Supporting Information
Plasmid Construction
All primers are listed in Table S1.
The pCU plasmid was constructed for the gene knockout, which were based on a markerfree method (Shi et al. 2013). The uracil ribose transferase encoding gene upp from B. subtilis
168 and chloramphenicol acetyltransferase encoding gene cat from pC194 were amplified
using primers Kit-fsn-uppU/L and Kit-fsn-catU/L respectively. The two fragments were fused
and amplified by fusion PCR using primers Kit-fsn-U/L, and then digested by AatII and ligated
to the matching sites of pUC18 to create pCU.
For bdhA deletion, the bdhA upstream and downstream fragments were amplified from the
B. subtilis genome using primers D-bdhA-FU/FL and D-bdhA-BU/BL respectively. The two
fragments were fused and amplified by fusion PCR with primers D-bdhA-FsnU/L. The fused
fragment (bdhA-FB) was then digested with SphI-KpnI, and was ligated into the same digested
sites of pCU to create pCU-bdhAFB. The acoAFB fused fragment was amplified by the same
procedure with corresponding primers, digested with SalI-KpnI, and ligated separately into the
corresponding sites of pCU to yield plasmids pCU-acoAFB
The shuttle vectors were constructed for bdhA and udhA overexpression, based on the B.
subtilis-E. coli shuttle vector pHP13-P43 with the B. subtilis strong promoter P43. The bdhA and
udhA genes were amplified by PCR from B. subtilis 168 and E. coli W1485 chromosomal DNA
respectively, with primers E-bdhA-U/L and E-udhA-U/L. The two fragments were digested with
the same restriction sites KpnI-BamHI, and ligated to KpnI-BamHI digested pHP13-P43
respectively, yielding pHP13-P43-bdhA and pHP13-P43-udhA. For pHP13-P43-bdhA-udhA
construction, the udhA fragment was amplified by PCR with primers E1-udhA-U/L, digested
with restriction sites AflII-XhoI, and ligated to AflII-XhoI digested pHP13-P43-bdhA, yielding
pHP13-P43-bdhA-udhA.
Strains Construction
The upp gene of B. subtilis 168 was replaced by the neomycin resistance gene (neo) using
plasmid pUC18-uppFB-neo through the double-crossing recombination, and the resulting strain
BSF1 was used as the parent strain. The vector pCU-bdhAFB was integrated into BSF1
chromosome by the first single-crossover recombination and the transformant was selected by
chloromycetin. Next, the resulting transformant was cultured in LB liquid medium for 12h, then
the cells were spread on a 5FU agar plate. The bdhA-deleted strain denoted as BSF10 was
selected from the grown colonies by PCR verification using primers D-bdhA-FU/BL. The acoA
was knocked out in BSF10 using the same method, yielding BSF3. The set of shuttle vectors,
pHP13-P43-bdhA, pHP13-P43-udhA and pHP13-P43-bdhA-udhA were transformed into BSF3
and selected by chloromycetin, yielding BSF18, BSF19 and BSF20.
Table S1 Strains, Plasmids and Primers
Name
Strains
B. subtilis 168
E. coli DH5α
K. pneumoniae CICC
10011
BSF1
BSF3
BSF10
BSF18
BSF19
BSF20
Plasmids
pUC18
pC194
pUC18-uppFB-neo
pHP13-P43
pCU
pCU-acoAFB
pCU-bdhAFB
pHP13-P43-bdhA
pHP13-P43-udhA
pHP13-P43-bdhA-udhA
Primers 5’-3’
Kit-fsn-catU
Kit-fsn-catL
Kit-fsn-uppU
Kit-fsn-uppL
Kit-fsn-U
Kit-fsn-L
D-acoA-FU
D-acoA-FL
D-acoA-BU
D-acoA-BL
D-acoA-FsnU
D-acoA-FsnL
D-bdhA-FU
D-bdhA-FL
D-bdhA-BU
D-bdhA-BL
D-bdhA-FsnU
Relevant genotype
Source
/reference
Wide-type strain, trpC2
BGSCa
Invitrogen
CICCb
B. subtilis 168Δupp
B. subtilis 168ΔuppΔacoAΔbdhA
B. subtilis 168ΔuppΔbdhA
B. subtilis 168ΔuppΔacoAΔbdhA; pHP13-P43-bdhA
B. subtilis 168ΔuppΔacoAΔbdhA; pHP13-P43-udhA
B. subtilis 168ΔuppΔacoAΔbdhA; pHP13-P43-bdhA-udhA
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Ampr
Cmr, Bacillus cloning vector
Kanr
Cmr, Emr, E. coli–B. subtilis shuttle vector
puC18 containing cat-upp cassette
pCU containing acoA flanks
pCU containing bdhA flanks
Emr, Cmr, containing bdhA under P43 promoter
Emr, Cmr, containing udhA under P43 promoter
Emr, Cmr, containing bdhA and udhA under P43 promoter
Lab Stock
Lab Stock
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Lab Stock
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TGCTTTTTCCCCTTTCTATGTATG
TTCGTGGTTTTTCAATGCAAGAAGGATATGGATCTGGAGCTGT
ACAGCTCCAGATCCATATCCTTCTTGCATTGAAAAACCACGAA
ACCCTCGTTTAGCATACAATAG
GCGCGGGACGTCAAAGGATTTTTCGCTACGCTCA
ACATGCATGCACTTTACGTCTCCACAGAATG
ACGCGTCGACCGTCATCGGAGAACACCAGCAT
CTTTTTCAATCGCTCGAGCGGCCATGGGGACTTAAGTCAGTTAATGACAAGCCTTCTCG
TTGTCATTAACTGACTTAAGTCCCCATGGCCGCTCGAGCGATTGAAAAAGCCGTCTCGT
CGGGGTACCTACTTTTACCGCCATTTGTGTCC
ACGCGTCGACTCATCATACAAAGGCATAG
CGGGGTACCACAAGCGCAGCAATATCCG
ACATGCATGCAATGTGACTTCTTGCTTTCCAGA
TTCCTTTTACTGTACGCTCGAGCGGAGATCTGGACTTAAGGTGACAGGTGCCGTTTCAT
GGCACCTGTCACCTTAAGTCCAGATCTCCGCTCGAGCGTACAGTAAAAGGAATTATCGG
CGGGGTACCTTGTGTTTATTGGATTAGGGA
ACATGCATGCTTTTTAGGCTTCGCTTTTGCAT
D-bdhA-FsnL
E-bdhA-U
E-bdhA-L
E-udhA-U
E-udhA-L
E1-udhA-U
E1-udhA-L
CGGGGTACCTGAATTCGATTATGTATTATGGG
TCGGGGTACCAAAGGAGGAATTCAAAATGAAGGCAGCAAGATGGCATAAC
CGCGGATCCGCACTCGAGCGGACTTAAGCAAATTAGTTAGGTCTAACAAGG
TCGGGGTACCAAAGGAGGAATTCAAAATGCCACATTCCTACGATTACG
CGCGGATCCTTAAAACAGGCGGTTTAAACCG
CGGACTTAAGAAAGGAGGAATTCAAAATGCCACATTCCTACGATTACG
CGCGGATCC GCA CTCGAG CTGTGCCGCCGGAGCGCAAACG
a
b
Bacillus Genetic Stock Center
China Center of Industrial Culture Collection