Supplementary Table 1
Table 1 Bacterial strains and plasmids used in this study
Strains/Plasmids
Relevant Characteristics
Source
ZM4
Wild-type Zymomonas mobilis ZM4
ATCC 31821
JM110
Streptomycin resistant Escherichia coli strain
Stratagene
used as cloning host for preparation of nonmethylated plasmids.
ZM6014
ZM4 adapted for acetic acid tolerance
(Chen et al. 2009)
ZM4 ∆XR
ZM4 with a ZMO0976 knock out
This study
ZM6014 ∆XR
ZM6014 with a ZMO0976 knock out
This study
pTeasy
Commercial
vector
containing
ampicillin
Promega
resistance cassette and ColE1 ori
pTspecXR
pTeasy
vector
containing
spectinomycin
This study
resistance (SpecR) cassette and homologous
regions flanking ZMO0976
pSTV28
pSTlsXR
Commercial plasmid containing chloramphenicol
TaKaRa
resistance cassette and p15A ori
(Japan)
pSTV28 containing SpecR and homologous
This study
Bio
Inc.
regions flanking ZMO0976
pZMETX
Plasmid containing xylose metabolic genes
(Agrawal et al. 2011)
obtained from A1
pZMETX*
Mutated pZMETX containing xylose metabolic
genes obtained from A3
(Agrawal et al. 2011)
Supplementary Materials and Methods
Knockout of XR-gene (ZMO0976) in Z. mobilis
XR-gene knockout was carried out in Z. mobilis according to a published procedure
(Viitanen et al. 2011). pTspecXR was constructed for this purpose (Supplementary Fig. 1).
pTeasy backbone of the plasmid pTspecXR was obtained by EcoRI and SphI double digestion
of a plasmid pTadh-fba. pTadh-fba has an ~2 kb operon Padh-fba ligated to the commercial
pTeasy vector (Promega) using A-T cloning. Sequence of spectinomycin resistance (SpecR)
cassette which includes the promoter was obtained from GenBank (accession # X03043).
Synthetic DNA was used for SpecR cassette (Genscript). SpecR cassette was ligated to pSTV28
vector at BamHI site to obtain plasmid pSTVls. The 5’ homologous region (HR) for XR was
cloned
from
ZM4
genome
using
TAATGCAAGCATGCCCTTATATGGTCTGACGTTG-3’)
primers
and
–
(5’(5’-
GCGATTCGTCGACTGATATTGATACCAAGATCAATC-3’). Restriction sites are indicated in
bold underlined fonts. The 5’ homologous region extends from 1 kb upstream to 0.2 kb
downstream of the 5’-end of ZMO0976. 5’ HR was ligated to pSTVls vector at SalI and SphI
restriction sites to get pSTls5’XR. Similarly the 3’ HR was cloned using primers – (5’ATATTACCCGGGCTATCCGCTGGATGTTGGAG-3’)
and
(5’-
ATTATTGAATTCGGACGGACGATCAGAGAAGCG-3’). The 3’ HR extends from 0.2 kb
upstream to 1 kb downstream of the 3’-end of ZMO0976. 3’ HR was ligated to pSTls5’XR vector
at XmaI and EcoRI restriction sites to get the suicide vector pSTlsXR. EcoRI and SphI were
used to double digest pSTlsXR to obtain two DNA fragments of size 3.0 and 3.4 kb. The 3.4 kb
DNA fragment consisted of the SpecR cassette flanked by homologous regions of ZMO0976.
This fragment was ligated to the EcoRI and SphI digested pTeasy backbone mentioned above
to obtain the suicide vector pTspecXR (Supplementary Fig. 1). JM110 was used as cloning host
so that methylation-free plasmids can be obtained for efficient electroporation in Z. mobilis ZM4.
Several primers were used to confirm the knockout of ZMO0976 from ZM4 genome.
Primers
(5’-
GCTATTGACGGTACCATGAACACTTCTACGCAAAAACC-3’)
and
(5’-
TATTCGTACTAAGCTTTTATTTATCGCGTGGCGGGGGTG-3’) bind to the ends of ZMO0976
and
amplify
the
entire
ZMO0976
gene.
GCCATCAAAGGTCAACGCGATAATTTGATTATTGCG-3’)
Primers
and
-
(5’(5’-
CCAGATAATGTTCAAAGCGCGGTTTCTGGAATTTC-3’) amplify the middle 526 bp portion of
the ZMO0976 gene. Primers (5’-AAAGAAACCAATCCCGTTGTTGTCAAAGCCAAAGTC-3’) and
(5’- CCATTGAAGAATGGCATGCTTTGACCGATGAAGC-3’) amplified the genomic region
flanking ZMO0976.
Supplementary Fig. 1
Upstream of XR
1
5'-end of XR
R
Amp
pTspecXR
SpecR
6397bp
ColE1
3'-end of XR
Downstream
of XR
Plasmid map of pTspecXR, a pTeasy derived suicide vector for knockout of XR-gene