Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.
Fig. S1Diagram of plasmid pTS2. The tetracycline resistance gene (TcR) of plasmid pBR322 (lightly shaded) was PCR amplified (including a point mutation removing a HindIII site and enhancing the −10 sequence of its promoter) and inserted into the HindIII site of plasmid pUC8:16. PUC8:16 contains the Vitreoscilla hemoglobin gene (vgb) and about 800 adjacent bases of Vitreoscilla DNA (darkly shaded) cloned into plasmid vector pUC8. Sizes (bp) of regions of pTS2 are indicated (DOC 111 kb)
Fig. S2Diagram of plasmid pTS3. The Vitreoscilla hemoglobin gene (vgb) and tetracycline resistance gene (TcR) cassette (shaded) from pTS2 (Fig. S1) was PCR amplified and inserted into the EcoRI site of plasmid vector pKT230 to construct plasmid pTS3. Sizes (bp) of regions of pTS3 are indicated (DOC 107 kb)
Fig. S3Diagram of plasmid pTS4. The Vitreoscilla hemoglobin gene (vgb) and tetracycline resistance gene (TcR) cassette from pTS2 (shaded) was PCR amplified and inserted into the EcoRI site of plasmid vector pBBR1-MCS #5 to construct plasmid pTS4. Sizes (bp) of regions of pTS4 are indicated (DOC 105 kb)
Fig. S4Diagram of plasmid pTS5. The pdc + adhb cassette from plasmid pLOI297 (lighter shading) was PCR amplified and inserted into the SrfI site of plasmid vector PCR-Script-Amp (Stratagene, La Jolla, CA). Then vgb (darker shading) was inserted into the XhoI site of PCR-Script-Amp- pdc + adhb. Sizes (bp) of regions of pTS5 are indicated (DOC 114 kb)