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. 2021 Mar 2;6(10):7175–7180. doi: 10.1021/acsomega.1c00339

New Set of Yeast Vectors for Shuttle Expression in Escherichia coli

Jifeng Yuan 1,*, Qiwen Mo 1, Cong Fan 1
PMCID: PMC7970545  PMID: 33748631

Abstract

graphic file with name ao1c00339_0005.jpg

Promoters that play an essential role in the gene regulation are of particular interest to the synthetic biology communities. Recent advances in high-throughput DNA sequencing have greatly increased the breadth of new genetic parts. The development of promoters with broad host properties could enable rapid phenotyping of genetic constructs in different hosts. In this study, we discovered that the GAL1/10 bidirectional promoter from Saccharomyces cerevisiae could be used for shuttle expression in Escherichia coli. Further investigation revealed that the GAL1/10 bidirectional promoter is subjected to catabolite repression in E. coli. We next constructed a set of Golden-Gate assembly vectors for shuttle expression between S. cerevisiae and E. coli. The utility of shuttle vectors was demonstrated for rapid phenotyping of a multigene pathway for cinnamyl alcohol production. Taken together, our work opens a new avenue for the future development of broad host expression systems between prokaryotic and eukaryotic hosts.

Introduction

Microbial synthesis of natural products and fine chemicals from renewable feedstocks is emerging as a sustainable alternative to traditional chemical synthesis and plant extraction. By simply reconstructing the biosynthetic pathways in microbes, these compounds can be synthesized from inexpensive and renewable feedstocks by large-scale fermentation operations.1−4Saccharomyces cerevisiae, a “generally regarded as safe” microorganism, represents the eukaryotic “workhorse” for many biotechnological applications. When compared to another model microorganism Escherichia coli, S. cerevisiae offers advantages such as inherent safety, better tolerance at acidic pH, insusceptibility to phage contaminations, and robustness under large-scale industrial fermentation conditions.5 Recently, an in vivo method called “DNA assembler” enables rapid construction of large biochemical pathways in S. cerevisiae.6,7 However, as S. cerevisiae does not readily express multigene (polycistronic) transcriptional units, an eight-gene pathway requires at least 24 fragments to be assembled, and the efficiency is reduced to 10–20%. Hence, there is a pressing need to develop more rapid multigene assembly and pathway elucidation approaches in yeast.

With the advancement of high-throughput DNA sequencing, many new genetic parts are generated, but their functionality remains poorly characterized. For example, promoters that play an essential role in the gene regulation are of particular interest to the synthetic biology communities.8,9 However, even for model microbes like S. cerevisiae, only a handful of promoter parts have been rigorously tested.10−13 Recently, there has been arising interest in the development of promoters with broad and host-specific expression properties,14,15 which could enable new types of synthetic circuits to engineer diverse microbial communities for industrial and therapeutic applications. However, majority of these studies only focused on the characterization of promoter behaviors between different bacteria. No report on eukaryotic promoters for direct gene expression in a prokaryotic host has been described. If such a prokaryote-to-eukaryote shuttle expression system is established, it could enable rapid phenotyping of the genetic constructs in both prokaryotic and eukaryotic hosts.

Currently, E. coli still represents the most widely used microbe for pathway elucidations. Even yeast plasmids are propagated in E. coli before being transformed into yeast for functionality tests. The GAL1/10 expression system represents one of the most widely used promoters in S. cerevisiae.10,11,16,17 The expression from the GAL1/10 bidirectional promoter in budding yeast is subjected to tight glucose repression by the GAL80 repressor under a glucose-containing medium, and it is de-repressed when galactose is used as the sole carbon source.18,19 During the cloning of plasmid P423-SMO/SpEH and P423-SMO/StEH in a previous study,20 we observed that E. coli colonies with styrene monooxygenase (SMO) under the GAL10 promoter would turn into blue color. E. coli naturally synthesized indole from L-tryptophan via the action of endogenous tryptophanase encoded by TnaA,21 and we suspected that the blue pigment of indigo was formed by SMO-mediated oxidation of indole.22,23 These findings suggested that the eukaryotic GAL1/10 bidirectional promoter from S. cerevisiae might have the ability to allow gene expressions in E. coli. In this study, we characterized the functionality of the GAL1/10 bidirectional promoter in E. coli. In addition, we also developed a new set of Golden-Gated assembly vectors for rapid pathway assembly and shuttle expression between S. cerevisiae and E. coli.

Results and Discussion

Evaluation of GAL1/10-Controlled Gene Expressions in E. coli

From the literature, the E. coli expression system typically requires −35 and −10 regions for the transcription24,25 and additional ribosome binding sites (RBSs) with approximately 5–10 bp spacers before AUG start codon for the translation initiation.26 Based on sequence analysis, the GAL1/10 bidirectional promoter used in this study had similar RBSs (AGGAG and AAGAA) with a suitable spacer before AUG start codon. Further promoter prediction by “PromoterHunter” (http://www.phisite.org/main/index.php?nav=tools&nav_sel=hunter) revealed a number of prokaryotic promoters located on the 668 bp GAL1/10 bidirectional promoter (Figure 1). Overall, four predicted prokaryotic promoters are located on the “+” strand; six predicted prokaryotic promoters are located on the “–” strand. Although E. coli strains containing P423-SMO/SpEH or P423-SMO/StEH with SMO under the GAL10 promoter could synthesize the blue color pigment of indigo, it requires further evidence to confirm whether the GAL1 promoter could be used to drive gene expression in E. coli. To further characterize the behavior of the GAL1/10 bidirectional promoter in E. coli, we constructed a dual reporter system that contains SMO-mediated indigo formation and enhanced green fluorescent protein (eGFP). Interestingly, we found that SMO-mediated indigo formation was completely suppressed when glucose was supplemented in culture media (Figure 2A). This feature is of particular interest for the future applications such as toxic gene expressions in E. coli, as the targeted genes could be switched off by glucose supplementation.

Figure 1.

Figure 1

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Prediction of prokaryotic promoters in the eukaryotic GAL1/10 bidirectional promoter. The GAL1/10 sequence was analyzed by the online software “PromoterHunter”. Representative predicted prokaryotic promoters are shown on the top. Overall, four predicted prokaryotic promoters are located on the “+” strand; six predicted prokaryotic promoters are located on the “–” strand.

Figure 2.

Figure 2

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Characterization of the S. cerevisiae GAL1/10 bidirectional promoter in E. coli. (A) Dual reporter system revealed the behavior of glucose repression under the GAL1/10 bidirectional promoter in E. coli. SMO, styrene mono-oxygenase from P. putida; eGFP, enhanced green fluorescent protein. Indigo formation was imaged after 24 h. The eGFP signal was measured by setting 480/520 nm as the excitation/emission wavelength. MG1655 (DE3) with pCDF–eGFP induced with 100 μM IPTG used as control. “+” indicates the LB medium with 2% glucose. “o” indicates the LB medium without glucose supplementation. P-values were determined by a two-tailed t-test. T7 indicates MG1655 (DE3) with pCDF–eGFP induced with 100 μM IPTG in the LB medium. (B) Styrene production in E. coli via PAL2- and FDC1-mediated biocatalytic routes. PAL2, phenylalanine-ammonia lyase from A. thaliana; FDC1, ferulic acid decarboxylase from S. cerevisiae.

In contrast, eGFP under the control of the GAL1 promoter was only slightly repressed by glucose in E. coli (Figure 2A). Further compared to the most studied T7 expression system, the strength of the GAL1 promoter in our system could reach ∼35% of that of the T7 expression system. We further transformed E. coli with P425-PAL2/FDC120 for detecting styrene production in E. coli (Figure 2B). In particular, the plasmid contains PAL2, which is phenylalanine-ammonia lyase from Arabidopsis thaliana under the GAL10 promoter, and FDC1, which is a decarboxylase from S. cerevisiae under the control of the GAL1 promoter. As can be seen from Figure 2B, when compared to the authentic standard, a styrene peak in E. coli strain harboring plasmid P425-PAL2/FDC1 was detected during GC-FID analysis, whereas the E. coli strain with the empty plasmid did not exhibit any peak. These findings suggested that the yeast vector could allow multigene pathway expression in E. coli.

Build a Set of Golden-Gate Assembly Vectors for Multigene Expressions

Many commercial vectors such as pESC-URA, pESC-LEU, pESC-HIS, and pESC-TRP are based on the GAL1/10 system, and these vectors have been extensively used in metabolic engineering and fundamental yeast-related studies.28,29 However, the traditional stepwise restriction enzyme-based cloning technique is a time-consuming process. In contrast, Golden-Gate assembly that utilizes type IIS restriction enzymes is arising as a more efficient and convenient cloning method.30 In this study, we decided to construct a set of yeast vectors that can simultaneously assemble multiple genes via the BsaI-mediated Golden-Gate method. In particular, the pre-existing BsaI site in pESC-URA, pESC-LEU, pESC-HIS, and pESC-TRP was removed by site-directed mutagenesis, multicloning sites (MCSs) were replaced with four BsaI sites via yeast-mediated homologous recombination, and the ampicillin resistant markers in pESC-LEU, pESC-HIS, and pESC-TRP derived plasmids were replaced with chloramphenicol, kanamycin, and spectinomycin resistant markers (Figure 3A). We sought to take the advantage of highly efficient Golden-Gate method to achieve multigene assembly in a single step followed by one-step co-transformation for rapid characterization of the genetic constructs in both E. coli and S. cerevisiae. Therefore, the broad host shuttle vector system is expected to greatly simply the experimental procedure with reduced costs.

Figure 3.

Figure 3

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Simplified experimental workflow for elucidating multigene metabolic pathways. (A) Experimental procedure for constructing a set of Golden-Gate assembly yeast vectors. MCSs, multicloning sites; Ap*, ampicillin selection marker with the BsaI site. (B) Schematic diagram of the biocatalytic route toward cinnamyl alcohol synthesis. CAR, carboxylic acid reductase from S. rotundus; PPTase, phosphopantetheinyl transferase from N. iowensis; ADH7, alcohol dehydrogenase from S. cerevisiae. (C) Cinnamyl alcohol production in E. coli and yeast.

As a proof-of-concept, we attempted to evaluate a four-gene metabolic pathway for cinnamyl alcohol production (Figure 3B). The cinnamyl alcohol biosynthetic route contains phenylalanine ammonia lyase (PAL2) from A. thaliana,(31) carboxylic acid reductase (CAR) from Segniliparus rotundus,32 phosphopantetheinyl transferase (PPTase) from Nocardia iowensis,(32) and alcohol dehydrogenases such as ADH7 from S. cerevisiae.(16,33) As shown in Figure 3C, the four-gene pathway was recast into two plasmid modules, which were co-transformed into E. coli during the cloning step. Next, cinnamyl alcohol-producing E. coli strain was identified by GC-FID analysis (Figure 3C), and 21.88 mg/L of cinnamyl alcohol was produced in E. coli. Subsequently, both pRS424S-PAL2/ADH7 and pRS423K-CAR/PPTase were extracted from the positive E. coli strain and directly transformed into the yeast host. As evidenced by the GC-FID analysis, cinnamyl alcohol was also produced in the engineered yeast (Figure 3C), reaching 19.52 mg/L cinnamyl alcohol. In the future, it would also be possible that Golden-Gate assembled plasmids might be directly transformed into yeast, which is expected to further shorten the experimental period.

Conclusions

With the recent development of metagenomics and meta-transcriptome, there is an urgent need to establish more rapid pathway assembly and functionality characterization methods. The traditional workflow requires multiple steps of verifications before the genetic constructs are ready for characterization. The new shuttle vector system could omit the labor-intensive steps of colony PCR, restriction enzyme digestion analysis, and sequencing verification involved in the traditional procedure for multigene pathways in yeast. We envision that mining more broad host expression systems is conducive to promote the development of synthetic biology. In the future, the simplified experimental procedure might allow the design and construction of an automated workflow for pathway elucidation based on the broad host expression system. Taken together, our work highlights a new experimental regime for the discovery of natural product biosynthetic pathways.

Methods

Strains and Reagents

E. coli strain DH5α was used for plasmid construction purposes, and the strains harboring plasmids were cultivated at 37 °C in the LB medium with appropriate antibiotics (kanamycin 50 μg/mL, ampicillin 50 μg/mL, spectinomycin 50 μg/mL, and chloramphenicol 34 μg/mL). S. cerevisiae BY2M20 was cultured in the rich YPD medium (10 g/L yeast extract, 20 g/L tryptone, and 20 g/L glucose), and the engineered strains were grown in the synthetic complete (SC) medium with appropriate dropouts to compensate with different auxotrophic selection markers. All restriction enzymes, T4 ligase, Taq polymerase, and high-fidelity Phusion polymerase were obtained from New England Biolabs (Beverly, MA, USA). A gel extraction kit and plasmid purification kit were purchased from BioFlex (Shanghai, China).

Characterization of the GAL1/10 Bidirectional Promoter in E. coli

To characterize the behavior of the GAL1/10 bidirectional promoter in E. coli strain, we developed a dual reporter system that contains SMO for indigo formation and eGFP for green fluorescence analysis. The plasmid P423-SMO/eGFP was constructed in a similar way using the method described previously.20 In brief, we first amplified and purified the eGFP fragment, then mixed together with P423-SMO, and performed a second round of circular PCR to assemble eGFP into plasmid P423-SMO. The oligonucleotides used in this study are provided in Supplementary Table S1. Next, plasmid P423-SMO/eGFP was transformed into E. coli DH5α. Single colonies were picked from the plate and inoculated into 2 mL of LB medium with or without 2% glucose and further cultivated for 24 h. The indigo formation was imaged, and the eGFP signal was measured using a microplate reader (Synergy H1, Biotek, USA).

Plasmid P425-PAL2/FDC120 was transformed into E. coli DH5α. Single colonies were inoculated into 2 mL of LB medium. 20% of dodecane was added for the extraction of styrene from the fermentation broth. After 12 h cultivation, the organic phase was acquired by phase separation via centrifugation at 13,000 rpm for 10 min. The organic layer (100 μL) was sampled and diluted in 900 μL of ethyl acetate. The styrene peak was determined using gas chromatography – flame ionization detector (GC-FID) (Shimazu, GC-2030) equipped with an Rtx-5 column (30 m × 250 μm × 0.25 μm thickness). Nitrogen (ultra-purity) was used as the carrier gas at a flow rate 1.0 mL/min. GC oven temperature was initially held at 40 °C for 2 min, increased to 45 °C with a gradient of 5 °C/min, and held for 4 min. Then, it was increased to 230 °C with a gradient 15 °C/min. The authentic standard from Sigma-Aldrich was used for determining the retention time of styrene.

Build a Golden-Gate Assembly Shuttle Vector System

The commercial vectors of pESC-URA, pESC-LEU, pESC-HIS, and pESC-TRP from Agilent Technologies were used as the starting materials. Next, the BsaI site in the ampicillin selection marker was removed by site-directed mutagenesis. The PCR mixture (50 μL) containing 1 unit of Phusion polymerase and 10 ng of vector template together with 0.5 μM primer Ap_SD_fwd/Ap_SD_rev (Supplementary Table S1) was cycled 16 times at 98 °C for 15 s, 56 °C for 30s, and 68 °C for 3 min. Next, 1 μL of DpnI was added to the PCR product to digest the vector template at 37 °C for 1 h. Then, the digested product was transformed into E. coli, and colonies were picked and verified by BsaI digestion. The multiple cloning sites of these vectors were then replaced with BsaI-mediated Golden-Gate cloning sites via yeast-mediated homologous recombination. In particular, each vector was first digested with EcoRI and XhoI to remove the GAL1/10 region, and the linearized vector backbone was co-transformed with the GAL1/10 promoter amplified by primer BsaI_MCS_fwd/BsaI_MCS_rev (Supplementary Table S1) into S. cerevisiae by electroporation. Yeast colonies were randomly picked, and the extracted plasmids were then subjected to BsaI digestion analysis to confirm the replacement of MCSs with BsaI sites. As listed in Supplementary Table S2, a set of pRS426A-GGA, pRS425A-GGA, pRS423A-GGA, and pRS424A-GGA were then constructed. The ampicillin selection marker in pRS425A-GGA, pRS423A-GGA, and pRS424A-GGA was further replaced with chloramphenicol, kanamycin, and spectinomycin selection markers using a method similar to that previously reported.20 For example, the chloramphenicol selection marker was amplified from pACYCDuet-1 using the primer Cm_SD_fwd/Cm_SD_rev (Supplementary Table S1). The purified PCR product was mixed with equimolar pRS425A-GGA and was cycled 30 times at 98 °C for 15 s, 54 °C for 30s, and 68 °C for 3 min. Next, 1 μL of DpnI was added to the PCR product to digest the vector template at 37 °C for 1 h, and the digested product was transformed into E. coli and selected on LB agar plates supplemented with chloramphenicol. Other vectors were constructed in a similar way, and the resulting new sets of yeast vectors were denoted as pRS425C-GGA, pRS423K-GGA, and pRS424S-GGA, respectively.

Cinnamyl Alcohol Production in E. coli

The PAL2 gene was amplified from the cDNA of A. thaliana, and the ADH7 gene was amplified from S. cerevisiae genomic DNA. Both fragments were assembled into pRS424S-GGA by the BsaI-mediated Golden-Gate method. In brief, equimolar of PAL2, ADH7, and BsaI-pretreated vector pRS424S-GGA in 20 μL of T4 buffer with 400 units of T4 ligase and 20 units of BsaI-HF were incubated on a thermocycler with 6 cycles of 10 min at 37 °C and 10 min at 16 °C. Similarly, both the CAR gene from S. rotundus DSM 44985 and the PPTase gene from N. iowensis were amplified from plasmid pETDuet-1-PPTase-CAR32 (a kind gift from Prof. Dunming Zhu from Tianjin Institute of Industrial Biotechnology) and assembled into pRS423K-GGA via the Golden-Gate approach. Both ligation mixtures were co-transformed into E. coli DH5α electrocompetent cells with high efficiency. After recovery on the SOC medium for 1–2 h, the cells were pelleted by centrifugation and plated on LB + 2% glucose with spectinomycin and kanamycin. Next day, the colonies were randomly picked and inoculated into 2 mL of LB medium. After 12 h cultivation, 100 μL of supernatant was extracted with 900 μL of ethyl acetate before being subjected to GC-FID analysis. The diluted sample (1 μL) was injected into a Shimadzu GC-2030 system equipped with an Rtx-5 column (30 m × 250 μm × 0.25 μm thickness). Nitrogen (ultra-purity) was used as the carrier gas at a flow rate of 1.0 mL/min. GC oven temperature was initially held at 40 °C for 2 min, increased to 45 °C with a gradient of 5 °C/min, and held for 4 min. Then, it was increased to 230 °C with a gradient of 15 °C/min. The authentic standard from Sigma-Aldrich was used to determine the cinnamyl alcohol level.

Cinnamyl Alcohol Production in S. cerevisiae

Upon confirming cinnamyl alcohol-producing E. coli strains, the cell pellets were next subjected to plasmid extraction. The extracted plasmids were transformed into yeast BY2M strain for the follow-up characterization of cinnamyl alcohol production in yeast. Small-scale shake-flask studies were carried out. In brief, 100 mL shake flasks containing 20 mL of SC medium (1.8% w/v galactose +0.2% w/v glucose) with appropriate dropouts were inoculated with fresh overnight yeast cultures to an initial OD600 of 0.1. After 24 h cultivation, 100 μL of supernatant was extracted with 900 μL of ethyl acetate for GC-FID analysis of cinnamyl alcohol as mentioned above.

Acknowledgments

This work was supported by Xiamen University under grant no. 0660-X2123310 and ZhenSheng Biotech, China. We would like to thank Prof. Dunming Zhu from Tianjin Institute of Industrial Biotechnology for providing pETDuet-1-PPTase-CAR.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c00339.

  • List of oligonucleotides used in the study (Table S1) and plasmids and strains used in the study (Table S2) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao1c00339_si_001.pdf (924.4KB, pdf)

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Associated Data

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Supplementary Materials

ao1c00339_si_001.pdf (924.4KB, pdf)

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