Adenosine Triphosphate and Carbon Efficient Route to Second Generation Biofuel Isopentanol

Abstract

Climate change necessitates the development of CO 2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO 2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization.

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

“…The prenol production could be improved by engineering the pathway from acetyl-CoA to prenol. Very recently, a new, redox balanced isopentanol biosynthetic pathway was developed (Eiben et al, 2020). The engineered E. coli produced 80 mg/L of isopentanol by using glucose at the microaerobic condition with mixed acids produced, providing another possibility to link the EUP developed in this study with prenol.…”

Diversifying Isoprenoid Platforms via Atypical Carbon Substrates and Non-model Microorganisms

“…Several other strategies utilize two upper MVA pathway genes ( E. coli AtoB, Staphylococcus aureus HMGS) prior to diverging with the expression of the hydratase LiuC and Myxococcus xanthus decarboxylase AibAB ( Clomburg et al, 2019 ; Eiben et al, 2020 ). From there, Eiben et al demonstrated 80mg/L isopentanol production through subsequent expression of M. xanthus AibC and Clostridium acetobutylicum AdhE2 ( Eiben et al, 2020 ).…”

“…Several other strategies utilize two upper MVA pathway genes ( E. coli AtoB, Staphylococcus aureus HMGS) prior to diverging with the expression of the hydratase LiuC and Myxococcus xanthus decarboxylase AibAB ( Clomburg et al, 2019 ; Eiben et al, 2020 ). From there, Eiben et al demonstrated 80mg/L isopentanol production through subsequent expression of M. xanthus AibC and Clostridium acetobutylicum AdhE2 ( Eiben et al, 2020 ). In a more holistic approach, AibAB was followed with expression of the Clostridium beijerinckii acyl-CoA reductase (cbjALD), and E. coli YahK to generate prenol at the highest titer reported for biological production ( Figure 1 ; Clomburg et al, 2019 ).…”

Synthetic Biology and Metabolic Engineering Employing Escherichia coli for C2–C6 Bioalcohol Production

“…In E. coli , iterative keto-acid elongation resulted in C5, C6, and even longer chain (C7–C8) alcohols ( Wang et al, 2017 ). Recently, Eiben et al (2020) constructed a new isopentanol production pathway in E. coli XX03 (BW25113 Δ adhE Δ ldhA Δ frdBC ) by introducing the isovaleryl-CoA pathway (LiuC, AibAB, and AibC) from Myxococcus xanthus and a butyryl-CoA reductase (AdhE2) from C. acetobutylicum , and the resulting strain produced 80.5 mg/L isopentanol after 36 h under microaerobic conditions. In addition, Chen et al (2017) constructed a 1-pentanol production pathway by controlling the keto acid elongation cycle [BW25113 Δ ilvB Δ ilvI Δ leuA expressing cimA Δ2, leuBCD , leuA (G462D), kivd (V461G), yqhD ] and identifying a new mutation in the ketoisovalerate decarboxylase, KivD V461D, that preferentially tuned the KivD from Lactococcus lactis specificity toward 1-pentanol synthesis.…”