FIG 2.

Strain construction and validation of isoprene production from methanol. (a) Plasmid map of pJA2 for constitutive expression of isoprene synthase IspS in Methanosarcina spp. (b) Validation of ispS transcription by RT-qPCR. Plasmid DNA from pJA2 was used as a positive control, while genomic DNA from the parent strain NB34 was used as a negative control. (c) Dimethylallyl pyrophosphate pyrophosphatase activity in cell extracts. (d) Isoprene production measured by gas chromatography. (e) Validation of isoprene production by mass spectrometry. (f) Endpoint methane production. (g) Growth curve of att:pNB730 and att:ispS strains. (h) Mass balance of M. acetivorans att:pNB730 (blue) and att:ispS (red) strains showing percent molar carbon fluxes. Standard deviations are shown in parentheses. Blue bars, att:pNB730 strain; red bars, att:ispS strain. Data presented in panels c, d, f, and h were obtained from quadruplicate biological and triplicate technical replicates (n = 12). Data presented in panel e were from a double-blinded experiment. Data from panel g were from five biological replicates.