. Schematic representation of the vector pZZ-Eva2 used for in-vivo cinnamyl-alcohol production.

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>pZZ-Eva2 (recombinant) ttatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgat cgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaa tccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatg tactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagct ccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatat tgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcc tggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattccc agcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttg cgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaa accggtaaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacatt gattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggattctacctgacgctttttatcgcaactctctactgtttctccatACCCGT TTTTTTGGGAATTCAAAAGATCtaggaggATAAAGAAATGCACACCATGGACACTGCCCTGGCAGCCAACGACAAGGCCGAGCT CCTCATCGACGGCCATACGCTGACGGTGGCCGATGTCGTCAGCGGCGCCCGCCCCGCCGACACCACTCGCGTCCGCGCCCGGC TCGCCGAAGGCGCGGTCCAGCGCATCGAGCAGTCCCTCGCGCTCAAGAACAAGGTCATCGAGGCCGGTCTGCCCGTCTACGG CGTCACCTCGGGCTTCGGCGACAGCAACACCCGGCAGATATCCGGCCTCAAGTCGGAGGCCCTGCAGACCAACCTCATCCGGT TCCTGTCCTGCGGCATCGGCCCCGTCGCCACCCCGGACGTCATCCGCGCCACCATGATCGTACGGGCGAACTGCCTGGCCCGG GGCGCCTCCGGGATCCGTACCGAGATCCTCGAACTGCTTCTGGACTGCCTCAACAACGATGTGCTGCCGCCCATCCCCGAGCG CGGCTCGGTCGGTGCGAGCGGCGACCTGGTACCGCTGAGCTACGTGGCCGCGCTGCTGACCGGACAGGGCAAGGCGCTGCA CCAGGGTGAGGAGAAGGACGCCAGCGCGGCACTGGCCGACGCCGGTCTCGGTGCGGTGGTGCTCGGGGCCAAGGAGGGCC TCGCGCTGGTCAACGGCACCTCGTTCATGTCGGGTTTCGCCACCCTCGCCGTCCACGACGCCACCGAACTGGCCTTCGCCGCCG ACCTGAGCACCGCGCTGGCCTCCCAGGTGCTCCAGGGCAACCCCGGCCACTTCGTCCCGTTCATTTTCGACCAGAAGCCGCAC ACCGGAACCCGCACCAGCGCCCGGACCATCCGCGAACTGCTCGGCAACCCCGAGGACTGCGACCCGTCCGTGGACCCCGAGG GCGCTGCCCTGACGGAGTCCGGTTTCCGGCAGCTGGAGGAGCCCATCCAGGACCGGTACTCGGTGCGCTGCGCGCCGCATGT GACCGGTGTGCTGCGTGACACCCTGGACTGGGCGAAGAACTGGGTCGAGGTCGAGATCAACTCCACCAATGACAACCCGCTG TTCGATGTGGAAGCGGGCATGGTCCGCAACGGCGGCAACTTCTACGGAGGCCACGTCGGTCAGGCCATGGACGCGCTCAAGA CCGCGGTGGCCAGTGTCGGCGACCTGCTGGACCGTCAGCTCGAACTGATCGTCGACGAGAAGTTCAACAACGGGCTCACTCC GAACCTGATCCCGCGGTTCGACGCCGACAGCTGGGAGGCCGGGCTGCACCACGGCTTCAAGGGCATGCAGATCGCCGCCTCC GGCCTCACCGCCGAGGCGCTGAAGAACACCATGCCGGCGACATCGTTCTCCCGGTCGACCGAGGCCCACAACCAGGACAAGG TCAGCATGGCCACCATCGCCGCGCGGGACGCCCGTACGGTCGTGGAACTGGTCCGCCAGGTCGCCaCCATCCACCTCCTGGCC CTGTGCCAGGCGGCGGACCTGCGCGGTCAGGAGTGCCTGAGCGCTCCGACGCGCGCCGCGTACGAGCTGATCCGCTCCGTCT CCGCCACGATGGACGGTGACCGGCCGCTGGCCCGGGACATCGAGCTCGTGGTCGGCCTCATCGCGTCCGGCGAGCTCCGCCG GGCCGTCGAGGACGCCGGGCGGGACtaaaggaggATAAAGAAATGCGGACGATGAAGGCAGTGCAGGTGGCAAAGGCTGGCG GACCCCTGGAACTCGTCGAACGGGACGTGCCGGAACCGGGCGCCGGACAGGTGCTCATCAAGATCCAGGCGTGCGGTATTTG TCACAGCGACGTGTTGACAAAAGAAGGGCAGTGGCCGGGCCTCGAATATCCGCGGGTGCCGGGGCACGAGATTGCAGGCGT CATCGATACGGTCGGCGCGGGCGTTGAAGGATGGGCGGCGGGGCAGCGCGTCGGCGTCGGCTGGCACGGCGGGCATTGCG GCCGGTGCGAGCATTGCCGTCGAGGCGACTTCGTTCTATGTCAGCGCGCACTCGTGCCGGGCATCAGCTACGACGGCGGCTAT GCCGAATTCATGGTGGCCCCGGTCGAAGCGCTGGCGCGCATTCCAGACGATCTTTCCGACGTCGATGCCGCACCGCTTCTGTG CGCGGGCATCACGACCTTCAACGCGCTGCGCAACAGCGGCGCACGCGCCGGGGATGTAGTCGCCGTGCTGGGCATCGGCGG ACTCGGTCACCTCGGCGTGCAGTTCGCGCGAAAGATGGGCTTCGTCACGGTCGCCATTGCGCGCGGGCAGGACAAGGCAAGT CTCGCGAAAGAGCTGGGCGCTCATCACTACATCGACAGCACGACGGCGAATGTCGCGCAAGCGCTGCAGGCGTTGGGCGGC GCTCGCGTCATTCTTGCAACCGTCACCAGCGGCAAGGCAATGAGTGCCGTGGTGGGCGGTCTGGGGTTGAACGGCAAGCTGA TCATGGTCGGACTCTCCGAAGAGCCCGTCGAGGTGCCGATTGCGCAGTTCATCATGGGGCGCAACTCGGTGCAGGGCTGGCC GTCGGGCACATCGGCGGATTCTCAGGACACGCTCGCCTTCAGCGCGCTATCAGGCATCAAGCCGATGATCGAAGAATTCCCGC TGACCAAAGCCGCCGAGGCCTACGACCGGATGATGAGCGGCGCTGCGCGATTCAGGGTTGTGCTGAACACGGGCCAATAAag gaggATAAAGAAATGGGCAGCAGtCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCAT GACTGAGTCGCAGAGCTACGAGACCAGGCAGGCCCGGCCGGCCGGACAGAGCCTCGCCGAGCGCGTCGCGCGCCTTGTCGC CATCGATCCGCAAGCCGCGGCCGCTGTGCCGGACAAGGCCGTCGCCGAGCGCGCGACGCAGCAGGGTTTGCGCCTCGCGCA GCGGATCGAAGCCTTCCTCTCCGGCTACGGAGACCGCCCGGCCCTCGCCCAGCGCGCTTTTGAGATCACAAAAGATCCCATCA CCGGACGGGCTGTCGCGACGCTGCTGCCGAAGTTCGAGACGGTGAGCTACCGCGAGCTGCTGGAGCGCTCGCACGCGATCGC GAGCGAGCTGGCGAACCACGCCGAGGCCCCGGTCAAGGCCGGGGAGTTCATCGCGACCATCGGGTTCACCAGCACCGACTAC ACCTCTCTCGACATCGCGGGCGTGCTGCTCGGGCTCACCTCGGTGCCGCTGCAGACCGGGGCGACGACCGACACCCTCAAGG CCATCGCCGAGGAGACCGCGCCCGCCGTGTTCGGCGCGAGCGTCGAACACCTCGACAACGCCGTGACGACCGCGCTCGCGAC CCCGTCGGTGCGCCGCCTGCTCGTGTTCGACTACCGCCAGGGCGTGGACGAGGACCGCGAGGCGGTCGAGGCCGCCCGAAG

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CCGGCTCGCCGAAGCGGGCAGCGCCGTCCTGGTGGACACGCTGGACGAGGTGATCGCCCGTGGCCGCGCCCTCCCCCGCGTG GCGCTCCCGCCCGCCACCGACGCGGGCGACGACTCCCTGTCCCTGCTCATCTACACCTCCGGGTCCACCGGCACCCCGAAGGG CGCGATGTACCCCGAGCGCAACGTCGCGCAGTTCTGGGGCGGCATCTGGCACAACGCCTTCGACGACGGCGACTCGGCCCCG GACGTTCCCGACATCATGGTCAACTTCATGCCGCTCAGCCACGTCGCCGGGCGCATCGGCCTGATGGGCACCCTCTCCAGCGG CGGCACCACGTACTTCATCGCCAAGAGCGACCTCTCCACGTTCTTCGAGGACTACTCGCTCGCCCGGCCCACCAAGCTCTTCTTC GTGCCGCGGATCTGCGAGATGATCTACCAGCACTACCAGAGCGAGCTGGACCGCATCGGCGCGGCGGACGGCTCGCCCCAGG CCGAGGCGATCAAGACCGAGCTGCGCGAGAAGCTCCTCGGCGGGCGGGTCCTCACGGCGGGCTCCGGCTCGGCTCCGATGTC CCCCGAGCTCACCGCTTTCATCGAATCCGTGCTGCAAGTCCACCTGGTGGACGGCTACGGGTCGACCGAGGCGGGCCCCGTGT GGCGCGACCGCAAGCTGGTCAAGCCGCCGGTGACCGAGCACAAGCTGATCGACGTGCCCGAACTCGGCTACTTCTCCACCGA CTCCCCGTATCCCCGAGGCGAGCTGGCGATCAAAACCCAGACCATCCTCCCCGGCTACTACAAGCGCCCGGAGACCACCGCCG AGGTCTTCGACGAAGACGGCTTCTACCTCACCGGCGACGTGGTCGCCGAGGTCGCCCCTGAAGAGTTCGTCTACGTGGACCG GCGCAAGAACGTCCTGAAGCTCTCGCAGGGCGAGTTCGTCGCGCTCTCGAAGCTGGAGGCGGCGTACGGCACGAGCCCGCTG GTGCGGCAGATCTCCGTCTACGGGTCGAGCCAGCGCTCGTACCTGCTCGCCGTCGTCGTCCCCACCCCGGAAGCCCTCGCGAA ATACGGCGACGGCGAGGCGGTCAAGTCGGCGCTCGGCGACTCGCTGCAGAAGATCGCGCGCGAGGAGGGCCTGCAGTCCTA CGAGGTGCCGCGCGACTTCATCATCGAGACCGATCCCTTCACCATCGAGAACGGCATCCTCTCCGACGCGGGCAAGACGCTGC GCCCGAAGGTGAAGGCGCGCTACGGCGAGCGGCTCGAAGCGCTGTACGCGCAGCTCGCCGAGACCCAGGCTGGCGAGCTGC GCTCGATCCGGGTCGGCGCGGGCGAGCGCCCGGTGATCGAGACCGTCCAGCGGGCCGCCGCCGCGCTGCTCGGAGCCTCCG CCGCAGAGGTCGACCCCGAGGCCCACTTCTCGGACCTCGGCGGCGACTCGCTCTCCGCGCTCACCTACTCCAACTTCCTGCACG AGATCTTCCAGGTCGAGGTGCCGGTGAGCGTCATCGTGAGCGCCGCGAACAACCTGCGCTCGGTTGCGGCGCACATCGAGAA GGAGCGCTCCTCCGGCAGCGACCGGCCCACCTTCGCGAGCGTGCACGGCGCGGGCGCGACGACGATCCGCGCGAGCGACCT GAAGCTGGAGAAGTTCCTCGACGCCCAGACCCTCGCCGCCGCCCCGTCCTTGCCCCGCCCGGCAAGCGAGGTCCGCACGGTG CTGCTCACCGGGTCCAACGGCTGGCTCGGGCGCTTCCTCGCCTTGGCCTGGCTGGAACGTCTGGTGCCGCAGGGCGGCAAGG TCGTCGTGATCGTGCGCGGCAAGGACGACAAGGCCGCCAAAGCCCGGCTGGACTCGGTCTTCGAGAGCGGCGACCCCGCGCT CCTCGCGCACTACGAGGATCTCGCCGACAAGGGCCTGGAAGTGCTCGCGGGCGACTTCAGCGACGCCGACCTCGGCCTGCGC AAGGCGGATTGGGACCGGCTCGCGGACGAAGTCGACCTCATCGTCCACTCCGGCGCGCTGGTGAACCACGTTCTGCCCTACA GCCAGCTGTTCGGCCCGAACGTGGTGGGCACGGCCGAGGTCGCCAAGCTCGCCCTCACCAAGCGGCTCAAGCCGGTCACCTA CCTCTCCACGGTGGCGGTGGCCGTCGGCGTGGAGCCCTCGGCCTTCGAGGAGGACGGCGACATCCGCGATGTGAGCGCGGT GCGCTCCATCGACGAGGGCTACGCGAACGGCTACGGCAACAGCAAGTGGGCGGGCGAGGTGCTGCTGCGCGAGGCATACGA GCACGCGGGCCTGCCGGTCCGGGTGTTCCGCTCGGACATGATCCTCGCGCACCGCAAGTACACCGGACAGCTCAACGTCCCG GACCAGTTCACCCGGCTCATCCTGAGCCTTTTGGCCACCGGCATCGCCCCGAAGTCCTTCTACCAGCTCGACGCGACGGGCGG GCGCCAGCGCGCGCACTACGACGGCATCCCGGTGGACTTCACCGCCGAGGCCATCACCACACTCGGCCTCGCCGGTTCGGAC GGCTATCACAGCTTCGACGTGTTCAACCCGCACCATGACGGGGTGGGCTTGGACGAGTTCGTGGACTGGCTCGTCGAGGCGG GGCACCCGATCTCGCGGGTCGACGACTACGCCGAGTGGCTGTCCCGGTTCGAGACTTCGCTGCGCGGCCTGCCGGAGGCGCA GCGCCAGCATTCGGTGCTCCCGCTGCTGCACGCGTTCGCCCAGCCCGCCCCGGCGATCGACGGCTCCCCGTTCCAGACCAAGA ACTTCCAGTCCTCGGTCCAGGAGGCCAAGGTCGGCGCGGAGCACGACATCCCGCATCTGGACAAGGCGCTCATCGTCAAGTA CGCCGAGGACATCAAGCAGCTCGGCCTGCTCTaaaaagtcaaaagcctccgaccggaggcttttgacttccacaattcagcaaattgtgaacatcatc acgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcgtattcaggcgctttttagactggtcgtaatgaaaggaggATAAAGAA ATGGGCAAGATTTACGGAATTTATATGGACCGCCCGCTTTCACAGGAAGAAAATGAACGGTTCATGTCTTTCATATCACCTGAA AAACGGGAGAAATGCCGGAGATTTTATCATAAAGAAGATGCTCACCGCACCCTGCTGGGAGATGTGCTCGTTCGCTCAGTCAT AAGCAGGCAGTATCAGTTGGACAAATCCGATATCCGCTTTAGCACGCAGGAATACGGGAAGCCGTGCATCCCTGATCTTCCTG ACGCCCATTTCAATATTTCTCACTCTGGCCGCTGGGTCATTTGCGCGTTTGATTCACAGCCGATCGGCATAGATATCGAAAAAA CGAAACCGATCAGCCTTGAGATCGCCAAGCGCTTCTTTGCAAAAACAGAGTACAGCGACCTTTTAGCAAAAGACAAGGACGA GCAGACAGACTATTTTTATCATCTATGGTCAATGAAAGAAAGCTTTATCAAACAGGAAGGCAAAGGCTTATCGCTTCCGCTTGA TTCCTTTTCAGTGCGCCTGCATCAGGACGGACAAGTATCCATTGAGCTTCCGGACAGCCATTCCCCATGCTATATCAAAACGTA TGAGGTCGATCCCGGCTACAAAATGGCTGTATGCGCCGCACACCCTGATTTCCCCGAGGATATCACAATGGTCTCGTACGAAG AGCTTTTATAAggatccaaactcgagtaaggatctccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtg aacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatacctagggcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaat acggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca taggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccc tcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggt gtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgac ttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaag gacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttg caagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggt catgactagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacagga gtccaagcgagctctcgaaccccagagtcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcac gaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatga

S11 atccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaac agttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtc gaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccg gcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgt cctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgt tgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatcaga tcatgatcccctgcgccatcagatccttggcggcaagaaagccatccagtttactttgcagggcttcccaaccttaccagagggcgccccagctggcaattccgacgtc

Figure S7. The FASTA format, representing the full size nucleic acid sequences (10446bp) of recombinant plasmid pZZ-Eva2, as it was used in the in-vivo experiment.

Table S1. Basic information on oligonucleotides used for polymerase chain reactions (PCR) in this study. Primer name Application Product Size (bp) Template srPAL+O_Fw PCR & OE-PCR 1638bp & 2704bp pET-16b srPAL_Rv PCR & Sequencing 1638bp pET-16b

KRED+O_Fw PCR 1081bp pET-28a

KRED+O_Rv PCR & OE-PCR 1081bp & 2704bp pET-28a

CAR11_Fw PCR & Sequencing 3636bp pET-21a

CAR11_Rv PCR & Sequencing 3636bp pET-21a

Rha+CAR_Fw PCR & OE-PCR 210bp & 881bp Synthetic

Rha+Sfp_Rv PCR 210bp Synthetic

Sfp_Fw PCR 692bp pCDF-1b

Sfp+O_Rv PCR 692bp pCDF-1b

Full-INs-Eva2_Rv OE-PCR & Sequencing 881bp Synthetic

Full-Ins_Fw Sequencing ND pZZ-Eva2

KRED-seq_Fw Sequencing ND pZZ-Eva2

KRED-seq_Rv Sequencing ND pZZ-Eva2

CAR-seq_2Fw Sequencing ND pZZ-Eva2

CAR-seq_3Fw Sequencing ND pZZ-Eva2

CAR-seq_4Fw Sequencing ND pZZ-Eva2

CAR-seq_5Fw Sequencing ND pZZ-Eva2

ND = not defined.

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Table S2. Oligonucleotides used in this study. Primer name Sequence 5'-3' srPAL+O_Fw ttgggaattcaaaaGATCTaggaggATAAAGAAATGCACACCATGGACACTGC srPAL_Rv ttaGTCCCGCCCGGCGT

KRED+O_Fw GCCGGGCGGGACtaaaggaggATAAAGAAATGCGGACGATGAAGGCA

KRED+O_Rv atgaTGaCTGCTGCCCATTTCTTTATcctcctTTATTGGCCCGTGTTCAGC

CAR11_Fw ATGGGCAGCAGtCATCATCAT

CAR11_Rv ttAGAGCAGGCCGAGCTG

Rha+CAR_Fw CAGCTCGGCCTGCTCTaaaaagtcaaaagcctccgaccg

Rha+Sfp_Rv AATTCCGTAAATCTTGCCCATTTCTTTATcctcctttcattacgaccagtctaaaaagcgc

Sfp_Fw ATGGGCAAGATTTACGGAATT

Sfp+O_Rv tcgaGtttGGATCCTTATAAAAGCTCTTCGTACGAGACCA

Full-INs-Eva2_Rv tcgaGtttGGATCCTTATAAAAGCT

Full-Ins_Fw ttgggaattcaaaaGATCTagg

KRED-seq_Fw AATTCATGGTGGCCCCG

KRED-seq_Rv AACTGCACGCCGAGGTG

CAR-seq_2Fw GCTCGTGTTCGACTACCGC

CAR-seq_3Fw ACTCCCCGTATCCCCGA

CAR-seq_4Fw ATCTTCCAGGTCGAGGTGC

CAR-seq_5Fw TGGAGCCCTCGGCCTT

>AvPAL(Anabaena variabilis)

MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTLVSLTNNTDILQGIQASCDYINNAVESGEPIYGV TSGFGGMANVAISREQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRLELIKRMEIFLNAGVTPY VYEFGSIGASGDLVPLSYITGSLIGLDPSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCVY DTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQMISLLANSQLVRDELDGKHDYRDHELIQDRYS LRCLPQYLGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYIGLLAKHLDVQIALLASP

S13

EFSNGLPPSLLGNRERKVNMGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQNY VAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVGQKPTSDRPYIWNDNEQGLDEHIARISADIAAGG VIVQAVQDILPCLH

>MCAR(Mycobacterium marinum) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRL LPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPT MIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGT DVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKRTWFEGGYEPSITLNFMPMSHVMGRQILYGTLCNGGTAYFV AKSDLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALTGSAPIS DEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAE VTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPT QEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHG QADELRSLRQSGADAPVLVTVCRAAAALLGGSASDVQPDAHFTDLGGDSLSALSFTNLLHEIFDIEVPVGVIVSPANDLQ ALADYVEAARKPGSSRPTFASVHGASNGQVTEVHAGDLSLDKFIDAATLAEAPRLPAANTQVRTVLLTGATGFLGRYLA LEWLERMDLVDGKLICLVRAKSDTEARARLDKTFDSGDPELLAHYRALAGDHLEVLAGDKGEADLGLDRQTWQRLADT VDLIVDPAALVNHVLPYSQLFGPNALGTAELLRLALTSKIKPYSYTSTIGVADQIPPSAFTEDADIRVISATRAVDDSYANG YSNSKWAGEVLLREAHDLCGLPVAVFRCDMILADTTWAGQLNVPDMFTRMILSLAATGIAPGSFYELAADGARQRAH YDGLPVEFIAEAISTLGAQSQDGFHTYHVMNPYDDGIGLDEFVDWLNESGCPIQRIADYGDWLQRFETALRALPDRQR HSSLLPLLHNYRQPERPVRGSIAPTDRFRAAVQEAKIGPDKDIPHVGAPIIVKYVSDLRLLGLL

>BsSfp(Bacillus subtilis) MGKIYGIYMDRPLSQEENERFMTFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQYQLDKSDIRFSTQEYGKPCIPD LPDAHFNISHSGRWVIGAFDSQPIGIDIEKTKPISLEIAKRFFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSL PLDSFSVRLHQDGQVSIELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELL

Figure S8. The FASTA format amino acid sequences of recombinant AvPAL, MCAR and BsSfp enzymes as used in biotransformation and biocatalyst preparation procedures.

S14

1H NMR of compound 4 from biotransformation

4,CDCl3,400 MHz

13C NMR of compound 4 from biotransformation

S15

4, CDCl3, 100 MHz

References

1. S. L. Lovelock, R. C. Lloyd and N. J. Turner, Angewandte Chemie, 2014, 53, 4652-4656. 2. F. W. Studier, Protein expression and purification, 2005, 41, 207-234. 3. N. J. Weise, S. T. Ahmed, F. Parmeggiani, E. Siirola, A. Pushpanath, U. Schell and N. J. Turner, Catalysis Science & Technology, 2016, 6, 4086-4089. 4. Z. Zebec, I. A. Zink, M. Kerou and C. Schleper, G3, 2016, 6, 3161-3168. 5. J. C. Anderson, J. E. Dueber, M. Leguia, G. C. Wu, J. A. Goler, A. P. Arkin and J. D. Keasling, Journal of biological engineering, 2010, 4, 1. 6. D. E. Tribe, Google Patents, 1987. 7. N. Yakandawala, T. Romeo, A. D. Friesen and S. Madhyastha, Applied microbiology and biotechnology, 2008, 78, 283-291.

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