Results
Overview
We chose Saccharomyces cerevisiae (yeast) to express enzymes that catalyze the production of seven specific terpenoids which are pleasant-smelling, distinctive, and safe compounds that could serve as olfactory signals. The selected terpenoids also have antibacterial properties, adding a functional benefit beyond their fragrance. Such as Limonene(tLsCl), ocimene (CcOCS), cineole (HpyCins, StrCins, SfCins), nerol (PgfB), nerolidol (AcNES1), Myrcene (AmMYS), borneol (CbTPS1).In this section, we will present all the results that are important checkpoints in our experiments. These include successful plasmid construction verification, successful transformation into yeast strain BY4741, and successful protein expression in yeast strain BY4741.
Figure 1: Plates of microbial medium used during the experiment
Verifying the Construction of plasmid in E.coli
Colony PCR of E.coli single colonies
Below are the results of the gel electrophoresis of colony PCR of transformed single DH5α E. coli colonies with our constructed plasmids. Two tubes of the sizes that are the same as the expected size are sequenced. The constructed plasmids were initially assembled from the terpene synthase gene segments and the HcKan-O vector. Subsequently, we combined these genes with our shuttle vector, along with the promoters and terminators.
The colony PCR Gel Electrophoresis Results of AmMYS, CbTPS1 and tLsCl:
Figure 2: The growth of POT-AmMYS, POT-CbTPS1 and POT-tLsCl colonies on petri dish.
Figure 3: HcKan-AmMYS, HcKan-CbTPS1, POT-AmMYS, POT-CbTPS1 and POT-tLsCl Colony PCR Gel Electrophoresis Results. A: HcKan-AmMYS and HcKan-CbTPS1. B: Enzymatic digestion result of HcKan-AmMYS and HcKan-CbTPS1. C: POT-AmMYS, POT-CbTPS1 and POT-tLsCl.
The colony PCR Gel Electrophoresis Results of CcOCS:
Figure 4: Single colonies of plasmid transformants on LB Ampicillin plate
Figure 5: DH5α E.coli colony PCR results of CcOCS A: HCKAN-O-CcOCS, B: POT2_pGPM1_CcOCS_PGK1t, C: POT2_pINO1_CcOCS_TEF1t, D: POT2_pTDH3_CcOCS_ADH1t（M=DL5000 marker）
Figure 6: Enzymatic digestion results of CcOCS
The colony PCR Gel Electrophoresis Results of StrCins, HpyCins and SfCins:
Figure 7: The growth of HcKan-HpyCins, HcKan-SfCins, POT-HpyCins, POT-StrCins and POT-SfCins colonies on petri dish.
Figure 8: DH5α E.coli colony PCR results of HpyCins, StrCins and SfCins A: HCKAN-O-SfCins, B: HCKAN-O-StrCins, C: HCKAN-O-HpyCins（M=DL5000 marker）
Figure 9: DH5α E.coli colony PCR results of HpyCins, StrCins and SfCins A: POT2_pTDH3_HpyCins_ADH1t, B: POT2_pINO1_HpyCins_TEF1t, C: POT2_pGPM1_StrCins_PGK1t（M=DL2000 marker）
Figure 10: Enzymatic digestion result of StrCins
The colony PCR Gel Electrophoresis Results of PgfB:
Figure 11: The growth of HcKan-PgfB and POT-PgfB colonies on petri dish. HCKAN-O-HpyCins（M=DL5000 marker）
Figure 12: DH5α E.coli colony PCR results of PgfB A: HCKAN-O-PgfB, B: POT2_pINO1_PgfB_TEF1t, C: POT2_pGPM1_PgfB_PGK1t, D: POT2_pTDH3_PgfB_ADH1t
Figure 13: Enzymatic digestion result of PgfB
The colony PCR Gel Electrophoresis Results of AcNES1:
Figure 14: The growth of HcKan-AcNES1 and POT-AcNES1 colonies on petri dish. HCKAN-O-HpyCins（M=DL5000 marker）
Figure 15: DH5α E.coli colony PCR results of AcNES1 A: HCKAN-O-AcNES1. B: POT2_pINO1_AcNES1_TEF1t, POT2_pGPM1_AcNES1_PGK1t, POT2_pTDH3_AcNES1_ADH1t. C: Enzymatic digestion result of AcNES1. （M=DL5000 marker） D: POT2_pTDH3_PgfB_ADH1t（M=DL5000 marker）
The colony PCR Gel Electrophoresis Results of PgfB and AcNES1 with pGAL1 promoter:
Figure 16: Single colonies of plasmid transformants on LB ampicillin plate HCKAN-O-HpyCins（M=DL5000 marker）
Figure 17: DH5α E.coli colony PCR results of PgfB and AcNES1 A: POT2_pGAL1_PgfB_PGK1t. B: POT2_pGAL1_AcNES1_PGK1t. C: Enzymatic digestion result of PgfB and AcNES1. （M=DL5000 marker）
Sequencing results
Sequencing of the plasmids can test whether our constructed plasmid is exactly the same as what we designed and inserted. Below are the diagrams of the sequencing results. The solid red arrows present at the top of the diagrams mean that the sequencing is correct.The sequencing result of AmMYS, CbTPS1 and tLsCl:
Strain 2 has been confirmed positive by sequencing
Strain 1 has been confirmed positive by sequencing
Strain 3 and 4 have been confirmed positive by sequencing
Strain 2 and 7 have been confirmed positive by sequencing
Strain 1 and 3 have been confirmed positive by sequencing
Strain 9 has been confirmed positive by sequencing
Strain 1 and 7 have been confirmed positive by sequencing
The sequencing result of CcOCS:
Strain 2 and 16 have been confirmed positive by sequencing
Strain 4 and 7 have been confirmed positive by sequencing
Strain 6 and 8 have been confirmed positive by sequencing
Strain 1 and 5 have been confirmed positive by sequencing
The sequencing result of HpyCins, StrCins and SfCins:
Strain 10 and 13 have been confirmed positive by sequencing
Strain 2 and 3 have been confirmed incorrect by sequencing
Strain 6 and 8 have been confirmed positive by sequencing
Strain 3 and 4 have been confirmed positive by sequencing
Strain 3 and 10 have been confirmed positive by sequencing
Strain 1 and 3 have been confirmed positive by sequencing
The sequencing result of PgfB:
Strain 2 and 3 have been confirmed positive by sequencing
Strain 8 and 9 have been confirmed positive by sequencing
Strain 1 and 2 have been confirmed positive by sequencing
Strain 1 and 2 have been confirmed positive by sequencing
The sequencing result of AcNES1:
Strain 4 and 8 have been confirmed positive by sequencing
Strain 3 and 10 have been confirmed positive by sequencing
Strain 2 and 4 have been confirmed positive by sequencing
Strain 1 has been confirmed positive by sequencing
The sequencing result of PgfB and AcNES1 with pGAL1 promoter:
Strain 2 and 6 have been confirmed positive by sequencing
Strain 1 and 2 have been confirmed positive by sequencing
Primers used for monoclonal colony PCR
| Terpene Synthase Gene | primer-F | primer-R | Expected Size (bp) | Results |
|---|---|---|---|---|
| HcKan-O-CcOCS | CcOCS-seq-F | CcOCS-seq-R | 971 | Success |
| HcKan-O-StrCins | StrCins-seq-F | StrCins-seq-R | 925 | Success |
| HcKan-O-AmMYS | AmMYS-seq-F | AmMYS-seq-R | 914 | Success |
| HcKan-O-CbTPS1 | CbTPS1-seq-F | CbTPS1-seq-R | 1610 | Success |
| HcKan-O-pgfB | pgfB-seq-F | pgfB-seq-R | 986 | Success |
| HcKan-O-AcNES1 | AcNES1-seq-F | AcNES1-seq-R | 970 | Success |
| HcKan-O-HpyCins | HpyCins-seq-F | HpyCins-seq-R | 1039 | Success |
| HcKan-O-SfCins | SfCins-seq-F | SfCins-seq-R | 1574 | Failed |
| POT2-pINO1-tLsCl-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1936 | Failed |
| POT2-pGPM1-tLSCl-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1842 | Failed |
| POT2-pTDH3-tLSCl-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2073 | Success |
| POT2-pINO1-CcOCS-TEF1 | pINO1-seq-F | TEF1t-seq-R | 2052 | Success |
| POT2-pGPM1-CcOCS-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1965 | Failed |
| POT2-pTDH3-CcOCS-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2148 | Success |
| POT2-pGPM1-StrCins-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1167 | Failed |
| POT2-pINO1-AmMYS-TEF1t | pINO1-seq-F | TEF1t-seq-R | 2014 | Failed |
| POT2-pGPM1-CbTPS1 -PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1902 | Failed |
| POT2-pTDH3-pgfB-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1368 | Success |
| POT2-pGPM1-pgfB-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1185 | Success |
| POT2-pINO1-pgfB-TEF1t | pTDH3-seq-F | ADH1t-seq-R | 1270 | Success |
| POT2-pGAL1-pgfB-PGK1t | pgfB-seq-F | PGK1t-seq-R | 1069 | Success |
| POT2-pTDH3-AcNES1-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2079 | Success |
| POT2-pINO1-AcNES1-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1990 | Failed |
| POT2-pGPM1-AcNES1-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1896 | Success |
| POT2-pGAL1-AcNES1-PGK1t | AcNES1-seq-F | PGK1t-seq-R | 1432 | Success |
| POT2-pTDH3-HpyCins-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1476 | Success |
| POT2-pINO1-HpyCins-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1387 | Success |
Verifying the transformation of plasmid in yeast
Colony PCR of Yeast single colonies
Below are the gel electrophoresis pictures of the colony PCR of transformed strain BY4741 with fully constructed plasmids, which are detected positive by colony PCR. The single colonies are shown in figure 18. If we see that the gene segment is the correct size, then we know that the plasmid is inside the yeast, and we can begin fermenting them for Gas chromatography–mass spectrometry check.
Figure 18: Streak monocultures of transformed strain BY4741 with terpene synthase genes.
The colony PCR Gel Electrophoresis Results of tLsCl:
Yeast colony PCR results confirmed that the plasmid POT2_pTDH3_tLsCl_ADH1t were positive in Saccharomyces cerevisiae.
Figure 19: Streak monocultures of transformed stain BY4741 with terpene synthase genes tLsCl.
Figure 20: Yeast colony PCR result performed to screen strain
The colony PCR Gel Electrophoresis Results of CcOCS:
Yeast colony PCR results confirmed that two plasmids POT2_pINO1_CcOCS_TEF1t and POT2_pTDH3_CcOCS_ADH1t were positive in Saccharomyces cerevisiae.
Figure 21: Streak monocultures of transformed stain BY4741 with terpene synthase genes CcOCS.
Figure 22: Yeast colony PCR result performed to screen strain A: BY4741-POT2_pGPM1_CcOCS_PGK1t, B: BY4741-POT2_pINO1_CcOCS_TEF1t, C: BY4741-POT2_pTDH3_CcOCS_ADH1t（M=DL5000 marker；P=Positive; N=Negative）
The colony PCR Gel Electrophoresis Results of StrCins, HpyCins:
Yeast colony PCR results confirmed that two plasmids POT2_pTDH3_HpyCins_ADH1t and POT2_pINO1_HpyCins_TEF1t were positive in Saccharomyces cerevisiae.
Figure 23: Streak monocultures of transformed stain BY4741 with terpene synthase genes HpyCins, StrCins and SfCins.
Figure 24: Yeast colony PCR result of cineole performed to screen strain.（M=DL5000 marker；P=Positive; N=Negative）
The colony PCR Gel Electrophoresis Results of PgfB:
Yeast colony PCR results confirmed that three plasmids POT2_pINO1_PgfB_TEF1t, POT2_pGPM1_PgfB_PGK1t and POT2_pTDH3_PgfB_ADH1t, were positive in Saccharomyces cerevisiae.
Figure 25: Streak monocultures of transformed stain BY4741 with terpene synthase genes PgfB.
Figure 26: Yeast colony PCR result of nerol performed to screen strain. A: BY4741-POT2_pGPM1_PgfB_PGK1t. B: BY4741-POT2_pINO1_PgfB_TEF1t. C: BY4741-POT2_pTDH3_PgfB_ADH1t.（M=DL5000 marker；P=Positive; N=Negative）
The colony PCR Gel Electrophoresis Results of AcNES1:
Yeast colony PCR results confirmed that two plasmids POT2_pGPM1_AcNES1_PGK1t and POT2_pTDH3_AcNES1_ADH1t were positive in Saccharomyces cerevisiae.
Figure 27: Streak monocultures of transformed stain BY4741 with terpene synthase genes AcNES1.
Figure 28: Yeast colony PCR result of nerolidol performed to screen strain. A: BY4741-POT2_pGPM1_AcNES1_PGK1t. B: BY4741-POT2_pTDH3_AcNES1_ADH1t. C: BY4741-POT2_pINO1_AcNES1_TEF1t.（M=DL5000 marker；P=Positive; N=Negative）
The colony PCR Gel Electrophoresis Results of PgfB and AcNES1 with pGAL1 promoter:
Yeast colony PCR results confirmed that two plasmids BY4741-POT2_pGAL1_AcNES1_PGK1t and BY4741-POT2_pGAL1_PgfB_PGK1t were positive in Saccharomyces cerevisiae.
Figure 29: Yeast colony PCR results of nerolidol and nerol performed to screen strain. A: BY4741-POT2_pGAL1_AcNES1_PGK1t. B: BY4741-POT2_pGAL1_PgfB_PGK1t. （M=DL5000 marker；P=Positive; N=Negative）
Figure 30: Streak monocultures of transformed stain BY4741 with terpene synthase genes PgfB and AcNES1 with pGAL1 promoter.
Primers used for yeast colony PCR
| Terpene Synthase Gene | primer-F | primer-R | Expected Size (bp) | Results |
|---|---|---|---|---|
| BY4741-POT2-pINO1-tLsCl-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1936 | Failed |
| BY4741-POT2-pGPM1-tLSCl-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1842 | Failed |
| BY4741-POT2-pTDH3-tLSCl-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2073 | Success |
| BY4741-POT2-pINO1-CcOCS-TEF1 | pINO1-seq-F | TEF1t-seq-R | 2052 | Success |
| BY4741-POT2-pGPM1-CcOCS-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1965 | Failed |
| BY4741-POT2-pTDH3-CcOCS-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2148 | Success |
| BY4741-POT2-pGPM1-StrCins-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1167 | Failed |
| BY4741-POT2-pINO1-AmMYS-TEF1t | pINO1-seq-F | TEF1t-seq-R | 2014 | Failed |
| BY4741-POT2-pGPM1-CbTPS1 -PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1902 | Failed |
| BY4741-POT2-pTDH3-pgfB-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1368 | Success |
| BY4741-POT2-pGPM1-pgfB-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1185 | Success |
| BY4741-POT2-pINO1-pgfB-TEF1t | pTDH3-seq-F | ADH1t-seq-R | 1270 | Success |
| BY4741-POT2-pGAL1-pgfB-PGK1t | pgfB-seq-F | PGK1t-seq-R | 1069 | Success |
| BY4741-POT2-pTDH3-AcNES1-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 2079 | Success |
| BY4741-POT2-pINO1-AcNES1-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1990 | Failed |
| BY4741-POT2-pGPM1-AcNES1-PGK1t | pGPM1-seq-F | PGK1t-seq-R | 1896 | Success |
| BY4741-POT2-pGAL1-AcNES1-PGK1t | AcNES1-seq-F | PGK1t-seq-R | 1432 | Success |
| BY4741-POT2-pTDH3-HpyCins-ADH1t | pTDH3-seq-F | ADH1t-seq-R | 1476 | Success |
| BY4741-POT2-pINO1-HpyCins-TEF1t | pINO1-seq-F | TEF1t-seq-R | 1387 | Success |
Gas chromatography–mass spectrometry analysis
To evaluate the production of nerol in S.cerevisiae cells, a shake flask fermentation was conducted. After fermentation for 12 h, dodecane overlay culture was added. The fermentation process ended at 120 h.
GC-MS analysis of Limonene :
Flask fermentation of tLsCl yielded 3.17 ± 0.30 mg/l of Limonene with the pTDH3 promoter and ADH1t terminator. There was no significant difference in yield of empty shuttle vector strain.
Figure 31: The production of limonene in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae with POT vector. B: GC-MS analyses of cineole extracts of S. cerevisiae expressing tLsCl with the pTDH3 promoter and ADH1t terminator. C: the OD600 and Limonene yield by POT vector and gene tLsCl. The data represent the mean ± standard deviation of three independent experiments.
GC-MS analysis of ocimene :
The ocimene titer was 0.83 ± 0.06 mg/L with the pINO1 promoter and TEF1t terminator, while it was 0.33 ± 0.09 mg/L with the pTDH3 promoter and ADH1t terminator.
Figure 32: Heterologous expression of HpyCins in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae expressing HpyCins with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of cineole extracts of S. cerevisiae expressing HpyCins with the pINO1 promoter and TEF1t terminator. C: the OD600 and cineole yield by HpyCins under the control of different promoters and terminators. The data represent the mean ± standard deviation of three independent experiments.
GC-MS analysis of cineole:
Flask fermentation of HpyCins yielded 11.88 ± 2.03 mg/l of cineole with the pINO1 promoter and TEF1t terminator, compared to 17.58 ± 3.20 mg/l with the pTDH3 promoter and ADH1t terminator.
Figure 33: Heterologous expression of HpyCins in S. cerevisiae. A: GC-MS analyses of cineole extracts of S. cerevisiae expressing HpyCins with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of cineole extracts of S. cerevisiae expressing HpyCins with the pINO1 promoter and TEF1t terminator. C: the OD600 and cineole yield by HpyCins under the control of different promoters and terminators. The data represent the mean ± standard deviation of three independent experiments.
GC-MS analysis of nerol:
Flask fermentation of PgfB yielded 6.07 ± 0.23 mg/l of nerol with the pTDH3 promoter and ADH1t terminator, but no terpenes are produced with pINO1 and GPM1 promoter.
Figure 34: Heterologous expression of PgfB in S. cerevisiae. A: GC-MS analyses of nerol extracts of S. cerevisiae expressing PgfB with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerol extracts of S. cerevisiae expressing PgfB with the pINO1 promoter and TEF1t terminator. C: GC-MS analyses of nerol extracts of S. cerevisiae expressing PgfB with the pGPM1 promoter and PGK1t terminator. D: The OD600 and nerol yield by PgfB under the control of different promoters and terminators. The data represent the mean ± standard deviation of three independent experiments.
GC-MS analysis of nerolidol:
Flask fermentation of AcNES1 yielded 45.06 ± 1.68 mg/l of nerolidol with the pTDH3 promoter and ADH1t terminator, compared to 12.92 ± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator.
Figure 35: Heterologous expression of AcNES1 in S. cerevisiae. A: GC-MS analyses of nerolidol extracts of S. cerevisiae expressing AcNES1 with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerolidol extracts of S. cerevisiae expressing AcNES1 with the pGPM1 promoter and PGK1t terminator. C: The OD600 and nerolidol yield by AcNES1 under the control of different promoters and terminators. The data represent the mean ± standard deviation of three independent experiments.
GC-MS analysis of nerol and nerolidol:
Flask fermentation of AcNES1 with the pGAL1 promoter and PGK1t terminator yielded 4.78 ± 0.88 mg/l of nerolidol, while no nerol production was detected.
Figure 36: Heterologous expression of AcNES1 in S. cerevisiae A: GC results of AcNES1 with glucose under the control of the pGAL1 promoter and PGK1t terminator. B: GC results of AcNES1 with galactose under the control of the pGAL1 promoter and PGK1t terminator.
Figure 37: Heterologous expression of PgfB in S. cerevisiae. A: GC results of PgfB with glucose under the control of the pGAL1 promoter and PGK1t terminator.B: GC results of PgfB with galactose under the control of the pGAL1 promoter and PGK1t terminator.
Figure 38: The OD600 and terpenes production by AcNES1 and PgfB under the control of pGAL1 promoter and PGK1t terminator
Reference
Yakun Guo and others, ‘YeastFab: The Design and Construction of Standard Biological Parts for Metabolic Engineering in Saccharomyces Cerevisiae’, Nucleic Acids Research, 43.13 (2015), pp. e88–e88, doi:10.1093/nar/gkv464.
Hongting Tang and others, ‘Promoter Architecture and Promoter Engineering in Saccharomyces Cerevisiae’, Metabolites, 10.8 (2020), p. 320, doi:10.3390/metabo10080320.