Engineering a Short, Aldolase-Based Pathway for 1,3-Butanediol Production in Escherichia coli

Aldolases have received wide interest as biocatalysts due to their ability to form new carbon-carbon bonds with high stereoselectivity, thereby forming long complex molecules from simple building blocks. They exhibit broad substrate specificity, which increases their utility in catalyzing the production of a wide range of compounds¹. Of the Class I aldolases, deoxyribose-5-phosphate aldolase (DERA) naturally catalyzes the reversible aldol condensation of acetaldehyde and glyceraldehyde-3-phosphate to produce deoxyribose-5-phosphate. As of yet, efforts have been focused on increasing its activity and stability through enzyme engineering towards a wide range of aldehydes. Current DERA-based processes typically use the purified enzyme for biotransformation; however, using it to shorten synthetic pathways in whole-cell biotransformations has not been fully explored for the production of biochemicals directly from renewable resources. Shorter biosynthetic pathways reduce the complexity of strain and pathway optimization, including fine-tuning gene expression levels and metabolic burden on the cell. As such, we explored the possibility of producing chemicals with potentially high market value via shorter pathways using aldolases. We have used a pathway predictor algorithm tool (PPA), which employs generalized enzymatic reactions, and we included all known classes of aldolases. The addition of the aldolase class of reactions increased the number of unique chemicals by 5 fold, which can be produced from acetaldehyde using pathways composed of a maximum of three reaction steps. From these results, a short 2-step pathway from acetaldehyde to (R)-1,3-butanediol (BDO) was identified. (R)-BDO can be used to synthesize high-value compounds. Previous studies have demonstrated an acetyl-CoA based pathway for BDO production in E. coli via four enzymatic steps^2,3. Our unique approach demonstrates the production of BDO via a shorter, 2-step pathway. To test our aldolase-based pathway, we used a systematic approach to engineer E. coli by: (1) screening and identifying the required enzymes as predicted by the pathway predictor algorithm, DERA and aldo-keto reductase (AKR); (2) expressing the pathway enzymes on a plasmid expression system; and (3) using a model-based strategy to delete native genes from the host strain to eliminate by-products, such as ethanol, and increase carbon flux from the acetaldehyde branch-point to pathway products by 97%. Our current strain produces 1.44 g/L of BDO with a yield of 0.09 g/g of glucose (17% of the maximum theoretical yield) by two-phase batch fermentation. Future work entails additional strain modifications to increase flux to BDO. Since DERA can accept a wide range of aldehydes as substrates, there is potential to design short, synthetic pathways for other long-chain chemicals.

1.           Liu, J. & Wong, C.-H. Aldolase-catalyzed asymmetric synthesis of novel pyranose synthons as a new entry to heterocycles and epothilones. Angew. Chem. Int. Ed. Engl. 41, 1404–7 (2002).

2.           Kataoka, N., Vangnai, A. S., Tajima, T., Nakashimada, Y. & Kato, J. Improvement of (R) -1,3-butanediol production by engineered Escherichia coli. J. Biosci. Bioeng. 115, 475–480 (2013).

3.           Kataoka, N. et al. Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH. Biosci. Biotechnol. Biochem. 78, 695–700 (2014).