Bacterial strains and plasmids
Bacterial strains and deoxynucleic acids (DNAs) used in this study are listed Table 1. Genomic DNA from bacterial cells was isolated using the Blood & Tissue DNA Isolation Kit (Qiagen, Hilden, Germany). Standard recombinant DNA technologies were applied for molecular cloning (Miller 1992). Taq DNA polymerase was obtained from New England Biolabs (Ipswich, MA, USA). All synthesized oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA). DNA sequencing was conducted by the Centre for Applied Genomics at the Hospital for Sick Children (Toronto, Canada). E. coli BW25113 was the parental strain for derivation of all engineered strains in this study and E. coli DH5α was used as a host for molecular cloning. Note that the ldhA gene (encoding lactate dehydrogenase) was previously inactivated in BW25113, generating BW∆ldhA (Srirangan et al. 2014).
For genetic implementation of the Shemin pathway in E. coli , thehemA gene was first amplified by polymerase chain reaction (PCR) using the primer set g-hemA and the genomic DNA of wild-typeRhodobacter sphaeroides DSM 158 as the template. The amplifiedhemA gene was Gibson–assembled with the PCR-linearized pK184 using the primer set g-pK-hemA to generate pK-hemA. The expression of the cloned hemA gene in the pK184 vector was under the control of the Plac promoter.
Knockouts of the genes, including sdhA (encoding succinate dehydrogenase complex flavoprotein subunit A, SdhA) and iclR(encoding transcriptional AceBAK operon repressor, IclR), were introduced into BW∆ldhA by P1 phage transduction (Miller 1992) using the appropriate Keio Collection strains (The Coli Genetic Stock Center, Yale University, New Haven, CT, USA) as donors (Baba et al. 2006). To eliminate the co-transduced FRT-KnR-FRT cassette, the transductants were transformed with pCP20 (Cherepanov and Wackernagel 1995), a temperature sensitive plasmid expressing a flippase (Flp) recombinase. Upon Flp-mediated excision of the KnR cassette, a single Flp recognition site (FRT “scar site”) was generated. Plasmid pCP20 was then cured by growing cells at 42°C. The genotypes of derived knockout strains were confirmed by colony PCR using the appropriate verification primer sets listed in Table 1.
The expression of hemB was repressed by CRISPRi using various derived plasmids from pdcas9-bacteria (Addgene plasmid # 44249) and pgRNA-bacteria (Addgene plasmid # 44251). All synthesized oligonucleotide pairs have 60 nucleotides (nt), which includes 20 nthemB -targeting sequence, 20 nt upstream and 20 nt downstream sequences of pgRNA-bacteria vector (Fig. 2). They were ordered from Integrated DNA Technologies (Coralville, IA, USA) and annealed as described previously (Pengpumkiat et al. 2016), generating four double-stranded DNA fragments ofhemB -gRNA-L1, hemB -gRNA-L2, hemB -gRNA-L3, andhemB -gRNA-L4 (Table 1). All these four DNA fragments were individually Gibson-assembled with the PCR-linearized pgRNA-bacteria using the primer set g-pgRNA to generate pgRNA-L1, pgRNA-L2, pgRNA-L3, and pgRNA-L4, respectively (Table 1). The four hemB -repressed strains, i.e., DMH-L1, DMH-L2, DMH-L3, and DMH-L4, were developed by creating a triple-plasmid system (Fig. 2) containing pK-hemA, pdcas9-bacteria, and the gRNA-containing plasmid (i.e., pgRNA-L1, pgRNA-L2, pgRNA-L3, or pgRNA-L4). For the control strain DMH-CT, the original pgRNA-bacteria plasmid without any hemB -targeting sequence was used as the third plasmid.