ars.els-cdn.com · Web viewItalic letters represent the restriction enzyme sites, and underlined letters represent homologous sequences for fusion PCR, inverse PCR or One Step Cloning

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

1


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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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


middle of pfkA, and front of glmM This study


back of pfkA, and front of glmM This study

psga-zwf2-pfkA1-glmM1psga derivate, contain sgRNAs targeting to middle of zwf,



middle of pfkA, and front of glmM This study

3



back of pfkA, and front of glmM This study

psga-zwf3-pfkA1-glmM1psga derivate, contain sgRNAs targeting to back of zwf,



middle of pfkA, and front of glmMThis study


back of pfkA, and front of glmMThis study


front of pfkA, and middle of glmM This study


middle of pfkA, and middle of glmM This study


back of pfkA, and middle of glmM This study














front of pfkA, and back of glmM This study


middle of pfkA, and back of glmM This study


back of pfkA, and back of glmM This study



psga-zwf2-pfkA2-

glmM3

psga derivate, contain sgRNAs targeting to middle of zwf,


psga-zwf2-pfkA3-glmM3 psga derivate, contain sgRNAs targeting to middle of zwf, This study

4


back of pfkA, and back of glmM






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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AAATGCTAAAAAGCTTGGCGGAGCAGCACACGCACAGCCAATCAAACTCGATTATTGCAGCAATTGCAATGACGAGTTGAACACCCCTGAAGCATA

TGAAAAACTAATTCACGACTGTCTTCTTGGCGATGCAACAAACTTTGCACACTGGGATGAAGTTGCCCTTTCTTGGAGCTTTGTCGACTCTATTTCTG

AAACATGGGCAGCAAACAAAACCTTATCTCCTAACTACGAATCAGGCTCAATGGGACCGAAAGAATCTGATGATCTTTTGGTGAAAGACGGCTTAC

ACTGGTGGAACATATAA

pfkA:

ATGAAACGAATAGGGGTATTAACGAGCGGCGGGGATTCCCCGGGAATGAACGCAGCAGTTCGCGCAGTAGTCAGAAAAGCGATCTATCATGACGTTGAAGTTTACGGTATTTACAACGGATACGCGGGATTGATCAGCGGAAAGATTGAAAAGCTTGAACTCGGATCAGTAGGCGATATTATACATCGTGGAGGGACTAAGCTTTATACGGCGAGATGTCCTGAATTCAAAACAGTTGAAGGCCGTGAAAAAGGGATAGCAAACTTGAAGAAGCTTGGTATTGAAGGCCTTGTTGTTATCGGTGGAGACGGTTCCTATATGGGTGCGAAAAAATTAACGGAACACGGGTTTCCATGTGTAGGTGTACCGGGTACAATTGATAATGACATTCCGGGCACTGATTTTACAATCGGTTTCGATACAGCTTTAAATACAGTAATTGACGCAATTGATAAGATTCGCGATACAGCGACTTCTCATGAACGTACATATGTAATCGAAGTAATGGGCCGTCATGCCGGCGATATCGCATTGTGGGCCGGTCTTGCAGGGGGCGCAGAATCGATCTTAATCCCTGAGGCAGACTATGACATGCACGAAATCATTGCCCGCTTAAAACGCGGCCACGAACGCGGCAAGAAGCACAGTATTATTATTGTTGCCGAAGGTGTAGGCAGCGGTGTTGAATTCGGGAAACGCATTGAAGAAGAAACAAATCTTGAAACTAGGGTATCTGTATTGGGCCATATCCAGCGCGGAGGTTCTCCGAGTGCTGCTGACCGTGTGTTGGCAAGCCGTCTCGGCGCATATGCAGTTGAACTGCTGCTTGAAGGAAAAGGCGGACGCTGTGTAGGTATACAAAACAATAAGCTTGTAGACCATGATATTATAGAAATACTTGAGACAAAACACACAGTTGAGCAAAACATGTATCAGCTTTCAAAAGAACTGTCTATCTAA

glmM：ATGGGCAAGTATTTTGGAACAGACGGTGTAAGAGGTGTCGCCAATAGTGAGCTTACACCTGAGCTGGCCTTTAAAGTCGGACGTTTCGGCGGTTATGTGCTGACAAAAGACAAACAACGTCCAAAAGTGCTGATAGGCCGCGATACACGCATCTCCGGCCATATGCTGGAGGGAGCCCTTGTCGCCGGACTTTTATCCATTGGCGCAGAAGTCATGCGCCTGGGTGTCATTTCTACACCAGGTGTATCTTATTTGACAAAAGCGATGGATGCAGAGGCGGGCGTCATGATTTCCGCTTCTCATAACCCAGTGCAGGATAACGGCATCAAATTCTTTGGGGGAGATGGATTTAAGCTTTCTGATGAACAGGAGGCTGAAATTGAGCGCCTGATGGACGAACCTGAGGATAAGCTGCCAAGACCTGTCGGAGCAGACCTTGGACTTGTAAACGATTATTTTGAAGGCGGACAAAAATATCTGCAATTCTTAAAACAGACAGCTGATGAAGATTTCACAGGCATTCATGTGGCATTGGACTGTGCCAATGGCGCAACGTCATCCTTGGCGACACACCTGTTTGCTGATTTAGATGCAGATGTTTCTACAATGGGGACTTCCCCGAACGGATTAAACATTAATGACGGCGTCGGTTCGACTCATCCCGAAGCGCTCAGCGCGTTTGTCAAAGAGAAAAACGCGGATCTCGGTCTTGCGTTCGACGGTGACGGCGACCGCCTGATTGCTGTCGATGAAAAAGGAAATATTGTAGACGGCGACCAAATCATGTACATATGCTCAAAACATCTGAAATCAGAGGGCCGTTTAAAGGATGATACAGTGGTTTCAACCGTGATGAGCAACCTCGGCTTCTATAAGGCGCTCGAAAAAGAAGGCATCAAAAGCGTGCAGACAGCTGTCGGCGACCGCTACGTAGTAGAAGCAATGAAAAAAGACGGCTACAACGTCGGCGGAGAGCAGTCAGGACATCTTATTTTCCTTGATTACAACACGACAGGGGACGGATTATTGTCTGCTATTATGCTGATGAACACTTTAAAAGCAACAGGCAAGCCGCTGTCAGAGCTTGCAGCTGAAATGCAGAAGTTCCCGCAGCTGTTAGTCAATGTGAGAGTGACTGATAAATATAAAGTTGAAGAAAATGAAAAAGTAAAAGCAGTTATTTCTGAAGTTGAAAAAGAAATGAACGGCGACGGCCGGATTTTGGTGCGCCCTTCAGGAACTGAACCGCTCGTCCGTGTCATGGCTGAAGCGAAGACGAAAGAGCTGTGCGATGAGTATGTCAATCGCATTGTTGAAGTCGTCCGGTCAGAAATGGGATTAGAGTAA

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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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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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Fig. S3. Stability verification of the sgRNA array in strain BNX122 after 60 generations by Sanger sequencing.

References

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CRISPR-Cas9 system. Appl. Environ. Microbiol. 81, 2506–2514.

Phan, T.T.P., Nguyen, H.D., Schumann, W., 2012. Development of a strong intracellular expression system for Bacillus

subtilis by optimizing promoter elements. J. Biotechnol. 157, 167–172.

Phan, T.T.P., Nguyen, H.D., Schumann, W., 2006. Novel plasmid-based expression vectors for intra- and extracellular

production of recombinant proteins in Bacillus subtilis. Protein Expr. Purif. 46, 189–195.

Qi, L.S., Larson, M.H., Gilbert, L. a., Doudna, J. a., Weissman, J.S., Arkin, A.P., Lim, W. a., 2013. Repurposing CRISPR as an

RNA-γuided platform for sequence-specific control of gene expression. Cell 152, 1173–1183.

Yan, X., Yu, H.J., Hong, Q., Li, S.P., 2008. Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl.

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ars.els-cdn.com · Web viewItalic letters represent the restriction enzyme sites, and underlined letters represent homologous sequences for fusion PCR, inverse PCR or One Step Cloning