To provide methods for effectively producing 1,4-butanediol (BOD) and chemical precursors thereof in commercial quantities.SOLUTION: Disclosed herein is a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding an enzyme in BOD production pathway to express the exogenous enzyme with a disrupted gene encoding an aerobic respiratory control regulatory system. The exogenous enzymes are succinyl-CoA synthetase, alpha-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and 4-hydroxybutyrate dehydrogenase and optionally 4-hydroxybutyryl-CoA/acetyl-CoA transferase, butyrate kinase and phosphotransbutyrase, 4-hydroxybutyryl-CoA reductase, 4-hydroxybutanal reductase, pyruvate dehydrogenase, NADH-insensitive citrate synthetase and phosphoenolpyruvate carboxykinase.SELECTED DRAWING: Figure 46

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.

Biotechnology offers a new sustainable approach to manufacturing chemicals, enabling the replacement of petroleum-based raw materials with renewable biobased feedstocks, thereby reducing greenhouse gas (GHG) emissions, toxic byproducts, and the safety risks associated with traditional petrochemical processing. Development of such bioprocesses is enabled by recent advances in genomics, molecular biology, and systems biology, and will continue to accelerate as access to these tools becomes faster and cheaper.

A sustainable bioprocess for the production of 1,4-butanediol (BDO) from carbohydrate feedstocks was developed. BDO is a chemical intermediate that goes into a variety of products including automotive parts, electronics, and apparel, and is currently manufactured commercially through energy-intensive petrochemical processes using fossil raw materials. This review highlights the development of an Escherichia coli strain and an overall process that successfully performed at commercial scale for direct production of bio-BDO from dextrose. Achieving such high level performance required an integrated technology platform enabling detailed engineering of enzyme, pathway, metabolic network, and organism, as well as development of effective fermentation and downstream recovery processes.

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.