Manipulation of the Precursor Supply in Yeast Significantly Enhances

21 Feb 2017 - Inorganic Chemistry; J; Journal of the American Chemical Society ..... We manipulated the biosynthetic pathways for two MTCA precursors,...
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Manipulation of the Precursor Supply in Yeast Significantly Enhances the Accumulation of Prenylated #-Carbolines Katja Backhaus, Lena Ludwig-Radtke, Xiulan Xie, and Shu-Ming Li ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00387 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Manipulation of the Precursor Supply in Yeast Significantly Enhances the Accumulation of Prenylated β-Carbolines Katja Backhaus, †,§ Lena Ludwig,† Xiulan Xie,‡ Shu-Ming Li*,†,§ †

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Robert-Koch-Straße 4, 35037 Marburg, Germany §

Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, Hans-Meerwein-Straße, 35032 Marburg, Germany



Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Straße, 35032 Marburg, Germany

KEYWORDS: β-carboline, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, fungal tryptophan prenyltransferase, regiospecific prenylation

ABSTRACT: The tryptophan derivative 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) is present in many plants and foods including fermentation products of the baker’s yeast Saccharomyces cerevisiae. MTCA is formed from tryptophan and acetaldehyde via a Pictet-Spengler reaction. In this study, up to 9 mg/l of MTCA were detected as a mixture of (1S,3S) and (1R,3S) isomers in a ratio of 2.2:1 in Saccharomyces cerevisiae cultures. To the best of our knowledge, this is the first report on the presence of MTCA in laboratory baker’s yeast cultures. Expression of three fungal tryptophan prenyltransferase genes, fgaPT2, 5-dmats, and 7-dmats in S. cerevisiae resulted in the formation of MTCA derivatives with prenyl moieties at different positions of the indole ring. Expression of these genes in dimethylallyl diphosphate and tryptophan overproducing strains led to generation of up to 400 mg/l of prenylated MTCAs as mixtures of (1S,3S) and (1R,3S) diastereomers in ratios similar to that of unprenylated MTCA. The structures of the described substances including their stereochemistry were unequivocally elucidated by mass spectrometry as well as one- and twodimensional NMR spectroscopy. The results of this study provide a convenient system for the production of high amounts of designed prenylated MTCAs in S. cerevisiae. Furthermore, our work can be considered as an excellent example for the construction of more complex molecules by introducing just one key gene.

β-carboline alkaloids are widely spread in nature and have been isolated from plants, marine tunicates, fungi and actinomycetes.1-3 They are considered to be important molecules in drug discovery and development due to a wide variety of biological and pharmacological activities including antiviral, antibacterial, fungicidal, insecticidal, antimalarial and antileishmanial activities.4-6 Reduction in alcohol self-administration, antiallergic effects, antiinflammatory and antitumoral activities have also been described for these compounds.7-9 1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) was detected in plants and foods such as smoked and cooked meat, soy sauce, cocoa, and citrus fruits, but also in alcoholic beverages like wine, beer, whiskey and sake.10-14 MTCA can also be found in biological tissues, urine and breast milk, probably originating from MTCA-containing food products.10,11 The detection of MTCA in food and drinks processed with Saccharomyces cerevisiae raises the question on its accumulation in laboratory yeast cultures, which has not been reported previously and will be addressed in this work.

In food products, the formation of MTCA occurs via a Pictet-Spengler reaction from L-tryptophan and acetaldehyde (Scheme 1). The extent of MTCA formation depends on the amount of the substrates as well as the pH value, temperature, storage time and other processing conditions of the respective food products. Upon the chemical reaction, two diastereomers, (1S,3S)- (2a) and (1R,3S)MTCA (2b), are formed with conservation of the Ltryptophan configuration (Scheme 1). The ratio of 2a to 2b was found to be 4:1 in food products, differing clearly from 12:1 for MTCA obtained by chemical synthesis.10 In general, the Pictet-Spengler reaction is a very successful strategy for the synthesis of isoquinoline as well as indole alkaloid frameworks.15 It comprises an acidcatalyzed condensation of an amine-containing substrate with an aldehyde generating an iminium ion. A subsequent electrophilic cyclization yields the ring system.15,16 Well-studied examples for enzyme-catalyzed PictetSpengler reactions are the formation of 3α-(S)strictosidine (Figure 1) from tryptamine and secologanin catalyzed by strictosidine synthase and (S)-norcoclaurine

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Scheme 1: Pictet-Spengler reactions in a laboratory S. cerevisiae culture (a) and in cultures of transformants with different DMATSs (b).

(Figure 1) from tyramine and 4-hydroxyphenyl acetaldehyde catalyzed by norcoclaurine synthase.17-19 Both enzymes are found in plants and catalyze the formation of their respective products in a stereoselective manner. McbB from the deep sea-derived microorganism Marinactinospora thermotolerans has been proven to be responsible for the formation of marinacarbolines A-C (Figure 1) and 1-acetyl-β-carboline, whereas (R)-salsolinol synthase was shown to catalyze the enantioselective formation of (R)-salsolinol (Figure 1) from dopamine and acetaldehyde in the human brain.1,20,21 In addition, Pictet-Spengler reactions are also involved in the formation of chaetoglines (Figure 1) in Chaetomium globosum as well as of the tetrahydroisoquinoline antibiotics saframycin (Figure 1) in Streptomyces lavendulae and safracin in Pseudomonas fluorescens.22-25 After detection of MTCA in yeast cultures, its chemical or enzymatic formation will also be addressed in this study. Prenylated natural products are widely distributed in nature and often carry biological and pharmacological activities distinct from their non-prenylated precursors, since prenylation often improves interactions of small molecules with proteins and biomembranes.26,27 The chemical synthesis of prenylated compounds requires extreme conditions and some prenylation positions are

favored above others. To achieve prenylation at the less favored positions, additional steps for protection and deprotection of functional groups in the reactants are usually required.28 Therefore, an enzymatic prenylation, NH O

HO O

NH

HO

N H O O

HO

OH

(S)-norcoclaurine

O

O

HO

OH OH 3 -(S)-strictosidine

O O N

CH3 HO

O

NH

O

N O CN

O HN

HO O

(R)-salsolinol

saframycin A

O NH N N H

COOH R

O

N

O

N COOMe

R=H marinacarboline A R=OH marinacarboline B R=OCH3 marinacarboline C

chaetogline A

Figure 1: Examples for Pictet-Spengler reaction products.

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which occurs in a regiospecific and stereospecific manner is a practical alternative to chemical prenylation. The enzymatic prenylation of indole derivatives including tryptophan is mainly performed by prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily. In the last years, more than 40 of such enzymes have been identified and characterized.29 However, direct prenylation of MTCA was unsuccessful.30 In case of a successful detection of MTCA in yeast culture, we aim at the production of prenylated derivatives by heterologous expression of different tryptophan prenyltransferases, e.g. FgaPT2 from Aspergillus fumigatus and 5-DMATS from Aspergillus clavatus, which catalyze the C4- and C5-prenylation of tryptophan, respectively. 31,32 Furthermore, a C7-prenylation of tryptophan can be achieved by 7-DMATS from Aspergillus fumigatus.33 These three fungal enzymes employ dimethylallyl diphosphate (DMAPP) as prenyl donor and catalyze regiospecific prenylations at the indole ring.29 Results and Discussion Formation of MTCA in S. cerevisiae cultures. As mentioned in the introduction, MTCA was detected in food products processed with S. cerevisiae. However, a direct involvement in the formation of the β-carboline derivative has not been reported for yeast cultures under laboratory conditions. Thus, we were interested in the presence of MTCA in yeast cultures under usual cultivation conditions and took the wild-type strain HOD114-2B, a CEN.PK derivative, as study subject, because this strain is physiologically well characterized and very suitable for biotechnological applications.34,35 Comparison of the LCMS chromatogram of a 24 h-old culture in synthetic complete medium containing 2% of glucose as a carbon source with that directly after inoculation revealed the presence of two peaks with the same retention times, UV spectra, [M+H]+ ions and MS2 fragmentation pattern as those of a commercial MTCA sample (Figure 2a, Figures S1 - S2 in the supplemental material). This proved unequivocally the formation of MTCAs as two diastereomers, (1S,3S) (2a) and (1R,3S) (2b, Scheme 1 and Figure 2a), during the cultivation of this yeast strain. The ratio of 2a to 2b was calculated to be 2.2:1. Thus, it differs only slightly from that of MTCA from food products at 4:1,10,11 but significantly from 12:1 reported previously10 and confirmed in this study (data not shown) for the samples from chemical syntheses. The accumulation of MTCAs reached 9 mg/l culture after 48 h and remained constant during further cultivation (Figure 2b). This may be due to the exhaustion of the glucose supply, upon which the yeast cells transiently reduce their growth rate to readjust their metabolism for a slower, respiratory growth on ethanol and thereby do not provide excess amounts of tryptophan and acetaldehyde for the formation of MTCA.36

creases from 6.25, which is set as the starting pH value of media, to approximate 2.6 after 24 hours (Figure S3 in the supplemental material). MTCAs in S. cerevisiae cultures are probably formed chemically by condensation of tryptophan and acetaldehyde, which are provided by the yeast, at the low pH values. Therefore, we investigated the formation of MTCAs upon addition of acetaldehyde and tryptophan to synthetic yeast media with different pH values. As shown in Figure 3, formation of 2a and 2b is clearly detected in the reaction mixtures with pH 2 and 3. The product yield decreased with the increasing pH value and no product formation was observed at pH 6.25. These results indicated that the detected MTCAs are very likely formed chemically and the yeast cells provide both reactants, tryptophan and acetaldehyde, as well as the suitable reaction condition by lowering the pH value during their growth.

The chemical formation of MTCA from tryptophan and acetaldehyde via a Pictet-Spengler reaction is acidcatalysed.10,11 The pH value of S. cerevisiae cultures de-

The yeast genome was searched for homologs of known Pictet-Spengler reaction catalyzing enzymes like McbB from Marinactinospora thermotolerans, stricto-

Figure 2. Formation of MTCAs (2a and 2b) in S. cerevisiae HOD114-2B cultures. (a) LC-MS analysis with UV-detection and extracted ions of samples taken directly after inoculation and after 24 h of incubation. (b) Yields of MTCAs (2a and 2b) in samples taken at different times. Three independent measurements were carried out for each time point. The areas in extracted ion chromatograms (EICs) were employed for quantification. The error bars indicate the standard deviation.

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sidine synthase and norcoclaurine synthase1,18,19 to address the possibility of an at least partial enzymatic contribution to the formation of MTCAs. However, our search did not result in promising hits. Results of enzyme assays containing tryptophan and acetaldehyde with crude protein extracts from S. cerevisiae in Tris-HCl or phosphate buffers at pH 7.0 and 7.5 did not support the hypothesis of an enzymatic formation of MTCAs (data not shown). Therefore, we assume a chemical formation of MTCAs in the yeast cultures similar to the pityriacitrin formation in Ustilago maydis.37 These results encouraged us to test S. cerevisiae as a convenient production system for prenylated MTCAs just by expression of tryptophan prenyltransferase genes. It can be expected that prenylated tryptophan can also effectively undergo the Pictet-Spengler reaction in the presence of acetaldehyde in S. cerevisiae cultures. The successful production of prenylated MTCAs in this way would expand our program for easy access to prenylated derivatives significantly, since MTCA is a very poor substrate for direct enzymatic prenylation,30 which was also confirmed in this study. No product formation was detected by incubation of MTCA with the purified tryptophan prenyltransferase FgaPT2 in the presence of DMAPP (data not shown). Expression of fgaPT2 led to the formation of dimethylallyl-MTCA in the yeast cultures. The tryptophan C4-prenyltransferase gene fgaPT2 was expressed with the construct pIU1131 in the wild-type strain HOD1142B under control of a galactose-inducible promoter. Formation of 4-dimethylallyltryptophan (4-DMAT, 3) was detected by LC-MS in cultures four hours after induction (Figure 4). The identity of 3 was proven by comparison of its retention time, UV spectrum, [M+H]+ ion and MS2 fragmentation pattern with those of an authentic sample from an enzyme assay with the purified FgaPT2 (Figures S4 and S5 in the supplemental material). The product yield of 3 in the culture was calculated to be 1.3 mg/l four hours after induction and increased to 5.3 mg/l eight hours after induction. An approximate product yield of 4 mg/l was observed for 3 during further cultivation of up to 168 h (Figure 5).

Figure 3: HPLC monitoring of the chemical formation of MTCAs in synthetic yeast medium without tryptophan (SCW), at different pH values. Tryptophan and acetaldehyde were added to the fresh media without yeast cells. Tryptophan and a commercial MTCA sample were used as standards. Detection was carried out on a photo diode array detector and illustrated for absorption at 277 nm.

No formation of dimethylallyl-MTCA (DMA-MTCA, 4) with an expected [M+H]+ at m/z 299.1754 was observed in the culture four hours after induction of the fgaPT2 expression (Figure 4). In comparison, an additional peak with the expected [M+H]+ for 4 was detected for the cultures eight hours after induction. Isolation and structure elucidation revealed that this peak comprises the two stereoisomers of the desired prenylated MTCAs 4a and 4b in a ratio of 2.5:1 in favor of the (1S,3S) diastereomer (Figure S6 in the supplemental material, see below for isolation and structure elucidation). For better comparison and conformity with data in the literature, we use different numbering systems for tryptophan and MTCA derivatives. Therefore, the numbering of the prenyl moiety at the indole ring of the prenylated tryptophan (4-DMAT, 3)

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ACS Synthetic Biology empty vector and its structure could not be elucidated in this study. The amount of 7-DMA-MTCAs reached 60 mg/l 48 hours after induction of fgaPT2 expression and remained nearly constant during further cultivation (Figure 5). The ratio of 7-DMA-MTCAs (4a and 4b) to 4-DMAT (3) is about 15:1, indicating an effective conversion of the prenylated tryptophan to MTCA derivatives. The amount of unprenylated MTCAs under the inducing conditions for fgaPT2, i.e. growth with galactose as a carbon source, is so low that no reliable data could be obtained. Increasing the product yields of 7-DMA-MTCAs by metabolic engineering. We manipulated the biosynthetic pathways for two MTCA precursors, tryptophan and DMAPP, to increase the productivity of S. cerevisiae cultures for its prenylated derivatives. For tryptophan overproduction, serine 76 in the anthranilate synthase was mutated to leucine. This amino acid exchange has been reported to abolish the feedback inhibition and thereby leads to an increased intracellular tryptophan concentration.38 The DMAPP overproduction strain KAB24 (Table 1) was achieved by the insertion of a truncated copy of the HMG-CoA reductase gene HMG1 in the yeast genome as described by Ro et al.39 In the strain KAB26 (Table 1), both manipulations were combined. The constructed strains harboring the fgaPT2 expression plasmid pIU11 were grown in shake flasks and the formation of 3 and 4 was monitored by HPLC. Slightly higher amounts of both products were detected in the tryptophan-overproducing strain KAB20 than in the wild type (Figure 5). Much higher amounts were found in the DMAPP overproduction strain KAB24, with up to 18.8 mg/l 3 eight hours after induction of fgaPT2 expression and 235 mg/l 4 after 168 h (Figure 5). In the DMAPP and tryptophan overproducing strain KAB26, about 250 mg/l of 4 were determined. Comparison of the product formation by the tested strains clearly revealed that overproduction of the prenyl donor DMAPP is more effective for the production of higher amounts of DMA-MTCA Table 1: S. cerevisiae strains used in this study strain

features

genotype

HOD114-2B

wild-type strain

MATa ura3-52 his3Δ1 leu23,112

KAB1

Figure 4: LC-MS monitoring of the formation of 4-DMAT (3) and 7-DMA-MTCAs (4a and 4b) in cultures of the S. cerevisiae wild-type strain HOD114-2B upon expression of fgaPT2.

differs from that in its Pictet-Spengler reaction products (7-DMA-MTCAs, 4a and 4b, Scheme 1).The peak eluted directly after 3 was also detected in transformants with

MATa ura3-52 his3Δ1 leu23,112 trp2frag::KlURA3

KAB20

tryptophan overproducer

MATa ura3-52 his3Δ1 leu23,112 trp2fbr

KAB24

DMAPP overproducer

MATa ura3-52 his3Δ1 leu23,112 GAL1p-HMG1t

KAB26

tryptophan and DMAPP overproducer

MATa ura3-52 his3Δ1 leu23,112 trp2fbr GAL1p-HMG1t

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capable to produce up to 450 mg of prenylated compounds per liter of culture. The ratios of 2a to 2b were found to be 2.7:1, 2.5:1 and 3:1 in KAB26 cultures expressing fgaPT2, 5-dmats and 7-dmats, respectively (Figure S7). Similarly, the ratio of 4a to 4b upon fgaPT2 or of 8a to 8b upon 7-dmats expression is about 2.5:1. The value for 6a to 6b at 1.8:1 in 5-dmats transformants differs slightly (Figure S6). However, the data from the transformants are very similar to that of MTCA in untransformed wild-type cultures. Hence, the prenylation of tryptophan does not influence the Pictet-Spengler reaction regarding the stereoselectivity, but significantly increases the product yields of this reaction by hijacking the tryptophan from the primary metabolism. LC-MS analysis revealed no difference on tryptophan content, at 0.1 µg/ml, between the wild type and the engineered strains.

Figure 5: Formation of 4-DMAT (dashed line, 3) and 7-DMAMTCA (solid line, 4a & 4b) upon fgaPT2 expression over time in different S. cerevisiae strains. Quantification was carried out on HLPC with absorptions at 277 nm. The data were obtained from three independent experiments. The error bars give the standard deviation.

than an increased supply of tryptophan. Integration of the additional truncated HMG1 allele in S. cerevisiae genome led to a four-fold increase in the formation of 4 compared to the wild type. This is similar to the five-fold increase observed by Ro et al. for the production of artemisinic acid with the same modification in the DMAPP biosynthetic pathway.39 The concentration of DMAPP itself in the cells is still very low, so that no DMAPP was detected by LC-MS with a detection limit of ca. 9.5 µg per ml cultures. Increasing the structural diversity of DMAMTCAs. After successful production of MTCA derivatives with a C7-prenyl moiety, the fungal tryptophan prenyltransferases 5- and 7-DMATS were employed to achieve prenylations at other positions of the MTCA molecule. The constructs pKAB24 and pKAB25 in pYES2-NT C were used for expression of 5- and 7-dmats in the DMAPP and tryptophan overproducing strain KAB26. Formation of prenylated tryptophan and MTCA derivatives was clearly detected by LC-MS in both transformants eight hours after induction. In analogy to the fgaPT2-transformant, the prenylated tryptophan derivatives were identified by comparison with products of both enzymes and the prenylated MTCA by isolation and structure elucidation with MS and NMR analyses. Approximately 400 mg/l of 8and 10-DMA-MTCA (6 and 8) were determined for 5- and 7-dmats transformants, respectively. This means that much higher product yields were obtained by 5- and 7dmats expression than in fgaPT2 transformants. Additionally, a maximum of 12 mg/l and 50 mg/l of the respective DMAT (5 and 7) was detected (Figure 6). In total, the DMAPP and tryptophan overproducing strain KAB26 is

Expression of the fungal DMATS genes does not influence the growth of the producing yeast cells (Figure S3 in the supplemental material). Therefore, DMA-MTCA derivatives are likely not toxic to the cells. Isolation and Structure elucidation of the DMAMTCA derivatives. For structure elucidation, the prenylated MTCAs were isolated on a CHIRALPAK Zwix(+) column as described in the method section. Two products each, 4a and 4b, 6a and 6b, as well as 8a and 8b, were obtained in high purity from transformants carrying fgaPT2, 5-dmats and 7-dmats, respectively. Highresolution mass spectrometry (Table S1) confirmed the monoprenylation in all the six structures by detection of the [M+H]+ ions, which are 68 daltons larger than those of 2a and 2b. Characteristic signals for one regular prenyl moiety at δH 3.3-3.6 (2H), 5.2-5.4 (1H) and approx. 1.7 ppm

Figure 6: Formation of DMAT (dashed lines; 3, 5 or 7) and DMA-MTCAs (solid lines; 4a and 4b, 6a and 6b or 8a and 8b) in the tryptophan and DMAPP overproduction strain KAB26 upon expression of fungal tryptophan prenyltransferase genes. The data are obtained from two independent experiments. The error bars give the standard deviation.

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(2x3H) were indeed observed in their 1H NMR spectra (Table S2, Figures S8, S11, S14, S27, S41, and S44 in the supplemental material). Furthermore, signals for three aromatic protons each were detected, indicating the prenylations at the benzene ring. Comparison of the coupling pattern and chemical shifts of these protons with those described in the literature revealed their regiospecific prenylations. The substitution position of 4a and 4b corresponded well to that of the enzyme product of tryptophan with FgaPT2,30,31 6a and 6b to that of 5-DMATS,32 and 8a and 8b to that of 7-DMATS.33 In addition, 1H13 C HSQC and 1H-13C HMBC NMR spectra were also obtained for 6a, 6b, and 8b (Figures S21, S24, S35, S38, S48, and S51). As given in Table S2, the substitution pattern in 6a, 6b, and 8b can be unequivocally confirmed by 1H-13C HMBC connectivities. This proved that each prenyltransferase catalyzes the same prenylation of tryptophan in S. cerevisiae and in its natural producer. Comparison of the 1H NMR data of the product pairs, i.e. 4a and 4b, 6a and 6b, or 8a and 8b, with each other, revealed clearly that the protons of the benzene ring and of the dimethylallyl moiety share similar chemical shifts. Significant differences were observed for the chemical shifts of H-1, H-3, and H-4, indicating their differences in the stereochemistry. More importantly, the resonances of H-1 at δH 4.43-4.45 (q) and H-3 at δH 3.52-3.54 ppm (dd) of 4a, 6a, and 8a are very similar to each other, but differ clearly from the corresponding signals of 4b, 6b, and 8b at δH 4.52-4.55 (q) and 3.67-3.73 ppm (dd), respectively. These data provide evidence for the same configuration of 4a, 6a, and 8a, whereas 4b, 6b, and 8b share another one. It can be expected that the stereochemistry at C-3 in the structures of the prenylated MTCAs remains unchanged from tryptophan. Thus, the configuration of 4a, 6a, and 8a differs from that of 4b, 6b, and 8b solely at C-1. For confirmation of the stereochemistry, 1H-1H NOESY NMR spectra were taken for all of the six products. In the spectra of 4a and 8a, clear correlations between H-1 (δH 4.45, q) and H-3 (δH 3.52-3.54, dd) were observed. In contrast, correlations between H-3 (δH 3.67-3.73, dd) and H-15 (δH 1.51-1.57, d), i.e. the methyl group at C-1, were detected in the NOESY spectra of their stereoisomeric counterparts 4b and 6b (Figure S54, Figures S9, S12, S32 and S42). These data prove the configuration of 4a, 6a, and 8a to be (1S,3S) and that of 4b, 6b, and 8b to be (1R,3S). Conclusions. In this study, we demonstrated that the β-carboline MTCA can also be found in S. cerevisiae cultures under laboratory conditions. This ability was successfully applied for the production of prenylated MTCAs by introducing just one gene encoding a fungal tryptophan prenyltransferase as the key enzyme into the yeast strain. The baker´s yeast provides not only the three precursors DMAPP, tryptophan and acetaldehyde for prenylated MTCAs, but also generates the acidic condition for the Pictet-Spengler reaction during its growth. Strain design, especially DMAPP overproduction, led to the accumulation of up to 400 mg of prenylated MTCAs per

liter in the yeast culture. These compounds can then be used as a starting point for further modifications such as decarboxylation, hydroxylation, O-methylation etc., leading to prenylated β-carboline derivatives like DMAharman, DMA-harmol or DMA-harmine. This study combined the advantage of the different tryptophan prenyltransferases, i.e their feature for regiospecific prenylation, with the unspecific non-enzymatic Pictet-Spengler reaction for production of different prenylated MTCAs. Only the two stereoisomers with the designed prenylation position, which is controlled by the prenyltransferases, were obtained from the respective transformants. Methods Materials. The E. coli strain DH5α (Invitrogen, Karlsruhe, Germany) was used for cloning. The genotypes of the employed S. cerevisiae strains are given in Table 1. All plasmids used in this study are given in Table S3 in the supplemental material. The oligonucleotide sequences are listed in Table S4. Culture conditions. E. coli cells were cultivated at 37 °C in LB medium. Carbenicillin was added to a final concentration of 50 µg/ml for selection. Yeast full medium contained 1% yeast extract, 2% peptone and 2% glucose. Synthetic medium comprises 0.67% yeast nitrogen base with ammonium sulfate, 0.065% CSM-his-leu-ura (MP Biomedicals, Illkirch, France) and histidine, leucine and uracil if necessary according to Sherman,40 with 2% glucose or galactose as a carbon source. To obtain synthetic medium without tryptophan, all required amino acids were added to yeast nitrogen base with ammonium sulfate according to Sherman.40 S. cerevisiae cultures were maintained at 30 °C. Strain construction. The tryptophan overproduction strain KAB20 was constructed by changing a cytosine to thymidine at position 227 within the genomic TRP2 gene according to Graf et al.34 For this purpose, the part of the TRP2 gene, which contains the position to be exchanged was replaced by a KlURA3 cassette. This marker was amplified from pUG7241 using the oligonucleotides dTRP2fFor and dTRP2fRev. Transformation of HOD114-2B with the PCR product yielded the strain KAB1. In parallel, a part of the TRP2 gene was PCR amplified with the oligonucleotides TRP2f-HindFor and TRP2f-EcoRev. The PCR product was inserted into pGEM-T Easy (Promega, Mannheim, Germany) yielding pKAB3, which was then subjected to site-directed mutagenesis PCR with the oligonucleotides TRP2mutFor and TRP2mutRev yielding pKAB6. The mutated part of TRP2 was then cut out with EcoRI and transformed into the strain KAB1, where it replaced the KlURA3 marker. The expected strain KAB20 carrying a feedback-resistant TRP2 allele was selected with 1 mg/ml 5-FOA and confirmed by PCR with the oligonucleotides contrTRP2for and contrTRP2rev as primers.

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For construction of the DMAPP overexpression strain S. cerevisiae KAB24, the plasmid pδ-HMGt containing a truncated allele of HMG1 was linearized by restriction with XhoI and subsequently integrated into the yeast genome at δ-sites following the procedure described by Ro et al.39 The transformants were tested for the formation of prenylated products after expression of the tryptophan prenyltransferase gene fgaPT2. The transformant with the highest product yield was chosen for all further experiments. S. cerevisiae KAB26 carrying the mutated TRP2 and the truncated HMG1 allele was used as DMAPP and tryptophan overproducing strain. Construction of DMATS expression plasmids. pIU11 for fgaPT2 expression in S. cerevisiae was described previously.31 The gene encoding 5-DMATS was amplified from pYL932 with the oligonucleotides 5DMATS-Eco and 5DMATS-Xba. The gene encoding 7-DMATS was amplified with the oligonucleotides 7DMATS-Eco and 7DMATS-Xba from pLW40.42 The resulting PCR products were cloned into pGEM-T Easy to get the plasmids pKAB20 and pKAB21, respectively. The coding sequences were then released from pKAB20 and pKAB21 by restriction with EcoRI and XbaI and subcloned into pYES2NT C, yielding the expression constructs pKAB24 and pKAB25, respectively. Detection of MTCA formation in yeast cultures. Synthetic complete medium was inoculated with an overnight culture to an absorption of 0.1 at 600 nm. Culture samples were taken at the different time points and mixed directly with the same volume of ice cold methanol. These mixtues were stored at -20 °C until further analysis by LC-MS. The amount of MTCA was quantified with a calibration curve obtained with commercial MTCA (SigmaAldrich, Steinheim, Germany) and the software Compass QuantAnalysis (Bruker Daltonik, Bremen, Germany). Cultivation for detection of the prenylated products. The yeast strains were transformed with the respective prenyltransferase-expressing constructs with the lithium acetate method according to Gietz and Woods.43 The transformants carrying the expression constructs were then precultured in 5 ml synthetic medium lacking uracil. These cultures were used to inoculate the main cultures to an absorption of 0.1 at 600 nm. For induction of gene expression, the main cultures were harvested 24 hours after inoculation, washed once with sterile H2O and resuspended in an equal volume of synthetic medium without uracil containing 2% galactose as a carbon source. Samples were taken at the indicated time points and mixed with an equal volume of ice cold methanol. These mixtures were stored at -20 °C until further analysis. Isolation of the prenylated products. The transformants were grown and induced as described above. Three days after induction, the cultures were mixed with an equal volume of 1-butanol and mixed on a shaker for 1 hour at room temperature. Afterwards, the butanol

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phase was evaporated until dryness and dissolved in methanol. After adjusting the pH value to 1.0 by addition of HCl, the extract was applied to cation exchange chromatography with Chromabond SA (Macherey-Nagel, Düren, Germany) following the procedure described in the publications by Adachi et al.10 and Herraiz.11 The eluate was extracted again with 1-butanol, evaporated to dryness and dissolved in methanol. The solutions were then applied to an Agilent HPLC 1200 series equipped with a CHIRALPAK Zwix(+) column (3 µm 0.4 cm x 15 cm, CHIRAL Technologies Europe, Illkrich, France) to separate the DMA-MTCA diastereomers. Elution was carried out with THF:MeOH:H2O (49:49:2) containing 25 mM diethylamine and 50 mM formic acid as solvent at a flow rate of 0.5 ml/min. The fractions of interest were collected, concentrated and subjected to reverse phase chromatography with a Multospher 120 RP 18 HP column (5 µm 250 x 10 mm, CS-Chromatographie Service GmbH, Germany) for further purification. Elution was performed with a gradient of 40% to 100% methanol in water in 30 min. Chemical formation of MTCA in synthetic yeast medium. The reaction mixtures were prepared in synthetic yeast media without tryptophan. The pH values of these media were set to 2, 3, 4 and 6.25, respectively, prior to sterilization. After sterilization, acetaldehyde and tryptophan were added to final concentrations of 44.5 mM and 1 mM, respectively. After incubation for 6 h at 18 °C, the reaction mixtures were mixed with an equal volume of ice cold methanol and subsequently analyzed on HPLC as described below. HPLC analysis. The samples were analyzed on an Agilent 1200 series HPLC equipped with an Agilent Eclipse XDB C18 column (5 µm 4.6 x 150 mm), unless otherwise indicated. Water and acetonitrile, both containing 0.1% formic acid, were adjusted to a flow rate of 0.5 ml/min. A gradient from 5% to 65% acetonitrile in water in 30 min was used for separation. LC-MS analysis. LC-MS analysis was carried out with an Agilent HPLC1260 series system equipped with a photodiode array detector and a Bruker micrOTOF-Q III mass spectrometer by using an Agilent Eclipse XDB C18 column (5 µm 4.6 x 150 mm). Water and acetonitrile, both containing 0.1% formic acid, were adjusted to a flow rate of 0.5 ml/min. A gradient from 5% to 65% acetonitrile in water in 30 min was used for separation. For data evaluation, Compass DataAnalysis software (Bruker Daltonik, Bremen, Germany) was employed. NMR analysis. The samples were dissolved in DMSOd6 and filled into Wilmad 3 mm NMR tubes (Rototec Spintec). One-dimensional (1D) 1H and 13C as well as twodimensional (2D) 1H-1H NOESY and 1H-13C HMBC experiments were performed on a Bruker Avance III 500 MHz spectrometer equipped with a 5 mm N2-cryo-probe Prodigy BBO. The 1D 13C spectra were acquired with 65536 data points and 24000 to 32000 transients, while the 2D HMBC

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spectra were collected with 4096 points and 32 to 64 transients in the F2 dimension and 512 increments in the F1 dimension. Mixing time for NOESY was 3 s. The spectra were processed with Bruker program package Topspin 3.1 and MestReNova version 6.0.2-5475. Chemical shifts of the 1H and 13C data were referenced to the solvent signal at 2.50 and 39.52, respectively.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. The plasmids and oligonucleotides used in this work, LC-MS analysis of MTCA and 4-DMAT; diastereomeric ratios of (1S,3S)- and (1R,3S)-MTCA and their prenylated derivatives in cultures expressing tryptophan prenyltransferases; growth curve and pH of a yeast culture expressiong tryptophan prenyltransferase; NMR spectra of 4a, 4b, 6a, 6b, 8a and 8b; selected NOESY correlations of 4a and 4b. Abbreviations MTCA - 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid; DMATS – dimethylallyltryptophan synthase, DMAMTCA – dimethylallyl-MTCA, DMAPP – dimethylallyl diphosphate

AUTHOR INFORMATION Corresponding Author * Tel +49 6421 28 22461. E-Mail: [email protected]

ORCID Shu-Ming Li: 0000-0003-4583-2655 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Katja Backhaus is partially financed by the LOEWE program of the state of Hesse (SynMikro to S.-M. Li). Shu-Ming Li acknowledges the Deutsche Forschungsgemeinschaft for funding of the Bruker micrOTOF QIII mass spectrometer. We thank Rixa Kraut and Florian Kindinger (both University of Marburg) for taking mass spectra and Jürgen J. Heinisch (University of Osnabrück) for the kind gift of the S. cerevisiae strain HOD114-2B. We are grateful to J. Keasling (University of California, Berkeley) for providing the plasmid pδ-HMGt.

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Manipulation of the Precursor Supply in Yeast Significantly Enhances the Accumulation of Prenylated β-Carbolines Katja Backhaus, Lena Ludwig, Xiulan Xie, Shu-Ming Li*

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