First, upstream and downstream regions (about 1 kbp) of cbbLS c were individually amplified by PCR with genomic DNA of R. eutropha H16 as a template and primer sets of cbbLSc-up5’/cbbLSc-up3’ and cbbLSc-down5’/cbbLSc-down3’, respectively. The second PCR with the amplified fragments

using cbbLSc-up5’/cbbLSc-down3’ primers gave a fused fragment of the upstream and downstream regions of cbbLS c . The resulting fragment was digested by EcoRI and HindIII and then ligated with pK18mobsacB [50] PLX4032 chemical structure at the corresponding sites to obtain pK18ms∆cbbLSc. pK18ms∆cbbLSp for deletion of cbbLS p from megaplasmid pHG1 was constructed in the same way using primer sets of cbbLSp-up5’/cbbLSp-up3’ and cbbLSp-down5’/cbbLSp-down3’. Transconjugation of mobilizable plasmids from E. coli S17-1 to R. eutropha and isolation of this website strains generated by pop in-pop out recombination using the pK18mobsacB-based

suicide plasmids were performed as described previously [13, 14]. The strains H16∆cbbLS c , H16∆cbbLS p , and H16∆∆cbbLS were obtained by single deletion of cbbLS c and cbbLS p , and double deletion of the genes in R. eutropha H16, respectively. Determination of the abundance of 13C in P(3HB) Cultivation of R. eutropha strains H16, H16∆cbbLS c , H16∆cbbLS p , and H16∆∆cbbLS were done in a 500 ml flask on a reciprocal shaker (115 strokes/min) at 30°C. Firstly, the strains were cultivated in 100 ml of a nutrient rich medium composed of 10 g/l tryptone, 2 g/l yeast extract, and 1 g/l meat extract in tap water for 12 h. The grown cells in 50 ml of the culture broth were harvested, washed with a salt solution (9 g/l Na2HPO4 · 12H2O, 1.5 g/l KH2PO4 in deionized water), and then transferred into 100 ml of a nitrogen-free MB medium (pH6.5 adjusted

with KH2PO4) containing 0.5% (w/v) fructose. The cells were further incubated for 24 h to promote P(3HB) biosynthesis. NaH12CO3 (1.08% 13C (natural abundance)) or NaH13CO3 (98% 13C) (Taiyo Nippon Sanso, Tokyo, Japan) 4-Aminobutyrate aminotransferase was added to a final concentration of 5 mM periodically every 2.5 h during the second stage, taking into consideration loss of dissolved CO2 to the atmosphere. The cells after the second stage cultivation were harvested, washed, and lyophilized as described above. The dried cells were subjected to methanolysis, and analyzed by GCMS-QC2010 system (Shimadzu, Kyoto, Japan) equipped with an Caspase Inhibitor VI ic50 InertCap 1 capillary column (ϕ0.25 mm, 30 m) (GL Science, Tokyo, Japan). 13C/12C ratios in the fragments of CH3–CH=OH+ (m/z 45), CH3–C(OH)H–CH3–C=O+ (m/z 87), and CH3–O–CO–CH2–CH=OH+ (m/z 103) derived from 3HB methyl ester were calculated from the respective isotopomer abundances, and the mean was referred as a abundance of 13C in the P(3HB) fraction. RNA-seq data accession number The RNA-seq data used in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under the accession number of GSE47759. Acknowledgement We thank Prof. K.