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Supporting Information 1
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Plasmid construction
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All the primers used for plasmid construction are shown in supplementary Table 1.
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Vectors pXMJ19 and pEC-XK99E carrying IPTG-inducible promoter were used for
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plasmid-based gene expression. In Addition, low-copy vector pEP2 without promoter
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was used for rhtA gene expression. The nonreplicating plasmid pDsacB derived from
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pK18mobsacB served as the integrative vector for genomic modifications. To enhance
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sacB expression, the trc promoter was inserted upstream of sacB gene (Zhu et al.,
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2013).
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To enhance expression of 5-aminolevulinic acid synthase in C. glutamicum, codons
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of hemA gene from R. sphaeroides representing rare variants in C. glutamicum were
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replaced by synonymous high-usage variants, and codon adaptation index was
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improved from 0.84 to 0.88. Additionally, GC content and secondary structures were
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also optimized to strengthen stability of mRNA (optimized sequence was shown in
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Supporting Information 2). The codon-optimized gene was synthesized by GENEWIZ,
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Inc. The fragment was amplified using primers hemA-1/hemA-2 and digested with
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PstI/XbaI. The digested fragment was inserted into pXMJ19 with PstI/XbaI sites to
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yield pXA.
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The constitutive tuf promoter (Woo and Park, 2014) was amplified from C.
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glutamicum genome using primer tuf-1 and tuf-2. The PCR product was digested with
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EcoRI/SalI and inserted into the same sites of pEP2 to yield pEP2tuf. The rhtA gene
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was amplified from E. coli genome using primers rhtA-1/rhtA-2, and the resulting
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PCR product was digested with XbaI/BamHI and inserted into XbaI/BamHI sites of
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pEP2tuf to give plasmid pEP2-rhtA.
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For pD-pck construction, upstream and downstream fragments of pck gene were
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amplified from C. glutamicum genome using primers pck-1/pck-2 and pck-3/pck-4,
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the upstream fragment was digested with EcoRI/XbaI and ligated into pD-sacB
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digested with the same enzymes to yield pD-pck (F). The obtained plasmid was
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digested with PstI/HindIII and ligated with downstream fragment digested with the
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same restriction enzymes to yield pD-pck.
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To knock out pbp1a, the upstream and downstream fragments of the target gene
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were amplified from C. glutamicum genome using pbp1a-1/pbp1a-2 and
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pbp1a-3/pbp1a-4. The two fragments were fused and amplified with pbp1a-1/
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pbp1a-4. The fused fragment was then digested with BamHI/HindIII and inserted into
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the same sites to create pD-pbp1a. pD-pbp1b, pD-pbp2a and pD-pbp2b were obtained
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by the same procedure using corresponding primers and restriction enzymes.
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Strain construction
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All strains in this study are listed in Table 1. Chromosomal inactivation of pck, pbp1a,
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pbp1b, pbp2a and pbp2b were performed via two-step homologous recombination
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using corresponding plasmids. The plasmids were transformed into C. glutamicum
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using electroporation, and the procedure in detail was achieved as described
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previously (Chen et al., 2015).
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Plasmid stability during fermentation
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To investigate the stability of pXA and pEP2-rhtA in C. glutamicum ALA7, we
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examined the percentage of chloramphenicol and kanamycin resistant cells at the end
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of cultivation. The culture was diluted and plated onto a nonselective BHI agar plate
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and a selective agar plate (containing 25 μg/mL kanamycin and 10 μg/mL
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chloroamphenicol). Following incubation for 48 h at 30 ℃ , the number of
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plasmid-carrying cells was calculated by comparing the number of colonies on a
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selective agar plate and nonselective agar plate.
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