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. 2024 Feb 12;13(3):921–929. doi: 10.1021/acssynbio.3c00718

Equipping Saccharomyces cerevisiae with an Additional Redox Cofactor Allows F420-Dependent Bioconversions in Yeast

Misun Lee 1, Marco W Fraaije 1,*
PMCID: PMC10949242  PMID: 38346396

Abstract

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Industrial application of the natural deazaflavin cofactor F420 has high potential for the enzymatic synthesis of high value compounds. It can offer an additional range of chemistry to the use of well-explored redox cofactors such as FAD and their respective enzymes. Its limited access through organisms that are rather difficult to grow has urged research on the heterologous production of F420 using more industrially relevant microorganisms such as Escherichia coli. In this study, we demonstrate the possibility of producing this cofactor in a robust and widely used industrial organism, Saccharomyces cerevisiae, by the heterologous expression of the F420 pathway. Through careful selection of involved enzymes and some optimization, we achieved an F420 yield of ∼1.3 μmol/L, which is comparable to the yield of natural F420 producers. Furthermore, we showed the potential use of F420-producing S. cerevisiae for F420-dependent bioconversions by carrying out the whole-cell conversion of tetracycline. As the first demonstration of F420 synthesis and use for bioconversion in a eukaryotic organism, this study contributes to the development of versatile bioconversion platforms.

Keywords: F420, F420 biosynthesis, S. cerevisiae, tetracycline biosynthesis, F420-dependent bioconversion

Introduction

F420 is a naturally occurring deazaflavin cofactor synthesized only by certain bacteria and archaea, such as actinobacteria and methanogenic archaea.1 While having a similar structure as the ubiquitous flavin cofactor FAD, the chemical properties of F420 are more like nicotinamide cofactors, as it exclusively performs hydride transfer reactions due to the C5 of the 5-deazaiso-alloxazine moiety. F420-dependent reductases catalyze the asymmetric reductions of imines, ketones, enoates, etc. and can potentially be used as an alternative to flavin-containing and other NAD(P)H-dependent reductases.2−4 Furthermore, the low redox potential of F420 compared to the flavin cofactors FMN and FAD, and even to NAD(P)H, allows the reduction of recalcitrant substrates, expanding the scope of the currently available applications of enzymatic reductions.2,5

Despite the potential use of F420 for various industrial applications, the biosynthesis of this cofactor is limited to the use of natural producers such as Mycobacterium smegmatis, which hinders the cofactor availability and thus the related research. Therefore, its heterologous production in more versatile organisms such as Escherichia coli and yeast can be an attractive solution for easy access to this deazaflavin cofactor. Previous studies have shown that it is possible to produce F420 and analogues in E. coli by heterologous expression of the F420 biosynthetic pathway, demonstrating the potential use of this organism for the cofactor production as well as F420-dependent bioconversion.6−9 As a substitute to F420, a synthesis of structurally much simpler and yet functional non-natural deazaflavin analogue FOP has also been explored using both E. coli and Saccharomyces cerevisiae, offering an attractive alternative solution.10

Either naturally or non-naturally, F420 has so far been synthesized only in prokaryotic organisms. In this study, we explored the biosynthesis of F420 in S. cerevisiae to extend the F420-dependent biosynthesis platform even further to eukaryotic organisms. S. cerevisiae is a widely used organism across laboratories and industries due to its robust and harmless nature as well as its well-understood biophysical properties and well-developed molecular biological tools. Therefore, producing F420 in this versatile organism can expand biotechnological means for related research and applications.

The biosynthetic pathway of F420 (Figure 1) is now well-elucidated and information on the chemical and structural properties of the involved enzymes from a few representative organisms such as M. smegmatis and Methanocaldococcus jannaschii are available.6,7,11−14 Using the available information, we explored the use of these enzymes for the production of F420 in S. cerevisiae by testing the expression and their in vivo functions. Initial studies were performed using plasmid-based expression of the involved enzymes, and we confirmed that expression of CofC (guanylyltransferase) and CofD (FO transferase) from M. jannaschii along with FbiB (glutamyl ligase) from M. smegmatis can produce F420 in a good yield when the precursor FO is provided. Based on this result, we constructed a F420-producing S. cerevisiae strain by CRISPR-Cas mediated genomic integration of the three genes. The F420 yield after some optimization was comparable to the yield from natural producer M. smegmatis.

Figure 1.

Figure 1

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Biosynthetic pathway of F420. The scheme is adapted from Bashiri et al.6 The pathway represents the F420 synthesis using 2-phosphenolpyruvate (PEP) as a precursor. F420 can also be synthesized using 2-phospho-l-lactate (2-PL) or 3-phospho-d-glycerate (3-PG), in which case the dehydro-F420-0 is not formed, therefore no reduction step to F420-0 is required.6,7 Generally, the Fbi-prefix is used for the enzymes from mycobacteria, and Cof-represents the homologues from archaea.

We then further explored the F420-producing S. cerevisiae strain for use in F420-dependent bioconversions. To demonstrate that the attained intracellular levels of F420 in S. cerevisiae can support new metabolic activities, we introduced several bacterial enzymes that catalyze the last steps of tetracycline synthesis. These involve the selective reduction of the C5a–C11a double bond of dehydrotetracycline, which requires reduced F420 as electron donor.15 By expressing the last two enzymes of the tetracycline biosynthesis as well as an F420-reducing enzyme from Cryptosporangium arvum (FSDcryar, an F420-dependent sugar-6-phosphate dehydrogenase)16 in the F420-producing S. cerevisiae strain, we could successfully convert anhydrotetracycline into tetracycline. This clearly shows the potential use of the strain for F420-dependent bioconversions.

Overall, the significance of the current study lies in the demonstration of the first eukaryotic production of F420 and the F420-dependent bioconversions using yeast. In addition to the previously reported E. coli-based production of the cofactor, this will expand the tools for F420-related research.

Results and Discussion

In Vivo FO Synthesis in S. cerevisiae is Hindered by Deficient Expression of FO Synthases

The catalytic core of F420, the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) moiety, is synthesized from tyrosine and 5-amino-6-(ribitylamino)-uracil. The reaction is performed by an FO synthase which mostly is (1) a bifunctional enzyme (FbiC) in actinobacteria, or (2) involves two separate enzymes (CofG and CofH) in Archaea.1 For in vivo production of FO, we attempted the expression of several FO synthases from different organisms including FbiCs from M. smegmatis (MsFbiC), M. tuberculosis (MtFbiC), and a eukaryote Chlamydomonas reinhardtii (CrFbiC)—some eukaryotes use FO as a chromophore in the DNA repair process17 —as well as CofG and CofH from M. jannaschii (mjCofG and mjCofH). The codon-optimized FO synthases were transformed in S. cerevisiae, and in vivo FO synthesis was analyzed. However, none of the FO synthases seem to express or function in S. cerevisiae, as no FO was detected in the cell extracts or in the culture media after the growth of the cells. Supplementing the growth media with tyrosine and methionine which are the precursors of FO and the cofactor SAM, respectively, did not change the result. SDS-PAGE analysis revealed no apparent protein bands for expressed FO synthases except for MjCofG (Figure S1). Previous studies showed that in E. coli very low expression of FO synthases was sufficient for the in vivo FO production.6,10 It is plausible that the expression of the SAM-dependent Fe–S cluster-containing enzymes can be problematic due to the different Fe–S cluster assembly pathways in prokaryotes and eukaryotes, causing the disturbed expression or malfunction of the enzyme.18,19 As the functional expression of the different FO synthases failed, we have focused on building the F420 pathway using the chemically synthesized FO. This would be analogous to the use of riboflavin as a precursor for flavin cofactors.

Coexpression of CofC and CofD Enables the In Vivo Production of Dehydro F420 (DF420) in S. cerevisiae

The precursor in F420 biosynthesis was initially identified to be 2-PL but in recent studies, PEP was also shown to be a precursor in some organisms.6,7 When PEP is used as a precursor, dehydroF420-0 (DF420-0) instead of F420-0 is produced and subsequently reduced to F420-0 by an FMN-dependent catalysis (Grinter 2020).11 In some organisms, yet another precursor, 3-PG, was found to be used as the precursor, which results in the production of a F420 analogue, 3-PGF420.7 The substrate specificities of the enzymes involved in attaching these moieties to FO, i.e., guanylyltransferase (CofC or FbiD) and FO transferase (CofD or FbiA), are therefore different between enzyme homologues.12 Since 2-PL is not known to be a common metabolite in S. cerevisiae, a guanylyltransferase and a FO transferase that are active on the more accessible substrates PEP and EGGP, respectively, seem more suited for in vivo F420 synthesis in S. cerevisiae.

Based on the available data from previous studies, we selected two guanylyltransferases from M. smegmatis and M. jannaschii (MsFbiD and MjCofC, respectively) as well as three FO transferases from M. smegmatis, M. jannaschii, and M. mazei (MsFbiA, MjCofD, and MmCofD, respectively). In order to select the best combination of two enzymes for the in vivo synthesis of DF420-0, we first expressed these enzymes individually in S. cerevisiae and performed reactions with mixtures of cell extracts. Among the possible six combinations of the cell extract mixtures of guanylyltransferase and FO transferase, the combination of MjCofC–MjCofD and MjCofC–MmCofD seemed to function, showing an additional peak when compared with the control reaction on HPLC analysis (Figure 2a). The F420 spiked reaction product of MjCofC–MmCofD shows that the additional peak is from a potentially F420-like product. The products were purified and further analyzed using LC–MS, which indeed showed the mass corresponding to that of DF420-0 (Figure 2b). Previously it was shown that MsFbiA exclusively uses EPPG as a substrate and MsFbiD preferably accepts PEP over 2-PL when studied in vitro.6,12 However, in this study, reactions with MsFbiD and/or MsFbiA did not show any products, which might be due to insufficient expression. SDS-PAGE analysis (Figure S2) showed no apparent overexpression of MsFbiA and visibly lower expression of MsFbiD compared to its homologous enzyme MjCofC. Based on this result we selected MjCofC and MjCofD for in vivo F420 production. When coexpressed in S. cerevisiae, these two enzymes produced DF420-0 in vivo using the FO provided in the media, which was confirmed by HPLC and LC–MS analysis (Figure 2c,d). Although it was previously suggested that mainly 2-PL was used as a precursor in archaea,12,20 the archaeal enzymes MjCofC and MjCofD expressed in S. cerevisiae seemed to exclusively use PEP, producing DF420-0 as the only detectable product. This result indicates that the in vivo PEP concentration in S. cerevisiae is sufficient for these enzymes to produce DF420-0 and that no or an insignificant amount of 2-PL is present in the yeast cells.

Figure 2.

Figure 2

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DehydroF420(DF420) production in S. cerevisiae. (a) HPLC analysis of in vitro reaction using S. cerevisiae cell extracts expressing various CofC (FbiD) and CofD (FbiA). The control reaction was performed with wild-type S. cerevisiae cells containing an empty plasmid. The orange chromatogram shows the reaction product of MjCofC and MmCofD, which was spiked with purified F420. (b) LC–MS analysis shows DF420-0 as the reaction product of MjCofC–MjCofD and its fragmented ion with m/z of 424.399 [M – H]−. (c) The HPLC result and d. LC–MS identification of in vivo DF420-0 production was performed using S. cerevisiae expressing MjCofC and MjCofD. The calculated mass of DF420-0 is 513.350. The ionized molecule with a m/z of 424.396 [M – H]− is expected to be a fragment of DF420-0 as shown in panel b.

S. cerevisiae Strain Expressing FbiB from M. smegmatis along with MjCofC and MjCofD Produces F420-n

The final step of F420 biosynthesis is the elongation of F420-0 with glutamyl tails in varying length by CofE or FbiB. When PEP is used as a precursor, the reduction of the intermediate product dehydroF420-0 (DF420-0) to F420-0 is additionally required. In mycobacteria, the bifunctional glutamyl ligase FbiB also performs the FMN-dependent reduction of DF420-0 to F420-011 in addition to the glutamyl ligation reaction. However, in organisms that use monofunctional glutamyl ligase CofE, such as methanogenic archaea, the reduction is possibly performed by a “stand-alone nitroreductase”21 or a yet-unknown enzyme. Considering the convenience of using the bifunctional enzyme for both the reduction of DF420-0 and the glutamyl ligation, we chose FbiB from M. smegmatis (MsFbiB) for the final step of F420 biosynthesis. Three enzymes, MjCofC, MjCofD, and MsFbiB, were successfully expressed in S. cerevisiae on two separate plasmids (MjCofC and MjCofD on one and MsFbiB on the other). Gratifyingly, when the strain was grown in the media containing FO in vivo, the production of F420 was detected (Figure S3a). The HPLC analysis showed several peaks of potential F420 species, which were confirmed by the LC–MS analysis. The mass spectroscopy data showed that the produced F420 species mostly contained five or six glutamyl moieties (Figure S3b).

After establishing a set of functional F420 pathway enzymes in yeast, we developed an S. cerevisiae strain that has a F420 synthetic pathway built in using CRISPR-Cas9-mediated genomic integration. One copy of each MjCofC, MjCofD, and MsFbiB genes was cloned at the HO locus as described in the Materials and Methods. The resulting strain is herein referred to as Sc-F420. In cell extracts of the Sc-F420 strain grown in synthetic defined (SD) media supplemented with FO, peaks corresponding to F420 were detected by HPLC-FLD, which were expectedly absent in the control sample of the wild-type S. cerevisiae strain (Figure 3). With the HPLC method used for verifying the in vivo F420 production, four apparent peaks for F420 were visible in both the standard samples purified from M. smegmatis and Sc-F420 samples. In order to identify the peaks, we purified the in vivo reaction products in two purification steps using anion exchange chromatography and reverse phase HPLC. The F420 species purified from the cell extracts using anion exchange chromatography were further separated by HPLC-FLD with an optimized elution method and collected manually. The purified F420 products were analyzed using LC–MS. The mass analysis confirmed that all of the peaks corresponded to F420 species ranging from F420-6 to F420-2 in the descending order of the retention time (Figure S4). Interestingly, the mass of one of the peaks corresponded to the unreduced (dehydro)F420 species with a single glutamyl tail, DF420-1. It shows that the glutamate ligation reaction of FbiB can occur before the reduction reaction. Confirming that all of the peaks that were detected in HPLC-FLD correspond to F420 species, the combined peak area was used for further quantification of F420 production.

Figure 3.

Figure 3

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F420 production in S. cerevisiae. F420 purified from M. smegmatis is used as the standard sample. The wild-type S. cerevisiae grown in media containing FO is used as the control. Only the samples from strain Sc-F420 shows the F420-corresponding peaks.

In order to optimize the F420 yield, we tested the effect of the growth media (Table 1). 80 mg/L glutamate was added in synthetic defined media (SD and Verduyn media) to support the glutamyl ligation reaction. Among the media tested, the highest yield per culture volume was shown when a rich medium (YPD) was used. When normalized by the amount of the biomass produced, Verduyn medium (VD) with the glutamate supplement was as effective as YPD yielding around 300 nmol/g dry biomass. SD medium was least favorable for F420 production, yielding about 100 nmol/g dry biomass. Furthermore, when SD media was used, the strain only reached an OD600 of ∼5.7, while OD600 values of 12.8 and 23.6 were reached when VD medium or YPD medium was used, respectively. The lower cell density also contributed to the low yield per volume of culture, which improved slightly when glutamate was supplemented. The estimated in vivo concentration of F420 in cells grown in YPD and VD media with glutamate was similarly high, reaching over 100 μM. Therefore, while YPD would be the best choice of media for the F420 production purpose, VD media would be the better choice for in vivo F420-dependent conversion as it offers more flexible options of using selective markers for expressing additional enzymes if required. The in vivo F420 concentration of 100 μM would be sufficient for most F420-dependent reactions considering the relatively high affinity of these enzymes toward the cofactor.22−25

Table 1. Effect of Media on the F420 Yielda.

  nmol/L nmol/g DWb in vivo conc (μM)c
SD + 2% glu 184 ± 16 95 ± 6 36 ± 2
SD + 2% glu + 80 mg/L Glu 232 ± 20 97 ± 7 37 ± 3
YPD 2590 ± 410 327 ± 52 124 ± 20
VD + 2% glu + 80 mg/L Glu 1250 ± 210 292 ± 50 110 ± 19
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a

The experiments were performed in biological duplicates.

b

The correlation between the cell dry weight (DW) and the OD600 was determined (1 OD600unit ≈ 0.37 mg/mL) to estimate the dry weight of the culture.

c

The in vivo F420 was estimated based on the reported cell volume per biomass of S. cerevisiae.26

Throughout the study, 200 μM FO was added in the media for F420 production. As the concentration of FO in the media can affect the physiological state of the cells as well as the F420 yield, we evaluated the effect of different FO concentrations on the cell growth and F420 production. The addition of up to 400 μM FO did not influence the growth of the Sc-F420 strain as the final OD600 upon harvest at 48 h incubation was similar, between 12 and 13, regardless of the FO concentrations added. The FO concentration showed positive correlation to the F420 yield as expected and this may be an indication of the low FO import efficiency into the cell (Figure 4). Even though we tested only up to 400 μM FO due to its poor solubility, it is possible that higher concentrations (if solubilized) of FO could further increase the F420 yield. For the purpose of F420 production, the highest FO concentration possible should be used, while it seems that a lower concentration (ex. 200 μM) is enough to produce sufficient in vivo F420 concentration for a F420-dependent bioconversion using cells.

Figure 4.

Figure 4

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Effect of the FO concentration on the F420 yield.

The experiments are performed in duplicate, and the error bas represent the standard deviation.

In the above-described end-point measurements, a rather long incubation time of 48 h was used in order to guarantee a sufficient time for the full growth and the maximum F420 yield. In order to improve the efficiency of F420 production of the Sc-F420 strain, we tested the F420 yield at different incubation times (Table 2). Between 24 and 48 h, the cells still seem to be growing as interpreted by the increasing OD600. The F420 yield per volume culture increased between 24 and 36 h incubation time, which coincide with the increasing cell biomass. However, despite the further increased OD600 at 48 h, the F420 yield decreased slightly compared to that at 36 h incubation time. The productivity (nmol/g dry biomass), on the other hand, was the highest at 24 h showing almost 500 nmol/g DW. As a result, it also showed the highest estimated in vivo F420 concentration of ∼180 μM. Therefore, for further experiments, we incubated the cells only for 24 h. The F420 yield achieved with Sc-F420 is comparable to the one with M. smegmatis but in a much shorter incubation time (Table 3).

Table 2. Incubation Time-Dependent F420 Yielda.

incubation time (h) OD600 DW (mg) nmol/L nmol/g DW in vivo conc (μM)
24 8 33 1326 ± 9 483 ± 5 183 ± 2
36 11 45 1455 ± 80 386 ± 21 146 ± 8
48 12 48 1443 ± 144 362 ± 33 137 ± 12
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a

The experiments were performed in triplicates. The values represent the average and the errors are the standard deviation. The yield per biomass and the in vivo F420 concentration were calculated as described in Table 1.

Table 3. F420 Yields Produced by Different Organisms and Conditions.

  μmol/L μmol/g DW refs
M. smegmatis WT 1.43 0.3 Isabelle et al.27
M. smegmatis engineered   3 Bashiri et al.28
E. coli 0.027   Bashiri et al.6
E. coli, condition optimized 2.3 1.6 Shah et al.8
E. coli, engineered and condition optimized 11.4   Last et al.9
Sc-F420 1.3 0.48 this study
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F420 Producing S. cerevisiae Strain Sc-F420 Can be Used for F420-Dependent Bioconversion

Upon confirmation that in vivo F420 production in yeast reached high F420 levels, we explored the possibility to build a Sc-F420-based bioconversion system. The final steps of the tetracycline synthesis involve a specific F420-dependent enzyme-catalyzed reaction. OxyR and CtcM produce tetracycline by catalyzing the reduction at 5a(11a) of dehydrotetracycline, which is essential for the potency of the bacterial antibiotic.29,30 In the natural biosynthesis in Streptomyces species, OxyR and CtcM are known to be involved in the synthesis of oxytetracycline and chlorotetracycline, respectively.15 However, in vitro studies showed that both OxyR and CtcM can reduce 5a(11a)-dehydrooxytetracycline as well as 5a(11a)-dehydrotetracycline, producing oxytetracycine and tetracycline, respectively (Figure 5).15,31 OxyS, a flavin-dependent enzyme in the pathway, performs a single or double hydroxylation on anhydrotetracycline producing 5a(11a)-dehydrotetracycline or 5a(11a)-dehydrooxytetracycline, respectively, which can be subsequently reduced by the above-described F420-dependent enzymes. As a proof of concept of a Sc-F420-based bioconversion, we set out to demonstrate the F420-dependent last step of tetracycline conversion using OxyR and CtcM. As anhydrotetracycline, and not the hydroxylated product, is commercially available, we also employed OxyS for the hydroxylation reaction.

Figure 5.

Figure 5

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Scheme of (oxy)tetracycline synthesis from anhydrotetracycline using OxyS, OxyR, or CtcM.

In order to produce tetracycline from anhydrotetracycline using in vivo-produced F420, we coexpressed OxyS together with either OxyR or CtcM on a plasmid in the Sc-F420 strain. For an F420-dependent reduction reaction, the cofactor needs to be reduced and we employed the F420-dependent glucose-6-phosphate dehydrogenase from C. arvum, expressed on a separate plasmid. This bacterial enzyme can conveniently use the available glucose-6-phosphate to generate reduced F420 (F420H2). After 24 h of cultivation in FO containing media and subsequent incubation with anhydrotetracycline, both OxyS_OxyR and OxyS_CtcM expressing Sc-F420 strains seemed to be able to produce tetracycline which was analyzed by HPLC and LC–MS methods (Figure 6). In the control reactions, wild-type CEN. PK yeast strain expressing OxyS_CtcM and FSDcryar as well as FSDcyar-absent Sc-F420 strain with or without OxyS_CtcM, no tetracycline related products were detected, indicating that the tetracycline was indeed produced by OxyS_OxyR or OxyS_CtcM using the in vivo produced and regenerated F420H2.

Figure 6.

Figure 6

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Biosynthesis of tetracycline using Sc-F420. HPLC (a) and LC–MS (b) analysis of production tetracycline. “SM’ and ‘SR” refer to the expression of OxyS_OxyM and OxyS_OxyR, respectively. (c) Mass spectrum of tetracycline products. The m/z of peaks 2 and 4 corresponds to tetracycline (exact mass: 444.435). Peaks 1 and 3 also show the mass that corresponds to tetracycline with slightly different m/z compared with peaks 2 and 4. This peak is also appearing in the standard tetracycline sample (b) as a minor peak. It is possible that tetracycline is epimerized in the acidic LC–MS analysis condition and eluted separately.33 OxyS_OxyR expressing strain also produces oxytetracycline, which is shown in peak 1, representing the [M + H]+ of 461.438. In contrast, OxyS_CtcM expressing strain did not produce any detectable amount of oxytetracycyline (peaks 3 and 4).

Both the Sc-F420 strains expressing OxyS_OxyR or OxyS_CtcM produced tetracycline but different analogues. While OxyR produced a significant portion of oxytetracycline along with the standard tetracycline, CtcM exclusively produced tetracycline and no detectable amount of oxytetracycline. This result is in line with a previous study where CtcM showed higher specificity toward dehydrotetracycline (single hydroxylation product of OxyS) thus producing tetracycline primarily.15 On the contrary, a previous study showed that in vitro reactions using FO and crude extract of yeast cells expressing OxyS, OxyR, and a F420-reducing enzyme FNO did not yield any tetracycline products.31 It is likely due to the nonactive OxyR when FO is used instead of F420. Even though it is possible to substitute F420 with FO or a non-natural analogue FOP for some F420-dependent reactions,32 the application is limited to the cofactor specificity of each enzyme. Therefore, conversion using the F420-producing system as described in this study is at an advantage for exploiting F420-dependent enzymes.

The production of tetracyclines in the Sc-F420 strain demonstrates that the strain can produce enough F420 for F420-dependent reduction and shows the potential for further exploration of the strain for F420-dependent bioconversions. Even though the F420 yield is lower than the previously constructed and engineered F420-producing E. coli strains,8,9 the development of the Sc-F420 strain as a first F420 producing eukaryotic organism expands the tools for F420 related research.

Conclusions

F420 is a unique cofactor that structurally resembles the canonical flavin cofactor FAD while having a similar chemical property as the nicotinamide cofactors NAD and NADP. Despite the potential value of F420 for biotechnological applications, research on this cofactor and the respective F420-dependent enzymes has been limited by the constrained production of the cofactor using unconventional laboratory organisms such as M. smegmatis. In this study, we tackled this problem by producing the cofactor in a robust industrial microorganism, S. cerevisiae, through heterologous expression of part of the F420 pathway. By optimizing the enzyme combination and the growth medium, we achieved comparable F420 yields to that of the natural production by M. smegmatis but in a much shorter production time. Furthermore, we demonstrated the use of the F420-producing S. cerevisiae strain for F420-dependent bioconversion by showing the successful bioproduction of tetracycline from anhydrotetracycline. Together with the previously developed F420-producing E. coli strain by others, the first F420 producing eukaryotic strain developed in this study extends the toolbox for further development in the field of F420-related research.

Materials and Methods

Strains and Plasmids

All plasmids used in this study are E. coli—yeast shuttle vectors assembled using a modular cloning kit, Moclo-YTK from Addgene and the assembly was performed as described previously34 with some modification in the Golden Gate assembly methods. E. coli NEB10-beta strain was used for cloning purposes. CEN. PK2-1C strain was purchased from Euroscarf and used for constructing the F420-producing yeast strain.

Growth Media

For the growth of E. coli, Lysogeny broth medium containing an appropriate type of antibiotic (100 μg/mL ampicillin, 50 μg/mL kanamycin, or 50 μg/mL chloramphenicol) was used. Media for S. cerevisiae used in this study are SD medium, YPD medium (formedium), and VD. The SD medium is composed of 6.9 g/L yeast nitrogen base without amino acids (formedium), 0.77 g/L complete supplement mixture (formedium) that is appropriate for the auxotroph markers, and 2% (w/v) glucose. VD containing 2% glucose was made according to Verduyn et al.35 For F420 production in S. cerevisiae, 72 mg/L FO was added to respective media prior to autoclave sterilization. FO was chemically synthesized as described in Drenth et al.32 For optimization of F420 production, 80 mg/L glutamate was added to the respective medium.

Cloning

All S. cerevisiae codon-optimized genes tested for F420 biosynthesis and F420-dependent bioconversion were purchased from Twist Bioscience. The gene fragments were first cloned into an entry vector and subsequently cloned into a preassembled E. coli-yeast shuttle vector by Goldengate assembly method according to the Moclo-YTK cloning protocols.34 All cloning products were initially transformed in E. coli NEB10b strain using the heat shock method, isolated, and analyzed by sequencing at Eurofins. The correct cloning products were then transformed to S. cerevisiae CEN. PK2-1C using the lithium acetate/single-stranded carrier DNA/PEG method that is optimized by Gietz et al.36

Construction of the F420-Producing Sc-F420 Strain

The genes encoding MjCofC, MjCofD, and MsFbiB were integrated in the HO locus using a Crispr-Cas9-mediated method. A vector containing an sgRNA sequence and Cas9 expression cassette was assembled using a Moclo YTK kit. The 20mer target sequence of sgRNA is 5′-GCTCCAGCATTATAGCATGC-3′. The vector contained CEN6/ARS4 origin, and pPGK1 promoter was used for Cas9 expression. The repair fragment was constructed via assembling the F420 pathway genes, HO locus homology fragments as well as a leu3 into a multigene plasmid and subsequently linearizing by NotI digestion. The resulting fragment contained 5′-HO homology sequence, cofD, cofC and fbiB, leu3, as well as 3′-HO homology sequence in this order. The transformation of CEN.PK21-C strain with Cas9_sgRNA plasmid and the repair fragment was performed following the protocol of Gietz et al.36 Approximately 500 ng of Cas9_gRNA plasmid and 5 μg of repair fragment was used for the transformation. The PCR verification of the correct integration was performed on the genomic DNA extracted from selected colonies. Cas9-gRNA plasmid was removed from the transformants by growing the cells in nonselective YPD media and confirming the loss of uracil selectivity that the plasmid was carrying.

In Vitro and In Vivo DF420 Conversion

In order to find a functional combination of PEP guanylyltransferase (FbiB/CofC) and FO transferase (FbiA/CofD) for in vivo DF420 production, reactions using crude extracts mixture of S. cerevisiae expressing each of the enzyme types were tested. Cells expressing FbiB from M. smegmatis, CofC from M. jannaschii, FbiA from M. smegmatis, CofD from M. jannaschii, or CofD from M. mazei were grown in 20 mL SD media with 2% glucose for 24 h at 30 °C. Cells were harvested and washed with ddH20 and resuspended in 1 mL of 50 mM KPi, pH 7.0 containing 1 mg/mL Zymolyase (Amsbio). The cell resuspension was then incubated for 20 min at 30 °C and subsequently vortexed in the presence of equal volume of glass beads (five repeats of 5 s vortexing and 10 s resting on ice). Total protein concentration in the crude extracts was measured by Bradford assay. One ml reaction mixtures in 50 mM KPi, pH 7.0 containing 1 mM GTP, 1 mM phosphoenolpyruvate, 5 mM MgCl2, 200 μM FO and mix of CofC (FbiD)- and CofD (FbiA)-containing crude extracts (normalized to 1 mg of total protein each) were incubated at 30 °C for 4 h. The reactions were stopped by heating at 95 °C for 10 min and subsequently centrifuged and filtered. The reaction samples were analyzed by HPLC method for DF420 production.

F420 Production Using S. cerevisiae

For in vivo production of F420 in S. cerevisiae, the respective cells were first grown in 5 mL of SD media with 2% glucose overnight at 30 °C. The precultures were then diluted to OD600 ∼ 0.2 in an appropriate FO-containing media and grown at 30 °C. Unless otherwise stated, the cells were harvested after 24 h and washed with ddH2O. In order to extract the in vivo produced F420, the cells were incubated with approximately 4× cell volume of 70% boiling ethanol at 95 °C for 5 min. The supernatants were collected after centrifugation at 8000g for 10 min, and the process was repeated once. The collected solutions were subjected to vacuum centrifugation at 60 °C for 1 h in order to remove ethanol and concentrate. The products were resuspended in ddH2O, centrifuged, and filtered prior to the analysis.

Tetracycline Conversion

F420-producing yeast strain Sc-F420 expressing OxyR or CtcM as well as OxyS and FSDcryar were grown in 20 mL VD media containing 80 mg/L glutamate and 72 mg/L FO for 24 h in order to accumulate the in vivo F420. Cells were collected by centrifugation at 5000g for 10 min and resuspended in 1 mL of reaction solution containing 1 mM anhydrotetracycline, 5 mM glucose, and 100 mM Tris·HCl, pH 7.5. The reactions were incubated for 24 h at 30 °C and subsequently extracted three times with 2 mL of EtOAc. The reactions were dried by vacuum centrifugation for 1 h, redissolved in 1 mL of ddH20, and centrifuged for 10 min at 11.000g to remove any debris prior to analysis.

Analytical Methods

For all HPLC analyses, samples were separated on a Phenomenex Germini C18 (4.6 × 250 mm, 5 μm) column using a JASCO LC-4000 system equipped with an UV–vis detector and a fluorescence detector. F420 and intermediates were detected using UV absorbance at 262 nm and fluorescence (ex: 400 nm and em: 470 nm). For the tetracycline conversion analysis, the reactions were monitored by UV absorbance at 265 nm. The mobile phase consisting of 50 mM ammonium acetate, pH 6.0 with 5% acetonitrile (A) and 100% acetonitrile (B) was applied with a flow rate of 1 mL/min for all analysis. Slightly different elution methods were applied for each analysis, which are as follows. In vivo and in vitro DF420 conversion: t = 0 min/100:0 (A/B), t = 16 min/80:20 (A/B), t = 19 min/5:95 (A/B), t = 22 min/5:95 (A/B), t = 26 min/95:05 (A/B), and t = 28 min/100:0 (A/B). In vivo F420 production: t = 0 min/100:0 (A/B), t = 5 min/90:10 (A/B), t = 16 min/90:10 (A/B), t = 19 min/5:95 (A/B), t = 22 min/5:95 (A/B), t = 26 min/100:0 (A/B), and t = 28 min/100:0. For improved separation of F420 species: t = 0 min/100:0 (A/B), t = 5 min/92:08 (A/B), t = 16 min/92:08 (A/B), t = 19 min/5:95 (A/B), t = 22 min/5:95 (A/B), t = 26 min/100:0 (A/B), and t = 28 min/100:0. For the tetracycline conversion analysis, the analytes were separated using the following elution method: t = 0 min/100:0 (A/B), t = 16 min/80:20 (A/B), t = 19 min/05:95 (A/B), t = 22 min/5:95 (A/B), t = 26 min/100:0 (A/B), and t = 28 min/100:0.

All LC–MS analysis described in this study were performed on ACQUITY TQD UPLC–MS system (Waters) using the ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 150 mm, Waters). 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) were applied at a flow rate of 0.31 mL/min as the mobile phase. Following gradient methods were used for the respective analysis: DF420 production—t = 0 min/100:0 (A/B), t = 5 min/75:25 (A/B), t = 6.12 min/5:95 (A/B), t = 7.14 min/5:95 (A/B), t = 8.16 min/75:25 (A/B), and t = 9.18 min/100:0 (A/B); F420 production—t = 0 min/100:0 (A/B), t = 2 min/90:10 (A/B), t = 4 min/90:10 (A/B), t = 4–6 min/85:15 (A/B), t = 6–8 min/80:20 (A/B), t = 12 min/5:95 (A/B), t = 14 min/5:95 (A/B), t = 16 min/100:0 (A/B), t = 17 min/100:0 (A/B); and tetracycline conversion—t = 0 min/100:0 (A/B), t = 16 min/5:95 (A/B), t = 18 min/95:5 (A/B), and t = 20 min/100:0 (A/B). The electrospray ionization (ESI) in the negative ion mode for DF420 as well as F420 detection and positive ion mode for tetracycline detection were used.

Acknowledgments

This work was supported by the Dutch research council (NWO) and project partners DSM and Syngenta through the NWO-LIFT project YeastPlus.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.3c00718.

  • SDS-PAGE analyses of enzyme expression and LC/MS analyses of the deazaflavins produced by recombinant yeast (PDF)

Author Present Address

† CJ CheilJedang Corp., CJ Blossom Park, 1356 Iui-dong, Yeongtong-gu, Suwon, Gyeonggi-do, Korea

Author Contributions

M.L. and M.W.F. designed this study. Experimental work was performed by M.L. M.L. wrote the manuscript, and M.W.F. contributed to manuscript editing.

The authors declare no competing financial interest.

Supplementary Material

sb3c00718_si_001.pdf (256.7KB, pdf)

References

  1. Ney B.; Ahmed F. H.; Carere C. R.; Biswas A.; Warden A. C.; Morales S. E.; Pandey G.; Watt S. J.; Oakeshott J. G.; Taylor M. C.; Stott M. B.; Jackson C. J.; Greening C. The Methanogenic Redox Cofactor F420 Is Widely Synthesized by Aerobic Soil Bacteria. ISME J. 2017, 11 (1), 125–137. 10.1038/ismej.2016.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Greening C.; Ahmed F. H.; Mohamed A. E.; Lee B. M.; Pandey G.; Warden A. C.; Scott C.; Oakeshott J. G.; Taylor M. C.; Jackson C. J. Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions. Microbiol. Mol. Biol. Rev. 2016, 80 (2), 451–493. 10.1128/MMBR.00070-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Shah M. V.; Antoney J.; Kang S. W.; Warden A. C.; Hartley C. J.; Nazem-Bokaee H.; Jackson C. J.; Scott C. Cofactor F420-Dependent Enzymes: An Under-Explored Resource for Asymmetric Redox Biocatalysis. Catalysts 2019, 9 (10), 868. 10.3390/catal9100868. [DOI] [Google Scholar]
  4. Mathew S.; Trajkovic M.; Kumar H.; Nguyen Q. T.; Fraaije M. W. Enantio- and Regioselective Ene-Reductions Using F420H2-Dependent Enzymes. Chem. Commun. 2018, 54 (79), 11208–11211. 10.1039/C8CC04449J. [DOI] [PubMed] [Google Scholar]
  5. Wolfe R. S. Unusual Coenzymes of Methanogenesis. Trends Biochem. Sci. 1985, 10 (10), 396–399. 10.1016/0968-0004(85)90068-4. [DOI] [Google Scholar]
  6. Bashiri G.; Antoney J.; Jirgis E. N. M.; Shah M. V.; Ney B.; Copp J.; Stuteley S. M.; Sreebhavan S.; Palmer B.; Middleditch M.; Tokuriki N.; Greening C.; Scott C.; Baker E. N.; Jackson C. J. A Revised Biosynthetic Pathway for the Cofactor F420 in Prokaryotes. Nat. Commun. 2019, 10 (1), 1558–1612. 10.1038/s41467-019-09534-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braga D.; Last D.; Hasan M.; Guo H.; Leichnitz D.; Uzum Z.; Richter I.; Schalk F.; Beemelmanns C.; Hertweck C.; Lackner G. Metabolic Pathway Rerouting in Paraburkholderia Rhizoxinica Evolved Long-Overlooked Derivatives of Coenzyme F420. ACS Chem. Biol. 2019, 14 (9), 2088–2094. 10.1021/acschembio.9b00605. [DOI] [PubMed] [Google Scholar]
  8. Shah M. V.; Nazem-Bokaee H.; Antoney J.; Kang S. W.; Jackson C. J.; Scott C. Improved Production of the Non-Native Cofactor F420 in Escherichia Coli. Sci. Rep. 2021, 11, 21774. 10.1038/s41598-021-01224-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Last D.; Hasan M.; Rothenburger L.; Braga D.; Lackner G. High-Yield Production of Coenzyme F420 in Escherichia Coli by Fluorescence-Based Screening of Multi-Dimensional Gene Expression Space. Metab. Eng. 2022, 73, 158–167. 10.1016/j.ymben.2022.07.006. [DOI] [PubMed] [Google Scholar]
  10. Lee M.; Drenth J.; Trajkovic M.; de Jong R. M.; Fraaije M. W. Introducing an Artificial Deazaflavin Cofactor in Escherichia Coli and Saccharomyces Cerevisiae. ACS Synth. Biol. 2022, 11 (2), 938–952. 10.1021/acssynbio.1c00552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grinter R.; Ney B.; Brammananth R.; Barlow C. K.; Cordero P. R. F.; Gillett D. L.; Izoré T.; Cryle M. J.; Harold L. K.; Cook G. M.; Taiaroa G.; Williamson D. A.; Warden A. C.; Oakeshott J. G.; Taylor M. C.; Crellin P. K.; Jackson C. J.; Schittenhelm R. B.; Coppel R. L.; Greening C. Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F420-0 in Mycobacteria. mSystems 2020, 5 (3), e00389-20 10.1128/mSystems.00389-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hasan M.; Schulze S.; Berndt L.; Palm G. J.; Braga D.; Richter I.; Last D.; Lammers M.; Lackner G. Diversification by CofC and Control by CofD Govern Biosynthesis and Evolution of Coenzyme F420 and Its Derivative 3PG-F420. mBio 2022, 13 (1), e03501 10.1128/mbio.03501-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graupner M.; Xu H.; White R. H. Characterization of the 2-Phospho-L-Lactate Transferase Enzyme Involved in Coenzyme F420 Biosynthesis in Methanococcus Jannaschii. Biochemistry 2002, 41 (11), 3754–3761. 10.1021/bi011937v. [DOI] [PubMed] [Google Scholar]
  14. Li H.; Graupner M.; Xu H.; White R. H. CofE Catalyzes the Addition of Two Glutamates to F420-0 in F420 Coenzyme Biosynthesis in < i > Methanococcus Jannaschii < i/>. Biochemistry 2003, 42 (32), 9771–9778. 10.1021/bi034779b. [DOI] [PubMed] [Google Scholar]
  15. Wang P.; Bashiri G.; Gao X.; Sawaya M. R.; Tang Y. Uncovering the Enzymes That Catalyze the Final Steps in Oxytetracycline Biosynthesis. J. Am. Chem. Soc. 2013, 135 (19), 7138–7141. 10.1021/ja403516u. [DOI] [PubMed] [Google Scholar]
  16. Mascotti M. L.; Kumar H.; Nguyen Q. T.; Ayub M. J.; Fraaije M. W. Reconstructing the Evolutionary History of F420-Dependent Dehydrogenases. Sci. Rep. 2018, 8 (1), 17571. 10.1038/s41598-018-35590-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Petersen J. L.; Ronan P. J. Critical Role of 7,8-Didemethyl-8-Hydroxy-5-Deazariboflavin for Photoreactivation in Chlamydomonas Reinhardtii. J. Biol. Chem. 2010, 285 (42), 32467–32475. 10.1074/jbc.M110.146050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Biz A.; Mahadevan R. Overcoming Challenges in Expressing Iron-Sulfur Enzymes in Yeast. Trends Biotechnol. 2021, 39 (7), 665–677. 10.1016/j.tibtech.2020.11.005. [DOI] [PubMed] [Google Scholar]
  19. Lill R.; Mühlenhoff U. Iron-Sulfur Protein Biogenesis in Eukaryotes: Components and Mechanisms. Annu. Rev. Cell Dev. Biol. 2006, 22, 457–486. 10.1146/annurev.cellbio.22.010305.104538. [DOI] [PubMed] [Google Scholar]
  20. Grochowski L. L.; Xu H.; White R. H. Identification and Characterization of the 2-Phospho-l-lactate Guanylyltransferase Involved in Coenzyme F420 Biosynthesis. Biochemistry 2008, 47 (9), 3033–3037. 10.1021/bi702475t. [DOI] [PubMed] [Google Scholar]
  21. Braga D.; Hasan M.; Kröber T.; Last D.; Lackner G. Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes. Appl. Environ. Microbiol. 2020, 86 (12), e00457-20 10.1128/AEM.00457-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnson E. F.; Mukhopadhyay B. A New Type of Sulfite Reductase, a Novel Coenzyme F420-Dependent Enzyme, from the Methanarchaeon Methanocaldococcus Jannaschii. J. Biol. Chem. 2005, 280 (46), 38776–38786. 10.1074/jbc.M503492200. [DOI] [PubMed] [Google Scholar]
  23. Nguyen Q. T.; Trinco G.; Binda C.; Mattevi A.; Fraaije M. W. Discovery and Characterization of an F420-Dependent Glucose-6-Phosphate Dehydrogenase (Rh-FGD1) from Rhodococcus Jostii RHA1. Appl. Microbiol. Biotechnol. 2017, 101 (7), 2831–2842. 10.1007/s00253-016-8038-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gurumurthy M.; Mukherjee T.; Dowd C. S.; Singh R.; Niyomrattanakit P.; Tay J. A.; Nayyar A.; Lee Y. S.; Cherian J.; Boshoff H. I.; Dick T.; Barry C. E.; Manjunatha U. H. Substrate Specificity of the Deazaflavin-Dependent Nitroreductase from Mycobacterium Tuberculosis Responsible for the Bioreductive Activation of Bicyclic Nitroimidazoles. FEBS J. 2012, 279 (1), 113–125. 10.1111/j.1742-4658.2011.08404.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Purwantini E.; Daniels L. Purification of a Novel Coenzyme F 420-Dependent Glucose-6-Phosphate Dehydrogenase from Mycobacterium Smegmatis. J. Bacteriol. 1996, 178 (10), 2861–2866. 10.1128/jb.178.10.2861-2866.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klis F. M.; de Koster C. G.; Brul S. Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces Cerevisiae and Candida Albicans. Eukaryot. Cell 2014, 13 (1), 2–9. 10.1128/EC.00250-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Isabelle D.; Simpson D. R.; Daniels L. Large-Scale Production of Coenzyme F420-5,6 by Using Mycobacterium Smegmatis. Appl. Environ. Microbiol. 2002, 68 (11), 5750–5755. 10.1128/AEM.68.11.5750-5755.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Bashiri G.; Rehan A. M.; Greenwood D. R.; Dickson J. M. J.; Baker E. N. Metabolic Engineering of Cofactor F420 Production in Mycobacterium Smegmatis. PLoS One 2010, 5 (12), e15803 10.1371/journal.pone.0015803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pickens L. B.; Tang Y. Decoding and Engineering Tetracycline Biosynthesis. Metab. Eng. 2009, 11, 69–75. 10.1016/j.ymben.2008.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Chopra I.; Roberts M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol. Mol. Biol. Rev. 2001, 65 (2), 232–260. 10.1128/MMBR.65.2.232-260.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Herbst E.; Lee A.; Tang Y.; Snyder S. A.; Cornish V. W. Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces Cerevisiae. ACS Chem. Biol. 2021, 16 (8), 1425–1434. 10.1021/acschembio.1c00259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Drenth J.; Trajkovic M.; Fraaije M. W. Chemoenzymatic Synthesis of an Unnatural Deazaflavin Cofactor That Can Fuel F420-Dependent Enzymes. ACS Catal. 2019, 9 (7), 6435–6443. 10.1021/acscatal.9b01506. [DOI] [Google Scholar]
  33. McCormick J. R. D.; Fox S. M.; Smith L. L.; Bitler B. A.; Reichenthal J.; Origoni V. E.; Muller W. H.; Winterbottom R.; Doerschuk A. P. Studies of the Reversible Epimerization Occurring in the Tetracycline Family. The Preparation, Properties and Proof of Structure of Some 4-Epi-Tetracyclines. J. Am. Chem. Soc. 1957, 79 (11), 2849–2858. 10.1021/ja01568a050. [DOI] [Google Scholar]
  34. Lee M. E.; DeLoache W. C.; Cervantes B.; Dueber J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 2015, 4 (9), 975–986. 10.1021/sb500366v. [DOI] [PubMed] [Google Scholar]
  35. Verduyn C.; Postma E.; Scheffers W. A.; van Dijken J. P. Effect of benzoic acid on metabolic fluxes in yeasts: A continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 1992, 8, 501–517. 10.1002/yea.320080703. [DOI] [PubMed] [Google Scholar]
  36. Gietz R. D.; Schiestl R. H. High-Efficiency Yeast Transformation Using the LiAc/SS Carrier DNA/PEG Method. Nat. Protoc. 2007, 2 (1), 31–34. 10.1038/nprot.2007.13. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

sb3c00718_si_001.pdf (256.7KB, pdf)

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