To be submitted to the section of “Microbial and Enzyme Technology”

High-level production of α-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis

Penghui He ·Zeying Zhang·DongboCai·Yaozhong Chen · Hao Wang ·Xuetuan Wei·Shunyi Li*· Shouwen Chen*

P. He · D.Cai· Y. Chen · H. Wang · S. Li ()· S. Chen ()

Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan 430062, PR China

E-mailaddress: (S. Li) and (S. Chen).

Z. Zhang

College of Food Engineering, Wuchang Institute of Technology, Wuhan, 430065, PR China

X. Wei

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China

Supplementary Materials

Supplementary Table 1 The strains and plasmids used in this study

Strains and plasmids / Relevant properties / Source of reference
Strains
Escherichia coliDH5α / supE44ΔlacU169 (f 80 lacZΔM15) hsdR17recA1gyrA96thi1relA1 / This study
Bacillus licheniformisWX-02 / Wide-type CCTCCM208065 / CCTCC
BL10 / WX-02(Δmpr, Δvpr, ΔaprX, Δepr, Δbpr, ΔwprA, ΔaprE, ΔbprA, Δhag, ΔamyL) / (Wei et al. 2015)
BL10/pP43SAT / BL10 harboring pP43SAT / This study
BL10D / BL10 (Δdal) / This study
BL10B / BL10 (ΔalrB) / This study
BL10D/pHY-Dal / BL10D harboring pHY-Dal / This study
BL10D/pP43SAT-P43Dal / BL10D harboring pP43SAT-P43Dal / This study
BL10D/pP43SAT-PdalDal / BL10D harboring pP43SAT-PdalDal / This study
BL10D/pP43SAT-PtetDal / BL10D harboring pP43SAT-PtetDal / This study
Plasmids
T2(2)-ori / Bacillus knockout vector; Kanr / This study
T2-dal / T2(ori)-dal(A+B);to knock out dal / This study
T2-alrB / T2(ori)-alrB(A+B);to knock out alrB / This study
pHY300PLK / E. coli and B. s shuttle vector; Ampr,Tetr / This study
pHY-Dal / Dal expression vector / This study
pP43SAT-P43Dal / Dal mediated by P43 was inserted into pP43SAT / This study
pP43SAT-PdalDal / Dal mediated by Pdal was inserted into pP43SAT / This study
pP43SAT-PtetDal / Dal mediated by Ptet was inserted into pP43SAT / This study

Supplementary Table 2 The primers used in this study.

Primers / Sequence
T2-F / ATGTGATAACTCGGCGTA
T2-R / GCAAGCAGCAGATTACGC
pHY-F / GTTTATTATCCATACCCTTAC
pHY-R / CAGATTTCGTGATGCTTGTC
dal-KF1 / GGGAGCTCTGGCGGTGAAAAATCCTTTACAT
dal-KR1 / CACCCTTGAACTTATCATACAGGTCGCCGATGTGCCGCTTCATATT
dal-KF2 / AATATGAAGCGGCACATCGGCGACCTGTATGATAAGTTCAAGGGTG
dal-KR2 / GCTCTAGACTGAGCCAACAACAGGAGATAG
dal-KYF / TTGATGAAAAGAAAAATATGACG
dal-KYR / CGAGCAATATAACGGAGTCTCT
alrB-KF1 / GGGAGCTCAACTTTAATTGGGGATTGCTC
alrB-KR1 / GCCGTAAATCACGACTTCCTCTCCTTGCTCCTCTTCGGAATATGCCGC
alrB-KF2 / GCGGCATATTCCGAAGAGGAGCAAGGAGAGGAAGTCGTGATTTACGGC
alrB-KR2 / GCTCTAGATTCTTCATCGTCTCCCTCGTA
alrB-KYF / CGACATCGGCGATAAAGTTTC
alrB-KYR / AACCTGCTTTGTCCAGCCTTTCC
P43-F1 / GCGAATTCTGATAGGTGGTATGTTTTCG
P43-R1 / AGAATGGTTTTAAGCTCATTTCATGTGTACATTCCTCTC
dal-F / GAGAGGAATGTACACATGAAATGAGCTTAAAACCATTCT
dal-R / AATCCGTCCTCTCTGCTCTTTCAGGATTGATCAGGCAAGA
TamyL-F1 / TCTTGCCTGATCAATCCTGAAAGAGCAGAGAGGACGGATT
TamyL-R1 / GGTCTAGACGCAATAATGCCGTCGCACT
P43-F2 / GCGAATTCTGATAGGTGGTATGTTTTCG
P43-R2 / AAGCCGTTTGTGTTGTTTCATTTCATGTGTACATTCCTCTCT
amyL-F / AGAGAGGAATGTACACATGAAATGAAACAACACAAACGGCTT
amyL-R / AATCCGTCCTCTCTGCTCTTTACCAATACCATTTGAAATC
TamyL-F2 / GATTTCAAATGGTATTGGTAAAGAGCAGAGAGGACGGATT
TamyL-R2 / GGTCTAGACGCAATAATGCCGTCGCACT
Pdal-F / GCGAATTCACTGACGGATAAATCGTTG
Pdal-R / CGGTTAACCATTTCTGCTCCCGTTAT
dal-F2 / GCGAATTCTGATAGGTGGTATGTTTTCG
dal-R2 / GAAATCCGTCCTCTCTGCTCTTCAGTGCGACGGCATTATTGCGGC
TamyL-F3 / GCCGCAATAATGCCGTCGCACTGAAGAGCAGAGAGGACGGATTTC
TamyL-R3 / CGGTTAACCCAGTGCGACGGCATTATTGCG
Ptet-F / GCGAATTCACAGATGATGACGAAAAAGC
Ptet-R / ATAGAATGGTTTTAAGCTCATATTTCACCCTCCAATAATG
dal-F3 / CATTATTGGAGGGTGAAATATGAGCTTAAAACCATTCTAT
dal-R3 / AAGTTGATCCCTTAACGATTCAGGATTGATCAGGCAAGATC
TamyL-F4 / GATCTTGCCTGATCAATCCTGAATCGTTAAGGGATCAACTT
TamyL-R4 / CGGTTAACCCAGTGCGACGGCATTATTGCG
RT-amP-F / GGGAACCGGAGCTGAATGAA
RT-amP-R / AATAAACCAGCCAGCCGGAA
RT-dal-F / GATGAAGCGCTTTCGCTGAG
RT-dal-R / TGCCCGTGTCGATCTTCAAA
RT-amyL-F / CTTTATGCCCGATTGCTGCC
RT-amyL-R / GTAAGCGCCGTAGCCTACAT

Supplementary Figure 1 Confirmation of gene deficient strains by PCR amplification. APCR product amplified from strain BL10B (Line 1 ) (1630 bp) and BL10 (Line 2) (2534 bp), 5000 DL Marker (M);BPCR product amplified from strain BLD (Line 1 ) (1441 bp) and BL10 (Line 2) (2424 bp), 5000 DL Marker (M).

Supplementary Figure 1

Supplementary Figure 2 A: Effects of dal or alrB deficiency on the cell growth in the medium without D-alanine. 1: BL10; 2: BL10D; 3: BL10B; 4: BL10D/pHY-dal; B: Effects of dal or alrB deficiency on the cell growth in the medium addition of 200 ug/mL D-alanine. 1: BL10; 2: BL10D; 3: BL10B; 4: BL10D/pHY-dal; C: Segregational stability of B. licheniformisBL10D/pHY-dal.

Supplementary Figure 2

Supplementary Figure 3 The flow chart for construction of the co-expression vectors.

Supplementary Figure 3