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Part Collection ¶

Utilized and characterized existing parts:

part name

part number

contribution

ERG20 (farnesyl diphosphate synthase)

BBa_K517004

We constructed several constructs with ERG20 to increase the expression of patchoulol in Saccharomyces cerevisiae. In our project, we also named ERG20 as FPS (farnesyl diphosphate synthase) because ERG20 and FPS both can catalyze IPP (isopentenyl diphosphate) to FPP (farnesyl pyrophosphate).[1]

pPGK1 promoter

BBa_K122000

As a constitutive promoter, we used it to express our patchoulol synthase (PTS) and farnesyl diphosphate synthase (ERG20).

pTEF1 promoter

BBa_K2765041

As an yeast promoter, we used it in expressing farnesyl diphosphate synthase (ERG20).

TADH1

BBa_K122004

As a terminator with strong termination efficiencies, we used it to avoid a transcription of genes downstream of this part.

T CYC1

BBa_K122003

We used this strong terminator to avoid a transcription of genes downstream of this part.

Parts ¶

New basic and Composite parts:

part name

part number

description

HCF173 promoter

BBa_K4270001

HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173), which is required for Arabidopsis thaliana PSII biogenesis. HCF173 transcript levels and expression were induced by light. HCF173 promoter contains ACE motif and G-box element.[2, 3]

PTS (patchoulol synthase)

BBa_K4270004

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes including patchoulol by conversion of the natural substrate FPP.[1, 4]

erg20 K197P

BBa_K4270007

A mutant of ERG20 gene; K197P substitution. Yeast strain K197P shows lower sterol content compared with the original strain K197K.[5] FPP is the precursor of yeast sterol and patchoulol biosynthetic pathway.[6, 7]

erg20 K197H

BBa_K4270008

A mutant of ERG20 gene; K197H substitution. Yeast strain K197H produces no statistically significant changes in recorded doubling time and sterol content compared with the original strain K197K.[5]

FPS-linker-PTS

BBa_K4270010

A gene fusion of PTS and FPS. The stop codon of FPS was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs.[1, 8]

pPGK1+PTS+TADH1

BBa_K4270011

This composite part consists of pPGK1 promoter, PTS gene, and TADH1 terminator. The product of this system is a patchoulol synthase.

pPGK1+ERG20+TADH1

BBa_K4270012

This composite part consists of pPGK1 promoter, ERG20 gene, and TADH1 terminator. The product of this system is a farnesyl diphosphate synthase.

pTEF1+ERG20+TCYC1

BBa_K4270016

This composite part consists of pTEF1 promoter, ERG20 gene, and TCYC1 terminator. The product of this system is a farnesyl diphosphate synthase.

HCF173 is the promoter of HIGH CHLOROPHYLL FLUORESCENCE173 (HCF173), which is required for Arabidopsis thalianaPSII biogenesis.HCF173 transcript levels and its expression were induced by light.HCF173 promoter contains ACE (HCF173PB) motif and G-box (HCF173PC) element.[1, 2]

2. erg20 K197P ¶

erg20K197P:a mutant of ERG20 gene; K197P substitution. Yeast strain K197P showslower sterol content compared with the original strain K197K[3].FPP is theprecursor of yeast steroland patchoulolbiosynthetic pathway.[4, 5]

3.erg20 K197H ¶

erg20K197H: a mutant of ERG20 gene; K197H substitution. Yeast strain K197H produces no statistically significant changes in in recorded doubling time and sterol content compared with the original strain K197K[3].

4. PTS ¶

PTS:patchoulol synthase.The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpenesynthase and produces more than 20 valuable sesquiterpenes including patchoulol by conversion of thenatural substrate farnesyl pyrophosphate (FPP)[6, 7].

5. FPS-linker-PTS ¶

FPS-linker-PTS:a gene fusion of PTS and FPS.The stop codon of FPS was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the FPS and PTS ORFs[7, 8].

Reference ¶

[1] Li X, Wang H B, Jin H L. Light Signaling-Dependent Regulation of PSII Biogenesis and Functional Maintenance [J]. Plant Physiol, 2020, 183(4): 1855-68.

[2] Schult K, Meierhoff K, Paradies S, et al. The nuclear-encoded factor HCF173 is involved in the initiation of translation of the psbA mRNA in Arabidopsis thaliana [J]. Plant Cell, 2007, 19(4): 1329-46.

[3] Fischer M J, Meyer S, Claudel P, et al. Metabolic engineering of monoterpene synthesis in yeast [J]. Biotechnol Bioeng, 2011, 108(8): 1883-92.

[4] Szkopinska A, D. P. Farnesyl diphosphate synthase; regulation of product specificity [J]. Acta Biochim Pol, 2005, 52: 45-55.

[5] Yu Z X, Wang L J, Zhao B, et al. Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-targeted SPL transcription factors [J]. Mol Plant, 2015, 8(1): 98-110.

[6] Ekramzadeh K, Bramer C, Frister T, et al. Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase [J]. Biotechnol Prog, 2020, 36(2): e2935.

[7] Wu S, Schalk M, Clark A, et al. Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants [J]. Nat Biotechnol, 2006, 24(11): 1441-7.

[8] Brodelius M, Lundgren A, Mercke P, et al. Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum [J]. Eur J Biochem, 2002, 269(14): 3570-7.