Table 1 Strains, gene cassettes, and primers used in this study
Strains/plasmids/primers | Genotype/sequences (5′ → 3′) | References/restriction sites |
|---|---|---|
E. coli strain | ||
BL21(DE3) | B F– ompT gal dcm lon hsdSB(rB–mB–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS) pLysS | New England BioLabs |
TOP10 | F– mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL endA1 nupG | Thermo Fisher Scientific |
BL21/AraDH1 | BL21(DE3) pET28a-AraDH1 | This work |
BL21/AraDH2 | BL21(DE3) pET28a-AraDH2 | This work |
BL21/MDH1 | BL21(DE3) pET28a-MDH1 | This work |
BL21/MDH2 | BL21(DE3) pET28a-MDH2 | This work |
BL21/XDH1 | BL21(DE3) pET28a-XDH1 | This work |
Y. lipolytica strains | ||
HCY107 (CGMCC7326, or ery929) | Suc-, Lac-, Mal- | Cheng et al., 2018 |
HCY109 | CGMCC7326 derivative, ery919â–³ku70 | This work |
HCY109-2 | HCY109 derivative, â–³ku70â–³ura3 | This work |
HCY110 | HCY109 derivative, â–³ku70â–³AraDH1 | This work |
HCY111 | HCY109 derivative, â–³ku70â–³AraDH2 | This work |
HCY112 | HCY109 derivative, â–³ku70â–³mdh1 | This work |
HCY113 | HCY109 derivative, â–³ku70â–³ura3â–³mdh2 | This work |
HCY114 | HCY109 derivative, â–³ku70â–³xdh1 | This work |
HCY115 | HCY113 derivative, â–³ku70â–³ura3â–³mdh2â–³eyd1 | This work |
HCY117 | HCY116 derivative, â–³ku70â–³mdh2â–³eyd1::Sc.rsp5 | This work |
HCY118 | HCY117derivative, â–³ku70â–³mdh2â–³eyd1::Sc.rsp5::zwf1::gnd1 | This work |
Plasmids | ||
pUC19 | LacZ, AmpR | |
pET28a | LacI, T7 promoter, KanR | Novagen |
pET28a-ylAraDH1 | Containing ylAraDH1 gene g1595.t1 (YALI0F02211g) | This work |
pET28a-ylAraDH2 | Containing ylAraDH2 gene g3858.t1 (YALI0E05643g) | This work |
pET28a-ylMDH1 | Containing ylMDH1 gene g5130.t1 (YALI0B16192g) | This work |
pET28a-ylMDH2 | Containing ylMDH2 gene g2069.t1 (YALI0D18964g) | This work |
pET28a-ylXDH1 | Containing ylXDH1 gene g4121.t1 (YALI0E12463g) | This work |
The above plasmids (except pUC19 and pET28a) were used to transform BL21(DE3) and in the production of ylAraDH1, ylAraDH2, ylMDH1, ylMDH2 and ylXDH1 recombinant enzymes | ||
pHB4-621XDH | pHB4-Cre derivative, hp4d-621XDH-TT-hp4d-Cre | This work |
pWSV-Ku70-loxP-hph-loxP | up Ku70-loxP-pLeu2-hph-TT-loxP-dw Ku70 | This work |
pWSV-AraDH1-loxP-hph-loxP | up AraDH1-loxP-pLeu2-hph-TT-loxP-dw AraDH1 | This work |
pWSV-AraDH2-loxP-hph-loxP | up AraDH2-loxP-pLeu2-hph-TT-loxP-dw AraDH2 | This work |
pWSV-MDH1-loxP-hph-loxP | up MDH1-loxP-pLeu2-hph-TT-loxP-dw MDH1 | This work |
pWSV-MDH2-loxP-hph-loxP | up MDH2-loxP-pLeu2-hph-TT-loxP-dw MDH2 | This work |
pWSV-XDH1-loxP-hph-loxP | up XDH1-loxP-pLeu2-hph-TT-loxP-dwXDH1 | This work |
pWSV-EYD1-loxP-hph-loxP | up EYD1-loxP-pLeu2-hph-TT-loxP-dw EYD1 | This work |
pINA1313-RSP5 | up zeta-ura3-hp4d-RSP5-TT–dw zeta | This work |
The above plasmids are used to delete ylAraDH1, ylAraDH2, ylMDH1, ylMDH2, ylXDH1, PGI genes and overexpress Sc.rsp5 gene for erythritol synthesis | ||
Primers | Sequences (5′ → 3′) | Restriction sites |
|---|---|---|
Primers used for amplification of ylAraDH1, ylAraDH2, ylMDH1, ylMDH2 and ylXDH1 genes and cloned to pET28a by Gibson assembly | ||
P28a-ylAraDH1-F | CTGGTGCCGCGCGGCAGCCATATGTCTCTCTTTTCACTCGCCAAGAAAAC | NdeI |
P28a-ylAraDH1-R | GTGGTGGTGGTGGTGGTGCTCGAGTTACCAGATGGTGTAACCTCCATCGAC | XhoI |
P28a-ylAraDH2-F | CTGGTGCCGCGCGGCAGCCATATGTCCAACTCCGCCAAAGCCGCTGTC | NdeI |
P28a-ylAraDH2-R | GTGGTGGTGGTGGTGGTGCTCGAGTTAAATAATAGTGTAACCACCATCAATG | XhoI |
P28a-ylMDH1-F | CTGGTGCCGCGCGGCAGCCATATGCCTGCACCAGCAACCTACGCTAC | NdeI |
P28a-ylMDH1-R | GTGGTGGTGGTGGTGGTGCTCGAGCTAAGGACAACAGTAGCCGCCATCAAC | XhoI |
P28a-ylMDH2-F | CTGGTGCCGCGCGGCAGCCATATGATGTCTGGACCTTCCACCCTCGCCAC | NdeI |
P28a-ylMDH2-R | GTGGTGGTGGTGGTGGTGCTCGAGCTAAGGAGCGCAGTAGCCACCATCGAC | XhoI |
P28a-ylXDH1-F | CTGGTGCCGCGCGGCAGCCATATGTCTTCTAACCCGTCATTTGTTCTTC | NdeI |
P28a-ylXDH1-R | GTGGTGGTGGTGGTGGTGCTCGAGCTACTCCTCCTCGGGACCGTCAATGATG | XhoI |
The below primers are used for verification of Ku70, ylAraDH1, ylAraDH2, ylMDH1, ylMDH2, ylXDH1, ylEYD1, pgi genes knockout in Y. lipolytica | ||
PKu70- knockout -F | ATGGAATGGATTTCACATCTGGAGAACG | |
PKu70- knockout -R | TCACTTCCCATAGTACTTTTTGACCAC | |
PAraDH1- knockout -F | ATGTCTCTCTTTTCACTCGCCAAGAAAAC | |
PAraDH1- knockout -R | CTCTTGTGGTGCTCTCGAATGGCCTTGA | |
PAraDH2- knockout -F | ATGTCCAACTCCGCCAAAGCCGCTGTC | |
PAraDH2- knockout -R | TTAAATAATAGTGTAACCACCATCAATG | |
PMDH1- knockout -F | ATGCCTGCACCAGCAACCTACGCTAC | |
PMDH1- knockout -R | CACTGTATTACATCGAGCGAATCCA | |
PMDH2- knockout -F | ATGTCTGGACCTTCCACCCTCGCCAC | |
PMDH2- knockout -R | CTAAGGAGCGCAGTAGCCACCATCGAC | |
PXDH1- knockout -F | ATG TCTTCTAACCCGTCATTTGTTCTTC | |
PXDH1- knockout -R | CTACTCCTCCTCGGGACCGTCAATGATG | |
PEYD1- knockout -F | CGAGACTCCCTCTGAGGAGTTCCTG | |
PEYD1- knockout -R | TTACCAGACGTGGTGGCCACCGCAGAC | |
The below primers are used to construct URA3 gene disruption cassette and verify the corrected URA3 gene mutant | ||
PURA3-upstream-F | CTGAAACGTTATCTTATATGAACTCC | |
PURA3-upstream-R | TTTGGTGGTGAAGAGGAGACTGAAATAAATTTAG | |
PURA3-downstream-F | CTAAATTTATTTCAGTCTCCTCTTCACCACCAAATATGTAATTTAACTGTGTATATAG | |
PURA3-downstream-R | CAGGCCAGTCCCGCCTCTCCTTTC | |
PURA3-verify-F | GTGTGCATGATCAAGACCCATATC | |
PURA3-verify-R | AGGTCGGTTCTGGGCAATGAAGCC | |
Primers used to detect the mRNA levels of ylAraDH1, ylAraDH2, ylMDH1, ylMDH2, ylXDH1 knockout mutants and mRNA levels of Sc.rsp5, ylTKL1 at 30–35 °C | ||
PAraDH1-qPCR-F | GGTATCGCAGCTGCCAAGCAGCTTC | |
PAraDH1-qPCR-R | GATTCTGTTTCTGTTTCAGAAAC | |
PAraDH2-qPCR-F | CGAGCCAACGCCGGATCCAAGGAAG | |
PAraDH2-qPCR -R | CTCAATGGCATCCAGACCCAGGTC | |
PMDH1-qPCR -F | GTATCCAAGAACATCATGGAGCG | |
PMDH1-qPCR-R | CATTTGTAAGCCTTAGATCGGACTCC | |
PMDH2-qPCR -F | TTCCCCACCAACATCATGGACCG | |
PMDH2-qPCR-R | CTTGTAGGCCTTGGCCTTGACGCC | |
PXDH1-qPCR-F | TGCGGCTCGGATGTCCACTACTATC | |
PXDH1-qPCR-R | GTTGTATCGTCCCTCCTTGTACTC | |
Prsp5-qPCR-F | TCCGCAGCGAAGAAAACGTTAAATC | |
Prsp5-qPCR -R | GTCTACCACTCGATGTAGCAGTATC | |
Ptkl-qPCR-F | AAGACTCCCGGCCACCCCGAGGCTG | |
Ptkl-qPCR-R | CTCCGAAGATGCAGTAAGTGTAGTT | |
Pβ-actin-up | ACTCTGGTGATGGTGTCACCCACG | |
Pβ-actin-down | TCGGACGATTTCTCGCTCGGCGGAG | |