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TRANSCRIPT
Supplementary material
Table S1 Primers used in this study
Primer Sequence
CRISPRi plasmids construction
Ori-Fe CACACATTAGGGGGTACCCCTCGAGTTCATGTGCAGCTC
Ori-Ra CGGAATCCATATGGAATTCCCGTAAGGAGAAAATACCGCATC
lacA -Fa′ GGAATTCCATATGGATTCCGTGATGTCAAAGCTTGAAAAAACG
lacA -Rb′ TGTATGCTATACGAACGGTATACGGGATCAAATCCGGTG
Cm-Fb TACCGTTCGTATAGCATACATTATACGAAGTTATGCCATAGTGACTGGCGAT
Cm-Rc TACCGTTCGTATAATGTATGCTATACGAAGTTATTTATTGGTATGACTGGTTTTAAGCG
PxylA-Fc CATACATTATACGAACGGTATTCAGAACGCTCGGTTGCC
PxylA-Rd ATGGAGCTCGGATCCCATTTCCCCCTTTGATTTTTAGATATCACTA
′lacA-Fd AAATGGGATCCGAGCTCCATTACCTGCAGCGTCATCACGAAAGAACAAGAC
′lacA-Re GGGGTACCCCCTAATGTGTGTTTACGACAATTCTC
dcas9-FEco311 ATCGGTCTCGGATCCATGGATAAGAAATACTCAATAGGCTTA
dCas9-RPStI AAGCGCTGCAGGCGGAATATATCCCTAGGTATAAACG
psga-L1-FBamH1 CGCGGATCCGCGATGTTTGCAAAACGATTCAAAAC
psga-L1-RH TGCTATACGAACGGTAGAATCTCATGCCATGGCATGTTCTGCGTGACATCCCATC
psga-L2-FH GATGGGATGTCACGCAGAACATGCCATGGCATGAGATTCTACCGTTCGTATAGCA
psga-L2-RBcuI
GAGC
GACTAGTACATTTATTGTACAACACGAGCCCATTTTTGTCAAATAAAATTTAAATTATATCAACGTTA
ATAAGCCTGCAGGTCGACG
psga-R-FEcoR1 ATCGCGGAATTCCGTCGACATGGATGAGCGATGA
psga-R-RHindIII CACGCAAGCTTGGGTCAATGGGGAAGAGAACCGC
sgRNA construction
sg-F GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
sg-GFP-R TTTCTAGCTCTAAAACGTTCCTTGGCCAACACTTGTACATTTATTGTACAACACGAGCC
sg-zwf1-R TTTCTAGCTCTAAAACTGGTCTAATGAGGATCTTCGACATTTATTGTACAACACGAGCC
sg-zwf2-R TTTCTAGCTCTAAAACGTTCCTGCTTATACAGATGAACATTTATTGTACAACACGAGCC
sg-zwf3-R TTTCTAGCTCTAAAACAACTACGAATCAGGCTCAATACATTTATTGTACAACACGAGCC
sg-pfkA1-R TTTCTAGCTCTAAAACCGGGAATGAACGCAGCAGTTACATTTATTGTACAACACGAGCC
sg-pfkA2-R TTTCTAGCTCTAAAACGGCACTGATTTTACAATCGGACATTTATTGTACAACACGAGCC
sg-pfkA3-R TTTCTAGCTCTAAAACAAGGTGTAGGCAGCGGTGTTACATTTATTGTACAACACGAGCC
sg-glmM1-R TTTCTAGCTCTAAAACATAGTGAGCTTACACCTGAGACATTTATTGTACAACACGAGCC
sg-glmM2-R TTTCTAGCTCTAAAACGATGGACGAACCTGAGGATAACATTTATTGTACAACACGAGCC
sg-glmM3-R TTTCTAGCTCTAAAACTGATTACAACACGACAGGGGACATTTATTGTACAACACGAGCC
Golden Gate Assembly
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Supplementary material
psga-liner-F-D GCCGCGGGTCTCACCGATCGACATGGATGAGCGATGATGATATCC
psga-liner-R-A GCCGCGGGTCTCAACCTGTCGACGATTTACCGTTCGTATAATGTATGC
sg-F-A GCCGCGGGTCTCAAGGTCGTCGACCTGCAGGCTTATTAAC
sg-R-B GCCGCGGGTCTCAGTGGCGGAATTCAAAAAAAGCACCGACTCG
sg-F-B GCCGCGGGTCTCACCACCGTCGACCTGCAGGCTTATTAAC
sg-R-C GCCGCGGGTCTCATACCGGAATTCAAAAAAAGCACCGACTCG
sg-F-C GCCGCGGGTCTCAGGTACGTCGACCTGCAGGCTTATTAAC
sg-R-D GCCGCGGGTCTCATCGGCGGAATTCAAAAAAAGCACCGACTCG
RT-qPCRhbsU-F(qPCR) ATCGGTTTTGGTAACTTCGAGG
hbsU-R(qPCR) GCAGGTACTTTGCTTGCTGGA
araE-F(qPCR) GCTTGGCGGCTTATTGTATGGaraE-R(qPCR) TATCCCGACGCCCACAACT
xylAB-F(qPCR) AGAGCCGACCACCCATCAA
xylAB-R(qPCR) TCAACAGAGCCAAGCAGACC
tkt-F(qPCR) TTCACTCCGTGCGATGCCTA
tkt-R(qPCR) TTGGGTGGTCAGTGCTTTGC
zwf-F(qPCR) CAATGGAGCCGCCTATCAAA
zwf-R(qPCR) CAGGAACCGGTACACCGTCA
pfkA-F(qPCR) ACGCGGCAAGAAGCACAGTA
pfkA-R(qPCR) GGTCAGCAGCACTCGGAGAA
glmM-F(qPCR) TCATCCTTGGCGACACACCT
glmM-R(qPCR) CCGAGATCCGCGTTTTTCTC
ywlF-F(qPCR) CAGGTCCGATCACCCGTT
ywlF-R(qPCR) GGCGAAGTTGACAGAGGCA
rpe-F(qPCR) TCTGCCGCTTGATGTCCATT
rpe-R(qPCR) ACTCCGGCCTTCACTCCTTG
ywjH-F(qPCR) CGGTGAAAATCCCAATGACG
ywjH-R(qPCR) ACATATGTTGCCCCCGCTCT
pgi-F(qPCR) GCGCGGATTTATGCAACAAC
pgi-R(qPCR) TGACAGCAATCGGCAAGAGA
sgRNA array sequencingamyE-F1 GCGAGTGCTGAAACGGCGAACA
amyE-R1 AAAGCCAGGCTGATTCTGACCG
amyE-R2 AAGAGGCGTACTGCCTGAACG
Italic letters represent the restriction enzyme sites, and underlined letters represent homologous sequences for fusion PCR, inverse PCR or One Step Cloning.
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Supplementary material
Table S2 Plasmids used in this study
Plasmid Characteristics Source or referencepTargetF pMB1 Specr (Jiang et al., 2015)
pdCas9-bacteria p15A Cmr dCas9 (Qi et al., 2013)
p7z6 ColE1 Ampr, lox71-Zeo-lox66 (Yan et al., 2008)
pTSCColE1 Ampr, RepF Emr Pspac-cre, E. coli-B. subtilis shuttle
vector(Yan et al., 2008)
pHT01 ColE1 Ampr, Cmr, E. coli-B. subtilis shuttle vector (Phan et al., 2012)
pAX01 ColE1 Ampr , Emr xylR-PxylA (Phan et al., 2006)
pLCxpMB1 Specr, Cmr xylR-PxylA integrant expression at lacA
locus of B.subtilisThis study
pLCx-dCas9 pLCx derived xylR-PxylA-dCas9 This study
psgapMB1 Specr, Zeor Pveg-sgRNA integrant expression at
amyE locus of B.subtilisThis study
psga-GFP psga derivate, contain sgRNA targeting to GFP This study
psga-zwf1 psga derivate, contain sgRNA targeting to front of zwf This study
psga-pfkA1 psga derivate, contain sgRNA targeting to front of pfkA This study
psga-glmM1 psga derivate, contain sgRNA targeting to front of glmM This study
psga-zwf1-pfkA1psga derivate, contain sgRNAs targeting to front of zwf
and front of pfkAThis study
psga-zwf1-pfkA1-glmM1psga derivate, contain sgRNAs targeting to front of zwf,
front of pfkA, and front of glmM This study
psga-zwf2 psga derivate, contain sgRNA targeting to middle of zwf This study
psga-zwf3 psga derivate, contain sgRNA targeting to back of zwf This study
psga-pfkA2 psga derivate, contain sgRNA targeting to middle of pfkA This study
psga-pfkA3 psga derivate, contain sgRNA targeting to back of pfkA This study
psga-glmM2psga derivate, contain sgRNA targeting to middle of
glmMThis study
psga-glmM3 psga derivate, contain sgRNA targeting to back of glmM This study
psga-zwf1-pfkA2-glmM1psga derivate, contain sgRNAs targeting to front of zwf,
middle of pfkA, and front of glmM This study
psga-zwf1-pfkA3-glmM1psga derivate, contain sgRNAs targeting to front of zwf,
back of pfkA, and front of glmM This study
psga-zwf2-pfkA1-glmM1psga derivate, contain sgRNAs targeting to middle of zwf,
front of pfkA, and front of glmM This study
psga-zwf2-pfkA2-glmM1psga derivate, contain sgRNAs targeting to middle of zwf,
middle of pfkA, and front of glmM This study
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Supplementary material
psga-zwf2-pfkA3-glmM1psga derivate, contain sgRNAs targeting to middle of zwf,
back of pfkA, and front of glmM This study
psga-zwf3-pfkA1-glmM1psga derivate, contain sgRNAs targeting to back of zwf,
front of pfkA, and front of glmM This study
psga-zwf3-pfkA2-glmM1psga derivate, contain sgRNAs targeting to back of zwf,
middle of pfkA, and front of glmMThis study
psga-zwf3-pfkA3-glmM1psga derivate, contain sgRNAs targeting to back of zwf,
back of pfkA, and front of glmMThis study
psga-zwf1-pfkA1-glmM2psga derivate, contain sgRNAs targeting to front of zwf,
front of pfkA, and middle of glmM This study
psga-zwf1-pfkA2-glmM2psga derivate, contain sgRNAs targeting to front of zwf,
middle of pfkA, and middle of glmM This study
psga-zwf1-pfkA3-glmM2psga derivate, contain sgRNAs targeting to front of zwf,
back of pfkA, and middle of glmM This study
psga-zwf2-pfkA1-glmM2psga derivate, contain sgRNAs targeting to middle of zwf,
front of pfkA, and middle of glmM This study
psga-zwf2-pfkA2-glmM2psga derivate, contain sgRNAs targeting to middle of zwf,
middle of pfkA, and middle of glmM This study
psga-zwf2-pfkA3-glmM2psga derivate, contain sgRNAs targeting to middle of zwf,
back of pfkA, and middle of glmM This study
psga-zwf3-pfkA1-glmM2psga derivate, contain sgRNAs targeting to back of zwf,
front of pfkA, and middle of glmM This study
psga-zwf3-pfkA2-glmM2psga derivate, contain sgRNAs targeting to back of zwf,
middle of pfkA, and middle of glmM This study
psga-zwf3-pfkA3-glmM2psga derivate, contain sgRNAs targeting to back of zwf,
back of pfkA, and middle of glmM This study
psga-zwf1-pfkA1-glmM3psga derivate, contain sgRNAs targeting to front of zwf,
front of pfkA, and back of glmM This study
psga-zwf1-pfkA2-glmM3psga derivate, contain sgRNAs targeting to front of zwf,
middle of pfkA, and back of glmM This study
psga-zwf1-pfkA3-glmM3psga derivate, contain sgRNAs targeting to front of zwf,
back of pfkA, and back of glmM This study
psga-zwf2-pfkA1-glmM3psga derivate, contain sgRNAs targeting to middle of zwf,
front of pfkA, and back of glmM This study
psga-zwf2-pfkA2-
glmM3
psga derivate, contain sgRNAs targeting to middle of zwf,
middle of pfkA, and back of glmM This study
psga-zwf2-pfkA3-glmM3 psga derivate, contain sgRNAs targeting to middle of zwf, This study
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Supplementary material
back of pfkA, and back of glmM
psga-zwf3-pfkA1-glmM3psga derivate, contain sgRNAs targeting to back of zwf,
front of pfkA, and back of glmM This study
psga-zwf3-pfkA2-glmM3psga derivate, contain sgRNAs targeting to back of zwf,
middle of pfkA, and back of glmM This study
psga-zwf3-pfkA3-glmM3psga derivate, contain sgRNAs targeting to back of zwf,
back of pfkA, and back of glmM This study
Table S3 Effects of nitrogen supplement on GlcNAc synthesis
ExperimentNitrogen source concentration (g/L)a Titer (g/L)b
CGlcNAc, maxc
CN1 CN2 CN3 CN4 DCW GlcNAc1 6 12 6 0 10 28 33
2 20 20 12 0 12 75 91
3 20 20 0 10 13 103 124
4 25 25 0 10 14 104 140
5 20 20 0 15 13 102 162a CN1, CN2, CN3, and CN4 represent the concentration of tryptone, yeast extract, (NH4)2SO4, and urea, respectively, in the fermentation medium.b The titers of DCW and GlcNAc in fed-batch culture of BNX122.c The theoretical maximum titer of GlcNAc in fed-batch culture calculated roughly using the following equation:
CN1×ωN1+ CN2×ωN2+ CN3×ωN3+ CN4×ωN4=DCW×ωNB+ CGlcNAc, max×ωNG (1)where, ωN1, ωN2, ωN3, ωN4, ωNB, and ωNG represent nitrogen content (w/w) in tryptone, yeast extract, (NH4)2SO4, urea, biomass, and GlcNAc, and are 0.12, 0.1, 0.21, 0.46, 0.12, and 0.06, respectively.
sgRNAs targeting sites on non-template strand of zwf, pfkA, and glmM
zwf:
GTGAAAACAAACCAACAACCAAAAGCAGTAATTGTCATATTCGGTGCAACTGGAGATTTAGCAAAACGAAAATTGTATCCGTCTATTCACCGTTTAT
ATCAAAACGGACAAATCGGAGAAGAGTTTGCAGTGGTAGGAGTTGGAAGAAGACCTTGGTCTAATGAGGATCTTCGCCAAACTGTTAAAACATCC
ATTTCCTCATCTGCAGATAAGCATATAGATGATTTCACGTCTCATTTTTACTATCACCCGTTTGACGTGACAAACCCTGGTTCTTATCAAGAGCTAAAC
GTATTGCTTAACCAGCTGGAAGATACATATCAAATTCCTAACAACAGAATGTTCTACTTGGCAATGGCTCCTGAATTCTTCGGAACGATTGCAAAAAC
ATTAAAATCAGAGGGTGTAACAGCTACAACCGGCTGGTCCCGCCTTGTCATCGAAAAACCGTTCGGCCATGATCTGCCAAGCGCACAGGCATTGAA
TAAAGAAATCCGCGAAGCATTTACGGAAGATCAAATTTACAGAATCGACCATTATCTAGGCAAACAAATGGTTCAGAACATTGAAGTGATTCGATTT
GCCAATGCGATTTTCGAACCGCTTTGGACAAACCGCTACATTTCAAACATTCAAATCACATCTAGCGAATCACTAGGCGTTGAAGACCGCGCAAGAT
ATTACGAAAAATCAGGCGCCCTTCGCGACATGGTGCAAAACCATATTATGCAGATGGTTGCCCTTCTTGCAATGGAGCCGCCTATCAAATTGAACAC
AGAAGAAATCCGCAGCGAGAAAGTGAAGGTGCTGAGAGCACTGCGTCCTATTGCAAAAGACGAAGTGGATGAATACTTTGTGCGCGGACAATATC
ATGCTGGTGAAATTGACGGTGTACCGGTTCCTGCTTATACAGATGAAGATAATGTCGCTCCTGACTCCAATACAGAAACCTTTGTTGCCGGCAAGCT
CTTGATCGACAACTTCAGATGGGCTGGTGTTCCATTCTACATCAGAACCGGAAAACGAATGAAAGAAAAGTCCACAAAAATTGTCGTTCAATTTAAG
GACATTCCGATGAACCTGTACTACGGTAATGAAAACAACATGAATCCGAACTTGCTTGTCATTCATATTCAGCCTGACGAAGGCATTACGCTTTACTT
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Supplementary material
AAATGCTAAAAAGCTTGGCGGAGCAGCACACGCACAGCCAATCAAACTCGATTATTGCAGCAATTGCAATGACGAGTTGAACACCCCTGAAGCATA
TGAAAAACTAATTCACGACTGTCTTCTTGGCGATGCAACAAACTTTGCACACTGGGATGAAGTTGCCCTTTCTTGGAGCTTTGTCGACTCTATTTCTG
AAACATGGGCAGCAAACAAAACCTTATCTCCTAACTACGAATCAGGCTCAATGGGACCGAAAGAATCTGATGATCTTTTGGTGAAAGACGGCTTAC
ACTGGTGGAACATATAA
pfkA:
ATGAAACGAATAGGGGTATTAACGAGCGGCGGGGATTCCCCGGGAATGAACGCAGCAGTTCGCGCAGTAGTCAGAAAAGCGATCTATCATGACGTTGAAGTTTACGGTATTTACAACGGATACGCGGGATTGATCAGCGGAAAGATTGAAAAGCTTGAACTCGGATCAGTAGGCGATATTATACATCGTGGAGGGACTAAGCTTTATACGGCGAGATGTCCTGAATTCAAAACAGTTGAAGGCCGTGAAAAAGGGATAGCAAACTTGAAGAAGCTTGGTATTGAAGGCCTTGTTGTTATCGGTGGAGACGGTTCCTATATGGGTGCGAAAAAATTAACGGAACACGGGTTTCCATGTGTAGGTGTACCGGGTACAATTGATAATGACATTCCGGGCACTGATTTTACAATCGGTTTCGATACAGCTTTAAATACAGTAATTGACGCAATTGATAAGATTCGCGATACAGCGACTTCTCATGAACGTACATATGTAATCGAAGTAATGGGCCGTCATGCCGGCGATATCGCATTGTGGGCCGGTCTTGCAGGGGGCGCAGAATCGATCTTAATCCCTGAGGCAGACTATGACATGCACGAAATCATTGCCCGCTTAAAACGCGGCCACGAACGCGGCAAGAAGCACAGTATTATTATTGTTGCCGAAGGTGTAGGCAGCGGTGTTGAATTCGGGAAACGCATTGAAGAAGAAACAAATCTTGAAACTAGGGTATCTGTATTGGGCCATATCCAGCGCGGAGGTTCTCCGAGTGCTGCTGACCGTGTGTTGGCAAGCCGTCTCGGCGCATATGCAGTTGAACTGCTGCTTGAAGGAAAAGGCGGACGCTGTGTAGGTATACAAAACAATAAGCTTGTAGACCATGATATTATAGAAATACTTGAGACAAAACACACAGTTGAGCAAAACATGTATCAGCTTTCAAAAGAACTGTCTATCTAA
glmM:ATGGGCAAGTATTTTGGAACAGACGGTGTAAGAGGTGTCGCCAATAGTGAGCTTACACCTGAGCTGGCCTTTAAAGTCGGACGTTTCGGCGGTTATGTGCTGACAAAAGACAAACAACGTCCAAAAGTGCTGATAGGCCGCGATACACGCATCTCCGGCCATATGCTGGAGGGAGCCCTTGTCGCCGGACTTTTATCCATTGGCGCAGAAGTCATGCGCCTGGGTGTCATTTCTACACCAGGTGTATCTTATTTGACAAAAGCGATGGATGCAGAGGCGGGCGTCATGATTTCCGCTTCTCATAACCCAGTGCAGGATAACGGCATCAAATTCTTTGGGGGAGATGGATTTAAGCTTTCTGATGAACAGGAGGCTGAAATTGAGCGCCTGATGGACGAACCTGAGGATAAGCTGCCAAGACCTGTCGGAGCAGACCTTGGACTTGTAAACGATTATTTTGAAGGCGGACAAAAATATCTGCAATTCTTAAAACAGACAGCTGATGAAGATTTCACAGGCATTCATGTGGCATTGGACTGTGCCAATGGCGCAACGTCATCCTTGGCGACACACCTGTTTGCTGATTTAGATGCAGATGTTTCTACAATGGGGACTTCCCCGAACGGATTAAACATTAATGACGGCGTCGGTTCGACTCATCCCGAAGCGCTCAGCGCGTTTGTCAAAGAGAAAAACGCGGATCTCGGTCTTGCGTTCGACGGTGACGGCGACCGCCTGATTGCTGTCGATGAAAAAGGAAATATTGTAGACGGCGACCAAATCATGTACATATGCTCAAAACATCTGAAATCAGAGGGCCGTTTAAAGGATGATACAGTGGTTTCAACCGTGATGAGCAACCTCGGCTTCTATAAGGCGCTCGAAAAAGAAGGCATCAAAAGCGTGCAGACAGCTGTCGGCGACCGCTACGTAGTAGAAGCAATGAAAAAAGACGGCTACAACGTCGGCGGAGAGCAGTCAGGACATCTTATTTTCCTTGATTACAACACGACAGGGGACGGATTATTGTCTGCTATTATGCTGATGAACACTTTAAAAGCAACAGGCAAGCCGCTGTCAGAGCTTGCAGCTGAAATGCAGAAGTTCCCGCAGCTGTTAGTCAATGTGAGAGTGACTGATAAATATAAAGTTGAAGAAAATGAAAAAGTAAAAGCAGTTATTTCTGAAGTTGAAAAAGAAATGAACGGCGACGGCCGGATTTTGGTGCGCCCTTCAGGAACTGAACCGCTCGTCCGTGTCATGGCTGAAGCGAAGACGAAAGAGCTGTGCGATGAGTATGTCAATCGCATTGTTGAAGTCGTCCGGTCAGAAATGGGATTAGAGTAA
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Supplementary material
Fig. S1. Relative mRNA expression of genes zwf, pfkA, and glmM in strain BNX111, BNX222, BNX333, and BNX122. Strain BNX000 was used as a control. All data were the average of three independent studies with standard deviations. The ** indicate p<0.01 relative to the control strain BNX000.
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Supplementary material
Fig. S2. Effect of xylose addition time on xylose consumption, glucose consumption, cell growth, and GlcNAc synthesis. (A) Xylose consumption. (B) Glucose consumption. (C) Cell growth. (D) GlcNAc synthesis. All data were the average of three independent studies with standard deviations.
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Supplementary material
Fig. S3. Stability verification of the sgRNA array in strain BNX122 after 60 generations by Sanger sequencing.
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