Extended Data Fig. 2: Construction and PCR verification of various mutants and stepwise enhancement of CPIII production in R. sphaeroides.
From: High-yield porphyrin production through metabolic engineering and biocatalysis
a, Construction and PCR verification 2.4.1-ΔfnrL. b, Construction and PCR verification of ZX-5-ΔfnrL. F1 and F2 are the PCR products that were amplified using ZX-5-ΔfnrL-up-ck-F/R and ZX-5-ΔfnrL-dn-ck-F/R, respectively. c. Construction and PCR verification of HY01-ΔfnrL. d. Construction and PCR verification of HY01-ΔhemN. F3 and F4 are the PCR products that were amplified using HY01-ΔhemN-up-ck-F/R and HY01-ΔhemN-dn-ck-F/R. e. Construction and PCR verification of HY01-ΔhemN-prrABpLB2. F5 and F6 are the PCR products that were amplified using ΔhemN-prrAB-up-ck-F/R and ΔhemN-prrAB-dn-ck-F/R. The PCR verifications in b-c were performed three times each (n = 3; replicates are shown in Source Data files; in each replicate, at least two double crossover conjugants were selected). f. Heat map depicts the differentially expressed PrrA target operons (selected) during fermentation in bioreactor at 16, 24, 40 and 60 h. This result indicated that three genes associated with the porphyrin metabolism pathway, namely hemA, hemC, and hemE, which are regulated by prrA, exhibited a substantial upregulation in transcription levels at the 40 h of fermentation compared to the 24 h. FPKM (fragments per kilobase of transcript per million fragments mapped) values were taken as generation of the heat map by origin 2022 software. g. Stepwise enhancement of CPIII production in R. sphaeroides (n = biologically independent replicates). *: Fed-batch fermentation in 5-L bioreactor; #: IPTG addition. Bar graph with error bars represents mean ± s. d.
