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A Combinatorial Approach To Study Cytochrome P450 Enzymes for

Nov 26, 2018 - Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid ...
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Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX

A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker’s Yeast Nicholas D. Gold,†,∇ Elena Fossati,†,∇,¶ Cecilie Cetti Hansen,†,∥,⊥ Marcos DiFalco,‡ Veronique Douchin,# and Vincent J. J. Martin*,†,‡,§ †

Centre for Applied Synthetic Biology, Concordia University, Montréal, Québec H4B 1R6, Canada Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec H4B 1R6, Canada § Department of Biology, Centre for Structural and Functional Genomic, Concordia University, Montréal, Québec H4B 1R6, Canada ∥ Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark ⊥ Center for Synthetic Biology, University of Copenhagen, DK-1871 Frederiksberg C, Denmark # Evolva, Lersø Parkallé 42-44, DK-2100 Copenhagen Ø, Denmark

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S Supporting Information *

ABSTRACT: Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPHdependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis. KEYWORDS: cytochrome P450 monooxygenase, cytochrome P450 reductase, steviol glucosides, diterpenoid biosynthesis

T

advantage for recombinant production. While the promiscuity of UGTs represents a challenging obstacle for recombinant SG production, Olsson et al. were able to tune the type and titer of SGs produced by generating UGT mutants selected for increased or specific accumulation of Reb D and Reb M in S. cerevisiae.1 Generating SG precursors up to steviol from glucose has been a matter of coordinating known bioactivities. Increasing isoprenoid levels in the mevalonate pathway is well established both in Escherichia coli and in S. cerevisiae as seen in artemisinin work.9,10 Geranylgeranyl pyrophosphate (GGPP) is formed from the condensation of farnesyl pyrophosphate and isopentenyl pyrophosphate by GGPP synthase (GGPPS). Copalyl pyrophosphate synthase (CPPS) converts GGPP to copalyl pyrophosphate (CPP), which is in turn converted to

he identification of the genes responsible for production of the sweet diterpene steviol glycosides in Stevia rebaudiana has led to the generation of steviol glucosides (SGs) in recombinant hosts.1−3 Using the GRAS approved synthetic biology chassis organism Saccharomyces cerevisiae, the sweetener stevia could soon be mass produced in bioreactors rather than extracted from agricultural crops. The stevia plant accumulates more than 30 different steviol glycosides, the majority being SGs, which are produced by the action of just four UDP glucose-dependent glucosyltransferases (UGTs) on the SG backbone steviol through a complex web of glucosylations.4−6 Some of these SGs, like the major components stevioside and rebaudioside A (Reb A), are undesirable because of their bitter aftertaste,7 while some minor SG components like Reb D and Reb M have shown promise as next generation sweeteners due to their sweetness and pleasant organoleptic properties.8 Thus, customizing SG production by selective UGT deployment is a potential © XXXX American Chemical Society

Received: November 9, 2018 Published: November 26, 2018 A

DOI: 10.1021/acssynbio.8b00470 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology

Figure 1. (a) Reaction scheme for conversion of the tetracyclic diterpene kaurene to steviol by two cytochrome P450 enzymes in succession. Kaurene oxidase (KO) hydroxylates the C19 position of kaurene, forming kaurenol, which it then further oxidizes to kaurenal and then to kaurenoic acid. Kaurenoic acid hydroxylase (KAH) then hydroxylates the C13 position of kaurenoic acid, giving rise to steviol. KO and KAH require the support of a NADPH-dependent cytochrome P450 reductase (CPR) to provide electrons for fixation of molecular oxygen. The affected carbon at each step is highlighted in blue. (b) Recombinant steviol glucoside pathway in Saccharomyces cerevisiae. Mevalonate pathway products farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP) are condensed by geranylgeranyl pyrophosphate synthase (GGPPS) to form GGPP. GGPP is then converted to copalyl pyrophosphate (CPP) by CPP synthase (CPPS), and then to kaurene by kaurene synthase (KS). Kaurene-to-steviol conversion is accomplished by KO and KAH with the support of a CPR, all of which are bound to the endoplasmic reticulum. Steviol can then be decorated by various UDP glucose-dependent glucosyltransferases (UGTs) to form steviol glucosides. Other abbreviations: Acetyl-CoA, acetyl coenzyme A; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; NADPH, nicotinamide adenine dinucleotide phosphate.

kaurene by kaurene synthase (KS)11 (Figure 1). Kaurene-tosteviol conversion requires the action of two cytochrome P450 enzymes (CYPs) in succession: kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO catalyzes three successive oxidations at the C19 of kaurene; first a hydroxylation to form kaurenol, followed by oxidation to kaurenal and then to kaurenoic acid. Subsequently, KAH hydroxylates the C13 position of kaurenoic acid, giving rise to steviol. Microsomal CYP monooxygenases are membrane-associated hemoproteins whose activity depend on the transfer of two electrons catalyzed by NADPH-dependent cytochrome P450 reductases (CPR), which are also membrane-bound.12,13 In the endoplasmic reticulum (ER) of plants, CYP and CPR function as a complex held together by protein−protein interactions, where the CPR is the node around which CYP subunits and other associated proteins organize themselves.14−19 Optimization of engineered CYP activity for recombinant production of marketable bioproducts is notoriously difficult.20 Benchmark work involving CYP optimization is demonstrated in the

artemisinin work by Paddon et al.10 CYPs can also function in a system with microsomal cytochrome b5 (CB5) membranebound hemoproteins, which require a NADH-dependent CB5 reductase (CBR) flavoprotein.21,22 The recent watershed work by Laursen et al. on the biosynthesis of the cyanogenic glucoside dhurrin in sorghum demonstrated copurification of CYPs with CPR, but also CB5 and CBR were enriched in the purified sample.15 Wang et al. recently produced SGs in E. coli, but reported a bottleneck at the KAH step, which they were able to improve upon using a KAH functional homologue from Arabidopsis thaliana rather than S. rebaudiana; however, kaurenoic acid was still found to accumulate.2 The steviol precursors kaurenol and kaurenoic acid can also be glucosylated by UGTs, diverting intermediates from the production of steviol,3 which is another reason for the need to maximize the efficiency of kaurene to steviol conversion. In engineering a recombinant pathway involving CYPs, choice of CPR for optimal CYP turnover, correct subcellular localization to the ER, and expression level or copy number modulation are important considerations.10,23 B

DOI: 10.1021/acssynbio.8b00470 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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driven by electrons donated by Ncp1 only as a baseline case. For the KOs in general, the plant CPRs did not contribute to significantly better activity than the endogenous Ncp1 (Figure 2a). SrKO showed only slightly higher activity with SrCPR1

The present work demonstrates the full reconstituted biosynthetic pathway to produce the SG precursor steviol from the simple sugar glucose in a S. cerevisiae host, focusing on optimization of the KO-KAH activities by testing, in batch growth, combinations of plant homologues of two KOs, two KAHs, and five CPR.



RESULTS Optimizing CYP-CPR Combinations. Our goal was to maximize kaurene-to-steviol conversion and limit side product accumulation in S. cerevisiae by identifying optimal KO-KAHCPR combinations from a small collection of functional homologues (Table 1). Our test proteins included two KOs, Table 1. Heterologous Genes Used in This Study gene GGPPS7 tCPPS5 AtKS5 55-CPPS-KS-6 SrKO (CYP701A5) KO75 (CYP701) SrKAH (CYP81) KAH82 (CYP72) SrCPR1 SrCPR7 SrCPR8 CPR12 ATR2

function or activity

promoter/ terminator

Geranylgeranyl pyrophosphate synthase Truncated copalyl pyrophosphate synthase Kaurene synthase CPPS-KS bifunctional Kaurene oxidase

pALD4

Kaurene oxidase Kaurenoic acid hydroxylase Kaurenoic acid hydroxylase Cytochrome P450 reductase Cytochrome P450 reductase Cytochrome P450 reductase Cytochrome P450 reductase Cytochrome P450 reductase

pTEF1/tPGI1 pFBA1/tADH1 pFBA1/tADH1 pPMA1/tTPI1 pPMA1/tTPI1 pPMA1/tTPI1 pPMA1/tTPI1 pPMA1/tTPI1

pTEF1 pPGK1 pHXT7 pTEF1/tPGI1

Figure 2. In vivo activity of individual CYP-CPR combinations by substrate supplementation at the time of inoculation. CYP or CYPCPR combinations were expressed in S. cerevisiae IS1 wild type. Strains were grown for 4 days in the presence of the appropriate substrate. Concentration values for the product (right y-axis) are averaged from clonal biological replicates (n = 3), with error bars representing ±95% confidence intervals. Percent yield values (left yaxis) were calculated as 100% × product concentration/substrate concentration. The endogenous yeast CPR Ncp1 was present in all strain iterations. (a) Strains expressing a KO alone or with a heterologous CPR were grown in the presence of 100 μM kaurene as substrate. Kaurenoic acid (KA) is the product of KO activity. (b) Strains expressing a KAH alone or with a heterologous CPR were grown in the presence of 150 μM kaurenoic acid as substrate. Steviol is the product of KAH activity.

two KAHs, and five CPRs. Five sequences originated from S. rebaudiana: SrKO (CYP701A5), SrKAH (CYP81), SrCPR1, SrCPR7, and SrCPR8. Our study also included Arabidopsis thaliana CPR ATR2, as well as three sequences from a patent3 designated here as KO75 (CYP701), KAH82 (CYP72), and CPR12. To the end of discovering the best KO-KAH-CPR combinations, we first sought to identify the most favorable CPR partner for each CYP individually. Individual CYP-CPR pairs were analyzed for in vivo activity in S. cerevisiae IS1, a wild-type haploid strain of baker’s yeast used in industrial applications. We also examined subcellular (co)localization by microscopy and protein production levels by immunoblot. These data informed our choices in creating productive KOKAH-CPR combinations, which were assembled into host strains also expressing constructs enabling production of kaurene from glucose. Expression cassettes were integrated into S. cerevisiae IS1, either alone or as CYP-CPR pairs in every permutation (Table S1). In all strain combinations, the endogenous S. cerevisiae genes ERG5 and ERG11, encoding CYPs involved in ergosterol biosynthesis were not deleted from the host strain, nor was the endogenous gene NCP1, encoding the CPR associated with the ergosterol pathway. In vivo activity of each CYP was tested with each heterologous CPR by growing strains to late stationary phase in medium supplemented at the point of inoculation with the appropriate substrate. Culture extracts were analyzed for KO activity by the production of kaurenoic acid from kaurene or for KAH activity by the production of steviol from kaurenoic acid. When no plant CPR was expressed, CYP activity was

and SrCPR7 compared to Ncp1, while the other heterologous CPRs contributed to lower kaurenoic acid production. KO75 had highest activity with Ncp1, while second best production was provided by SrCPR8. Lowest KO75 activity was obtained when coexpressed with SrCPR1. Conversely, the KAHs were generally more productive with plant CPRs than with Ncp1 (Figure 2b). While all S. rebaudiana CPRs were equally productive with KAH82, SrCPR1 was clearly the best coupling partner for SrKAH (Figure 2b). Both KAHs performed poorly in the absence of a plant CPR but also in the presence of ATR2. The best of the KO75-plant CPR combinations marginally outperformed the best of the SrKO combinations (∼10%). The best of the KAH82 combinations outperformed the best of the SrKAH combinations by around 30%. No significant differences in growth were observed between strains, and no significant toxicity effect was observed in wild-type cells when grown with 100 μM kaurene, kaurenoic acid, or steviol. Strains coexpressing pairs of CYP-mCherry and CPR-GFP fusions were examined by fluorescence microscopy in early log phase growth to assess each protein’s subcellular localization. Figure 3a shows the red and green channels corresponding to signals from CYP-mCherry and CPR-GFP fusions, respecC

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Figure 3. Evaluation of CYPs and CPRs coexpressed in S. cerevisiae IS1 wild type. (a) Subcellular (co)localization of CYP-mCherry fusions and CPR-GFP fusions in early log phase by fluorescence microscopy. For each CYP-CPR combination, the red (mCh) and green (GFP) channels are shown as grayscale images for the CYP and CPR fusions, respectively. Scale bar is 5 μm. White arrows indicate an example of the perinuclear space for the SrKO-SrCPR7 strains. A yellow arrow shows an example of a fraction of SrKO that did not coincide with SrCPR7 signal. (b) Protein expression and presence up to 3 days by immunoblot. Highest MW band(s) corresponding to the expected size for each full-length protein is shown over 4 time points: 8, 24, 48, and 72 h. Anti-PSTAIR for cyclin-dependent kinases was used as loading control (ctrl). Gray boxes indicate immunoblots collected in a separate experiment under identical conditions.

CPR sequences targeted their respective fusion proteins to the ER, even though in some cases a fraction of CYPmCherry fusion protein was observed outside of the ER where it did not colocalize with CPR-GFP fusion. Protein production of CYPs and CPRs, alone or coexpressed, was assessed by immunoblotting. Figure 3b shows the highest MW band(s) that corresponded to the expected sizes of the full-length proteins. In the cases of the KAH variants, both variants consistently exhibited a double banding pattern, possibly due to glycosylation. On the basis of band intensity, all the CYPs were produced to more or less the same level and were present up to 72 h, regardless of whether they were expressed alone or with a CPR. This stability of CYPs was

tively. Images for CYP-mCherry and CPR-GFP fusions expressed separately are shown in Figure S1. Signals for all CYP-mCherry and CPR-GFP fusions were observed to delineate the perinuclear space, indicating protein localization to the ER (Figure 3a; white arrows show example). In some instances, signal for CYP-mCherry fusion suggested protein aggregation in the cytoplasm (Figure 3a; yellow arrow shows example), which was not observed in the same area as the coexpressed CPR-GFP fusion. The S. rebaudiana CYPs provided fainter signals compared to KO75 and KAH82. No observations with respect to specific CYP-CPR combinations were noted. Generally, the predicted N-terminal transmembrane helices (TMH) on each of the native CYP and D

DOI: 10.1021/acssynbio.8b00470 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology observed in spite of the fraction of CYP protein observed by fluorescence microscopy as possibly aggregated outside of the ER (Figure 3a). Only KAH82 in coexpression with SrCPR1 exhibited a marked decrease in band intensity after 24 h: at 24 h, the lower of the two closely spaced bands became more intense, and at 48 and 72 h both bands were faint. The CPRs, when expressed alone, behaved differently. SrCPR1, CPR12 and ATR2 full-length protein bands were present up to 72 h, whereas the SrCPR7 and SrCPR8 bands were faint after 8 h. Protein production of SrCPR1 was marginally lower in the presence of a KO. SrCPR7 band intensity improved somewhat in the presence of SrKO and more so in the presence of either KAH. SrCPR8 production only seemed to improve slightly in the presence of a KAH. CPR12 band intensity decreased when paired with a KO, but increased in the presence of a KAH. Finally, ATR2 band intensity was constant over time in the absence of a heterologous CYP and with SrKAH, but decreased at 24, 48, and 72 h when coexpressed with the other CYPs. Taken together, our CYP-CPR coexpression data suggests that coexpression of the two proteins will influence their stability, sometimes showing a stabilizing effect, as with SrCPR7 combined with SrKO or KAH, or a destabilizing effect such as with CPR12 combined with KOs. KAH82-CPR Activity over Time Tested by Staggered Substrate Supplementation. In light of our CYP-CPR activity and production data we postulated that protein stability would affect CYP activity over an extended steviol production period. To test this postulate, kaurenoic acid was added at different times to cultures of strains expressing KAH82 with SrCPR1, SrCPR7, CPR12, or no plant CPR, and steviol production was analyzed after 6 days (Figure 4a). KAH82 paired with SrCPR1 or SrCPR7 produced high steviol amounts when substrate was provided at the time of inoculation, but production was highest when it was added to the culture broth at mid log growth (14 h). Addition of kaurenoic acid at 24 h produced 40−50% less steviol, and additions at later time points produced ∼90% less. KAH82CPR12, on the other hand, at best produced only about 60% compared to the SrCPR1 and SrCPR7 pairings, but that production was consistent whether the substrate was added at 0, 14, or 24 h. Even when kaurenoic acid was added at 48 h, KAH82-CPR12 still produced around 50% of its 0−24 h outputs, thereby doubling that of the SrCPR1 or SrCPR7 pairings. These activity patterns agree with the activity and immunoblot data (Figure 2B and 3b) for SrCPR1 and CPR12. The KAH82-SrCPR1 pair is highly active, but activity is reduced after 24 h, possibly due to degradation of the CYP protein. In the case of the KAH82-CPR12 pair, CYP activity is lower but sustained for a longer period, possibly due to a more stable CYP. Conversely, the staggered substrate assay results did not agree with our activity and immunoblot data (Figure 2b and 3b) for SrCPR7 and no CPR. With these two CPRs, the KAH82 appears to be stable up to 72 h even though activity is reduced after 24 h. KAH82-CPR Pairs Coexpressed with ATR2. Because NCP1 was not deleted from any of our test strains, the results from the initial activity assay suggested that in the presence of two different CPRs, the activity of a CYP is determined by one CPR more than the other. For example, the coexpression of each of SrKO, KO75, and SrKAH with ATR2 led to lower product yields than in the presence of the only native CPR Ncp1. In the case of KAH82, coexpression of ATR2 resulted in higher steviol yield than with the native Ncp1 only, but still

Figure 4. KAH82 substrate supplementation assays. Concentration values are averaged from clonal biological replicates (n = 3), with errors expressed as 95% confidence intervals. (a) KAH82-CPR stability by activity test with staggered addition of substrate. KAH82 paired with SrCPR1, SrCPR7, CPR12, or no CPR (native yeast CPR Ncp1) was grown in batch YPD. Substrate (100 μM kaurenoic acid) was added at either time 0, 14, 24, 41, or 48 h. Cultures were sampled at 6 days and analyzed for steviol production. (b) CPR competition for KAH82 at 4 days. Substrate (150 μM kaurenoic acid) was added at time of inoculation. The steviol production of strains coexpressing KAH82 and SrCPR1, SrCPR7, SrCPR8, or CPR12 was compared without and with a copy of ATR2 added. KAH82-ATR2 was included as control. Strains were grown as batch YPD cultures and sampled at 4 days.

lower steviol yield than with any of the other heterologous CPRs (Figure 2b). To further explore this phenomenon, a copy of ATR2 was added at the Fgf20 XVI site of strains expressing KAH82-CPR pairs (Table S1). ATR2 was chosen because it had produced the least amount of steviol of any CPR when paired with KAH82. These strains were grown alongside the original KAH82-CPR strains and tested as before with 150 μM kaurenoic acid as substrate (Figure 3b). As before, KAH82-ATR2 produced the least steviol compared to pairings with the other CPRs. In the case of both SrCPR1 and SrCPR7, the additional presence of ATR2 negatively affected steviol production, resulting in a steviol yield equal to or lower than that of KAH82-ATR2. KAH82-SrCPR8 with ATR2 produced the same activity as KAH82-CPR8. Finally, the presence of ATR2 did not change the apparent activity of KAH82-CPR12; however, KAH82-CPR12 activity was comparable to KAH82-ATR2 control to begin with. Coexpression of KOs, KAHs and CPRs in a KaureneProducing Cell. Even though the KOs were most productive with the endogenous yeast CPR in the substrate supplementation assays (Figure 2a), any KO-KAH combination would require coexpression with a heterologous plant CPR since KAH activity was nonoptimal with Ncp1 alone (Figure 2b). To identify the best CYPs-CPR combination, permutations of one KO, one KAH, and one CPR were tested for production of steviol in a kaurene producing host. Every permutation of KOKAH was combined with SrCPR1 or SrCPR7 (Table S2). SrCPR8 and CPR12, however, were only paired with E

DOI: 10.1021/acssynbio.8b00470 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Figure 5. Optimization strategies of KO-KAH-CPR combinations in S. cerevisiae strains producing kaurene in vivo. Strains were grown as YPD batch cultures and sampled at 7 days for production of steviol (red), kaurenoic acid (KA; yellow), kaurenal (green), and kaurene (blue). Values are averaged from clonal biological replicates (n = 3), with error bars representing ±95% confidence intervals. Red brackets show significant differences in steviol (p < 0.05) between adjacent columns. (a) KO-KAH-CPR strains in ascending order of steviol output, left to right. Combinations including SrKAH-SrCPR1 were significantly better than any other. (b) Modulation of gene copy number for the KO75-KAH82-SrCPR1 combination. Strains in ascending order of steviol output, left to right.

next four best strains were combinations with SrCPR7: KO75KAH82 produced 61 μM steviol, SrKO-SrKAH 53 μM, and KO75-SrKAH 43 μM. KO75-KAH82-SrCPR8 produced 49 μM. CPR12 combinations produced very little steviol (50% from 48 h onward. Kaurenoic acid percent yield from kaurene equivalents was ∼40% for SrKOSrCPR1 from 72 h onward, but with KAH82 added this dropped to 23−29%.



DISCUSSION Our work demonstrates de novo production of the SG precursor steviol from glucose by expression of a recombinant pathway in yeast. Using a combinatorial approach to test functional homologues from our collection of KO, KAH and CPR enzymes, we achieved optimization of kaurene to steviol conversion by the consecutive CYP activities in the SG pathway. The individual testing of CYP-CPR pairs was an imperfect predictor of KO-KAH-CPR behavior because it did not take into account the changes in CYP-substrate affinity24,25 and CYP-CPR affinity26,27 that can occur when multiple CYPs and CPR form a complex in the membrane. The 4 d substrate supplementation assays on individual CYP-CPR pairs correctly identified SrCPR1 as the best coupling partner for most CYPcatalyzed reactions in this study. CPR12 was also correctly identified as a nonoptimal partner. However, both SrCPR7 and SrCPR8 appeared to couple well with KAH82 activity, but the produced steviol only reached average levels when coexpressed with KAH82 in the presence of a KO. A multi-CYP complex is not quite the sum of its individual parts. The complexity of a multi-CYP complex where substrate binding, channelling and overall flux depend on relative expression levels of each component and protein−protein interactions between them (not to mention the membrane itself) makes predicting activity very difficult from the individual parts. Our test system did not take into account possible effects of endogenous yeast Erg5, Erg11 and Ncp1, which may have played a role in competing for and with the heterologous enzymes.28 The phenomenon of competition between two CYPs for a single CPR has been studied in vitro,27,29 but the present work may represent the first documented competition of CPRs for a single CYP in vivo. Coexpression of CPRs with ATR2 in the presence of KAH82 created an apparent competition with sometimes adverse effects on CYP activity. The KAH82-CPR plus ATR2 data imply varying levels of interaction between KAH82 and each CPR. In the case of SrCPR1 or SrCPR7 with ATR2, we can speculate that the affinity of KAH82 for ATR2 was greater than it was for either SrCPR1 or SrCPR7 since the observed KAH activity with either one combined with ATR2 was equal to or lower than that of KAH82-ATR2 control. Likewise, in the case of SrCPR8 (and possibly CPR12), its interaction with KAH82 appears to have been strong enough that it was not disrupted by the coexpression of ATR2. These results caution against pathway engineering strategies that coexpress multiple CPRs if one has a strong affinity for a CYP but provides poor electron transfer and hence activity. CYP-CPR complexes are known to function at CYP:CPR ratios ranging from 5:1 to 20:1 in the ER.15,19,30,31 Our copy number modulation test with KO75, KAH82 and SrCPR1 showed that two copies of KO75 was a requirement for an increase in steviol production pointing to a limitation at the KO step. However, addition of a copy of KO (KO:KAH:CPR ratio of 2:1:1) did not require addition of an extra copy of SrCPR1 to yield higher steviol than a 1:1:1 ratio, suggesting that the KO:CPR ratio is suboptimal at 1:1. In contrast, addition of an extra copy of KAH, at a KO:KAH:CPR ratio of 1:2:1 or 2:2:1, produced more steviol only when an extra copy



MATERIALS AND METHODS Strains and Genetic Materials. An industrial haploid strain of wild-type S. cerevisiae (MATα HOΔura3Δhis3Δleu2Δ), designated here as IS1, was used as the host for all gene integrations.39 Escherichia coli DH5α [F− Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK−, mK+) phoA supE44 λ− thi-1 gyrA96 relA1] was used for plasmid maintenance and storage. The heterologous genes used in this work are listed in Table 1. All nucleotide sequences were codon optimized for expression in S. cerevisiae. Kaurene production constructs used have previously been described in H

DOI: 10.1021/acssynbio.8b00470 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Kishore et al.40 and Houghton-Larsen et al.41 The test CYPs included two KOs and two KAHs, namely SrKO from S. rebaudiana (accession no. AAQ63464) as well as KO75, SrKAH and KAH82, described as polypeptide sequence no. 75, SrKAHe1 and polypeptide sequence no. 82, respectively, in Robertsen et al.3 CPRs included SrCPR1 (accession no. ABB88839) and ATR2 from Arabidopsis thaliana (accession no. CAA46815), as well as SrCPR7, SrCPR8, and CPR12 from Robertsen et al.3 Sequence Analysis. Phylogenetic analyses of CYPs and CPRs were done using MEGA7: protein sequences were aligned using MUSCLE (default settings) and the consensus trees were built using Neighbor-Joining Tree (default settings) with 1000 bootstrap replicates.42 The resulting phylogenetic trees are reported in Figure S3. Phylogenetic analysis of the KO75 sequence suggests that it belongs to the CYP701 family whereas SrKAH is likely a CYP81 member and KAH82 clusters with members of the CYP72 family (Figure S3a). The S. rebaudiana CPRs and ATR2 cluster with class II CPRs, while CPR12 clusters with class I CPRs and among rosid sequences (Figure S3b). Prediction of transmembrane helices (TMHs) was performed using TMHMM Server v. 2.0.43 All the CYP and CPR sequences have a predicted N-terminal TMH. Cloning and Integration of Pathway in S. cerevisiae. CYP and CPR genes were amplified using Phusion DNA polymerase (ThermoFisher) and cloned via restriction enzymes SapI (type IIs) and KasI into pBOT expression plasmids,44 all under constitutive S. cerevisiae promoters (Table 1). CYPs were tagged at their C-terminus with sequences for either mCherry or Myc epitope. CPRs were tagged at their Cterminus with sequences for either GFP or HA epitope. All start codons were preceded directly by Kozak sequence AAAACA and all constructs were verified by Sanger sequencing. Assembled expression cassettes were amplified by PCR from pBOT plasmids to generate DNA for homology directed repair (HDR) of double strand breaks in the chromosome of IS1 wild-type using CRISPR-Cas9. Integration cassettes for HDR were amplified with Phusion DNA polymerase with primers containing 60 bp of homology to either the targeted chromosomal locus and/or to adjacent cassettes being assembled and integrated simultaneously. Primers used to generate repair DNA for integrations are listed in Tables S4 and S5. Gene cassettes were integrated into chromosome XII or XVI, at sites respectively corresponding to USER site XII-1 from Mikkelsen et al.45 and site 20 from Flagfeldt et al.46 We refer to these sites as USER XII-1 and Fgf20 XVI, respectively. A 20-nt PAM sequence for each locus was cloned separately into a modified p426 gRNA plasmid, which had a kanMX selection marker in place of the URA3 cassette:47 GTTGGGAAGAGCCGCGAGTA for USER XII-1 or TCTTGGAATCAGTACATAGC for Fgf20 XVI. For a CRISPR genome editing experiment, host cells were pretransformed with a modified p414 Cas9 plasmid, which had hphNT1 and URA3 cassettes in place of the TRP1 marker.47 Competent cells were grown in the presence of 200 mg/L hygromycin. The gRNA plasmid (1.7−4.2 μg/mL) and each linear repair DNA component for HDR (as nonpurified PCR product, estimated at 2.8−5.6 μg/mL) were cotransformed by the Gietz method.48 Integration transformants were selected on solid standard yeast peptone dextrose (YPD) supplemented with 200 mg/L hygromycin and 200 mg/L G418. Integrants were screened by colony PCR using Phire DNA polymerase (ThermoFisher) to amplify entire integration

cassettes (up to >9 kb). PCR products of positive integrants were further confirmed by Sanger sequencing. Positive integrant strains were cured of the Cas9 plasmid and their growth was tested using a Tecan Sunrise 96-well plate reader to measure cell density at 595 nm at 20 min intervals over a 48 h period, with continuous shaking at 30 °C in between readings. Kaurene production constructs were cloned by the USER method49 into pX-XII integration vectors from Mikkelsen et al.45 (Table 1). For integration of these constructs, the resulting vectors were linearized by digestion with Not I restriction enzyme, purified and then transformed into IS1 as above. Constructs expressing the kaurene pathway were cotransformed, and double integration events were selected by growth on YPD with 200 mg/L hygromycin and 200 mg/L G418. Positive integrants were confirmed by colony PCR as above, and growth was also tested over 48 h. Growth Conditions. Cells were grown in liquid batch cultures in YPD for all experiments unless otherwise specified. Antibiotic G418 at a concentration of 200 mg/L was added to YPD for activity and immunoblot experiments. All liquid cultures were seeded to a starting OD660 of 0.1 from overnight culture, and then grown at 30 °C with 200 rpm shaking. For activity tests in which substrate needed to be supplemented to the medium, cultures were grown in a volume of 0.6 mL in a 10 mL glass tube with either kaurene or kaurenoic acid added to a concentration of 100 or 150 μM. For activity tests in which kaurene was produced from glucose, cultures were grown in a volume of 5 mL in a 25 mL glass tube. Kaurene was a gift from Evolva. Kaurenoic acid and steviol were obtained from Sigma-Aldrich (Oakville, Canada). Kaurene, Kaurenal, Kaurenoic Acid, Steviol, and Steviol Glucoside Analysis. For analysis of kaurenoic acid and steviol, 30 μL of cell broth (or supernatant) was sampled and extracted with 120 μL of acetonitrile (MeCN) into a polypropylene V-bottom 96-well plate sealed with an aluminum adhesive seal. After shaking the plate at 800 rpm and room temperature for 15 min, extracts were centrifuged at 2400g for 5 min. The MeCN extracts were removed to a clean plate and diluted 1/5 or more if necessary to fall within the linear range of the standard curve for either analyte (0.625−40 μM). Samples were analyzed using an Agilent (IM)Q-TOF 6560 MS equipped with a 1290 Infinity II LC system or a Thermo Finnigan LTQ FT MS equipped with a PerkinElmer Series 200 LC. Five μL of sample were separated over a Waters Acquity UPLC BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 50 mm) at a flow rate of 0.2 mL/min, using a mobile phase gradient as follows: 0−3 min 65−85% MeCN in water with 0.1% (v/v) formic acid, then hold at 85% for 2 min. MS data was acquired in negative mode, scanning from 100 to 1000 m/ z. Steviol and kaurenoic acid were quantified as the abundances of the 317.2139 m/z ion eluting at 1.705 ± 0.2 min and the 302.2170 m/z ion eluting at 4.494 ± 0.2 min, respectively, using external mixed standard curves run after every 48 injections. For analysis of kaurene and kaurenal, 30 μL of cell broth (or supernatant) was combined with 120 μL of ethyl acetate (EtOAc) in 1.7 mL polypropylene tubes and vortexed vigorously 3 × 30 s. Tubes were then centrifuged at 21 000g for 3 min and the EtOAc extracts were transferred to glass vials for analysis by GC−MS using an Agilent 6890N-5975C. Samples were analyzed as 1/4 dilutions. One-μL splitless injections were separated by temperature gradient (80 °C hold I

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membrane for detection of the specific epitopes. Fifteen μg instead of 5 μg were loaded in the case of the 8 h samples. The anti-HA (Abcam 18181) and the loading control anti-PSTAIR (Abcam 10345) primary antibodies are both mouse IgG monoclonal and are recognized by the same secondary antibody (IRDye 800CW donkey antimouse IgG polyclonal; LI-COR 926−32212), while the anti-Myc (Abcam 9106) is a rabbit IgG polyclonal that can be probed with IRDye 680RD goat antirabbit IgG polyclonal (LI-COR 926−68071). Each blot was first probed for Myc and HA, their secondary antibodies visualized on different channels using the Odyssey infrared imaging system (LI-COR Biosciences), allowing for individual or simultaneous visualization of the CYP and the CPR. The membrane was then stripped using the NewBlot nitrocellulose stripping buffer (LI-COR 928−40030) and probed for the loading control.

2 min, 80−170 °C at 30 °C/min, 170 °C hold 3 min, 170−300 °C at 30 °C/min, 300 °C hold 8 min) over a Supelco SLB-5 ms column (30 m × 0.25 mm × 0.25 μm), using hydrogen/ nitrogen as carrier/makeup gases at a flow rate of 1.2 mL/min. The MS scan range was 50−550 m/z with threshold set at 150 ion counts and an 8 min solvent delay. Kaurene was quantified as the abundance of the 91.1 m/z ion (molecular ion 272.3) eluting at 10.9 ± 0.1 min, using second order polynomial or quadratic fit to external standard curves (6.25−400 μM) run every 40 injections. An unknown peak eluting at 11.8 min presented a nearly identical mass spectrum to that of kaurene, but with a molecular ion (M+•) of 286.3 (Figure S4). We inferred the identity of this peak to be kaurenal, which has a molecular weight of 286.46 g/mol. Kaurenal was quantified as the abundance of the 91.1 m/z ion of the peak eluting at 11.8 min, reported in peak area. Cell density measurements were made at each sampling in order to normalize metabolite concentrations. Metabolite values are averaged from three clonal biological replicates, with uncertainties expressed as 95% confidence intervals. Percent product yields were calculated based on molar amounts, where a given product or reactant includes its quantified downstream products, the sum of which are referred to here as equivalents of that intermediate. Kaurene equivalents were thus calculated as the molar sum of kaurene, kaurenoic acid, and steviol, and kaurenoic acid equivalents as the molar sum of kaurenoic acid and steviol. Steviol percent yield was determined from kaurene equivalents and from kaurenoic acid equivalents, and kaurenoic acid percent yield was calculated from kaurene equivalents (see Supporting Information for details). Microscopy Imaging. To visualize the expression of tagged fluorescent proteins, cells from overnight YPD cultures were inoculated into fresh YPD medium at an OD660 of 0.4 and incubated at 30 °C with 200 rpm shaking for 4−6 h (approximately two doublings). Cells were washed with SC medium and then mounted unfixed on microscope slides. Images were captured using a Nikon Ti microscope with a 100× PlanAPO lens (NA 1.49). Cells were illuminated using high inclination laminated optical sheet TIRF illumination with either 488 nm (GFP) or 568 nm (mCherry) lasers, and their respective filter cubes (Chroma, USA). Images are of single planes. Image processing was done using FIJI (NIH, USA). Immunoblotting. Strains expressing Myc-tagged CYPs and/or HA-tagged CPRs were grown in 5 mL batch cultures and sampled for analysis by immunoblot over a 72-h period. To have sufficient cells for analysis at 8 h, a separate 40 mL culture was seeded. A cell equivalent of 1 mL of broth at an OD660 of 4 was collected for all samples. Cell pellets were washed in 0.1 mL of water, then collected by centrifugation 21 000g for 1 min and suspended in 0.15 mL of lysis buffer (1 tablet of Roche cOmplete Mini Protease Inhibitor Cocktail dissolved in 10 mL of 50 mM Tris-HCl pH 7.5, 2 mM MgCl2, 150 mM NaCl, NP40 0.1% (v/v)) and 50 μL of LI-COR 928− 40004 loading dye. Samples were denatured at 95 °C for 5 min and then centrifuged. Supernatants were divided into three aliquots of 90, 90, and 20 μL, which were snap-frozen in liquid nitrogen. The total protein per sample was quantified using the Pierce 660 nm protein assay reagent (Thermo Scientific 22660) with the ionic detergent compatibility reagent (Thermo Scientific 22663) and BSA as standard. Five μg of total protein per sample, unless otherwise specified, were resolved by SDS-PAGE and transferred to a nitrocellulose



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00470.



Tables S1−S5; Figures S1−S4 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Vincent J. J. Martin: 0000-0001-7511-115X Present Address ¶

Lallemand Inc., 6100 Royalmount Avenue, Montréal, Québec H4P 2R2, Canada. Author Contributions ∇

N.D.G. and E.F. have contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors acknowledge the Centre for Microscopy and Cell Imaging funded by Concordia University and the Canada Foundation for Innovation. This study was financially supported by Evolva. V.J.J.M. is supported by a Concordia University Research Chair.

■ ■

ABBREVIATIONS CYP, cytochrome P450; CPR, cytochrome P450 reductase; KO, kaurene oxidase; KAH, kaurenoic acid hydroxylase. REFERENCES

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