This project has demonstrated the level of commercial readiness for production of the industrial chemical, 1,4-butanediol (BDO), from lignocellulosic biomass by engineered E. coli. Targets were BDO titer, rate, and yield (TRY) and growth in lignocellulosic hydrolysates (Hz). A range of Hzs were used to assess limitations for biomass-to-BDO. Via adaptive evolution methods, whole-genome sequencing, and introduction of identified target genes, strains co-utilizing C5/ C6 sugars were made. The composition of Hz versus TRY led to a modified Hz composition. This was used in partnership with the DOE to redirect the project to focus on 1) several biomass Hz from new suppliers, 2) Hz specification due to the characteristics of the Genomatica BDO process, 3) a gene cassette to engineer any BDO producing strain for biomass, and 4) modified BDO recovery to more economically recover BDO at industry specifications. BDO TRY and growth of the E. coli strains were predictable based on Hz composition from several suppliers. This defined metrics for biomass Hz composition to achieve BDO TRY along with internal TEA to evaluate the economic potential of each modification to strain, Hz feed, and process. An improved biomass-to-BDO production strain reached BDO T-R in a 30 L fermentation above original objectives. Yield approached the proposed Y and modifications to BDO recovery were demonstrated. Genomatica is now in the position of being able to incorporate biomass feedstocks into the commercial GENO BDO process.