Synthetic Biology Journal ›› 2021, Vol. 2 ›› Issue (5): 792-803.DOI: 10.12211/2096-8280.2021-014
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Xiaodong LI1,2, Chengshuai YANG3, Pingping WANG1, Xing YAN1, Zhihua ZHOU1
Received:
2021-01-31
Revised:
2021-03-10
Online:
2021-11-19
Published:
2021-11-19
Contact:
Zhihua ZHOU
李晓东1,2, 杨成帅3, 王平平1, 严兴1, 周志华1
通讯作者:
周志华
作者简介:
基金资助:
CLC Number:
Xiaodong LI, Chengshuai YANG, Pingping WANG, Xing YAN, Zhihua ZHOU. Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae[J]. Synthetic Biology Journal, 2021, 2(5): 792-803.
李晓东, 杨成帅, 王平平, 严兴, 周志华. 构建酿酒酵母细胞工厂从头合成倍半萜类化合物α-新丁香三环烯和β-石竹烯[J]. 合成生物学, 2021, 2(5): 792-803.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2021-014
Name | Genotype | Source |
---|---|---|
BY4742 | MATa, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0 | EUROSCARF |
CEN.PK2-1 | MATa, wra3-52, trp1-289, leu2-3_112, hisΔ1 | EUROSCARF |
SQTBY01 | BY4742(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t) | this study |
SQTCEN01 | CEN.PK2-1(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t) | this study |
BY4742Δ | BY4742(ΔGAL80) | this study |
SQTBY02 | BY4742Δ(deltaDNA:: GAL1p-ERG10-TDH2t, GAL10p-ERG13-HXT7t, GAL1p-tHMGR1-tHMGR1t, GAL10p-ERG12-PYK1t, GAL1p-ERG8-PDC1t) | this study |
SQTBY03 | SQTBY02(rDNA:: GAL1p-tHMGR1-CYC1t, GAL10p-ERG19-ADH1t, GAL1p-IDI1-ENO2t, GAL10p-ERG20-PGIt) | this study |
NCVBY01 | SQTBY03(X-4:: TEF1p-synec38-cs-PRM9t) | this study |
CPLBY01 | SQTBY03(X-4:: TEF1p-synQHS1-PRM9t) | this study |
Tab. 1 Strains used in this study
Name | Genotype | Source |
---|---|---|
BY4742 | MATa, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0 | EUROSCARF |
CEN.PK2-1 | MATa, wra3-52, trp1-289, leu2-3_112, hisΔ1 | EUROSCARF |
SQTBY01 | BY4742(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t) | this study |
SQTCEN01 | CEN.PK2-1(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t) | this study |
BY4742Δ | BY4742(ΔGAL80) | this study |
SQTBY02 | BY4742Δ(deltaDNA:: GAL1p-ERG10-TDH2t, GAL10p-ERG13-HXT7t, GAL1p-tHMGR1-tHMGR1t, GAL10p-ERG12-PYK1t, GAL1p-ERG8-PDC1t) | this study |
SQTBY03 | SQTBY02(rDNA:: GAL1p-tHMGR1-CYC1t, GAL10p-ERG19-ADH1t, GAL1p-IDI1-ENO2t, GAL10p-ERG20-PGIt) | this study |
NCVBY01 | SQTBY03(X-4:: TEF1p-synec38-cs-PRM9t) | this study |
CPLBY01 | SQTBY03(X-4:: TEF1p-synQHS1-PRM9t) | this study |
Gene | Description | Accession No. |
---|---|---|
GAL80 | Coding gene of transcription regulator | NM_001182409 |
ERG10 | Coding gene of acetyl-CoA C-acetyltransferase | NM_001183842 |
ERG13 | Coding gene of hydroxymethylglutaryl-CoA synthase | NM_001182489 |
tHMGR1 | Coding gene of 3-hydroxy-3-methyl-glutaryl-CoA reductase | NM_001182434 |
ERG12 | Coding gene of mevalonate kinase | NM_001182715 |
ERG8 | Coding gene of phosphomevalonate kinase | NM_001182727 |
ERG19 | Coding gene of diphosphomevalonate decarboxylase | NM_001183220 |
IDI1 | Coding gene of isopentenyl-diphosphate delta-isomerase | NM_001183931 |
ERG20 | Coding gene of (2E,6E)-farnesyl diphosphate synthase | NM_001181600 |
ec38-cs | Coding gene of caryophyllene synthase Hypoxylon sp. EC38 | OTA59675 |
QHS1 | Coding gene of β-caryophyllene synthase from Artemisia annua | AF472361 |
Tab. 2 Genes used in this study
Gene | Description | Accession No. |
---|---|---|
GAL80 | Coding gene of transcription regulator | NM_001182409 |
ERG10 | Coding gene of acetyl-CoA C-acetyltransferase | NM_001183842 |
ERG13 | Coding gene of hydroxymethylglutaryl-CoA synthase | NM_001182489 |
tHMGR1 | Coding gene of 3-hydroxy-3-methyl-glutaryl-CoA reductase | NM_001182434 |
ERG12 | Coding gene of mevalonate kinase | NM_001182715 |
ERG8 | Coding gene of phosphomevalonate kinase | NM_001182727 |
ERG19 | Coding gene of diphosphomevalonate decarboxylase | NM_001183220 |
IDI1 | Coding gene of isopentenyl-diphosphate delta-isomerase | NM_001183931 |
ERG20 | Coding gene of (2E,6E)-farnesyl diphosphate synthase | NM_001181600 |
ec38-cs | Coding gene of caryophyllene synthase Hypoxylon sp. EC38 | OTA59675 |
QHS1 | Coding gene of β-caryophyllene synthase from Artemisia annua | AF472361 |
Strains | FOH/(mg/L) | Squalene/(mg/L) | Lanosterol/(mg/L) |
---|---|---|---|
BY4742 | 0.177±0.011 | 0.5±0.07 | 3.1±0.57 |
SQTBY01 | 4.556±0.245 | 51.1±4.55 | 46.0±9.24 |
CEN.PK2-1 | 0.044±0.002 | 0.3±0.05 | 3.5±0.16 |
SQTCEN01 | 0.504±0.093 | 32.0±6.23 | 26.8±1.66 |
Tab. 3 Production of FOH, squalene and lanosterol in different yeast strains
Strains | FOH/(mg/L) | Squalene/(mg/L) | Lanosterol/(mg/L) |
---|---|---|---|
BY4742 | 0.177±0.011 | 0.5±0.07 | 3.1±0.57 |
SQTBY01 | 4.556±0.245 | 51.1±4.55 | 46.0±9.24 |
CEN.PK2-1 | 0.044±0.002 | 0.3±0.05 | 3.5±0.16 |
SQTCEN01 | 0.504±0.093 | 32.0±6.23 | 26.8±1.66 |
Retention time/min | Products | Mass spectra match factors/% | Area/% |
---|---|---|---|
11.538 | (-)-cyperene | 92 | 21.62 |
12.359 | β-caryophyllene | 95 | 8.52 |
12.752 | humulene | 97 | 2.53 |
12.932 | AC1LBUZB | 96 | 5.02 |
13.067 | germacrene D | 96 | 3.54 |
13.395 | β-caryophyllene Fcc | 86 | 18.87 |
16.865 | α-neoclovene | 89 | 39.90 |
Tab. 4 GC/MS analysis for identification of by-products produced by NCVBY01
Retention time/min | Products | Mass spectra match factors/% | Area/% |
---|---|---|---|
11.538 | (-)-cyperene | 92 | 21.62 |
12.359 | β-caryophyllene | 95 | 8.52 |
12.752 | humulene | 97 | 2.53 |
12.932 | AC1LBUZB | 96 | 5.02 |
13.067 | germacrene D | 96 | 3.54 |
13.395 | β-caryophyllene Fcc | 86 | 18.87 |
16.865 | α-neoclovene | 89 | 39.90 |
Strain | Products | Yield/(mg/L) |
---|---|---|
NCVBY01 | α-neoclovene | 487.1 |
(-)-cyperene | 247.6 | |
β-caryophyllene Fcc | 221.2 | |
β-caryophyllene | 109.2 | |
CPLBY01 | β-caryophyllene | 2949.1 |
α-humulene | 109.8 |
Tab. 5 Yield of sesquiterpenes in NCVBY01 and CPLBY01 by fed-batch fermentation.
Strain | Products | Yield/(mg/L) |
---|---|---|
NCVBY01 | α-neoclovene | 487.1 |
(-)-cyperene | 247.6 | |
β-caryophyllene Fcc | 221.2 | |
β-caryophyllene | 109.2 | |
CPLBY01 | β-caryophyllene | 2949.1 |
α-humulene | 109.8 |
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