Developing an ethanol utilisation pathway based NADH regeneration system in Escherichia coli

Abstract

Many industrially relevant biotransformation in whole-cells are dependent on cofactors such as NADH or NADPH. Cofactor regeneration is an established approach for providing a cheap source of cofactors in support of the main biotransformation reaction in biocatalysis. In essence, cofactor regeneration uses a sacrificial substrate to help regenerate a cofactor consumed by the main biotransformation reaction. Enzymatic in nature, alternative cofactor regeneration systems with high efficiency and which utilises low cost sacrificial substrate are of interest. Glucose dehydrogenase system has been dominant in NADH regeneration. But, in its current incarnation, glucose dehydrogenase system is relatively inefficient in regenerating NADH with theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-gene ethanol utilisation pathway in NADH regeneration. Comprising the first step that takes ethanol to acetaldehyde, and a second step that converts acetaldehyde to acetyl-CoA, NADH from both steps could be mined for supporting biotransformation reaction in cofactor regeneration mode. Theoretically, ethanol utilisation pathway (EUP) affords a higher NADH yield of two NADH per ethanol molecule, and is therefore more efficient than glucose dehydrogenase (GDH) system. In this project, the EUP pathway was coupled to a cpsADH (an alcohol dehydrogenase from Candida parapsilosis) mediated ketone to alcohol anaerobic biotransformation with concentration of alcohol product as marker for efficiency of cofactor regeneration. Experiment tests showed that EUP was more efficient than GDH. Further, EUP could support biotransformation of both butanone and acetophenone in single and two-phase biotransformation, respectively. Additional work conducted to improve biotransformation efficiency revealed that ethanol provision positively correlated with biotransformation efficiency. Growing cell biotransformation was also found to improve biotransformation efficiency compared to resting cell due largely to the driving force generated by cell growth. Tests of a growth medium effect also found that cells cultivated in M9 ethanol medium delivered higher biotransformation efficiency compared to those cultivated in LB medium. This could arise due to the lower expression of NADH dependent enzymes during growth in M9 ethanol medium compared to LB medium that allowed more NADH to be diverted to support ketone biotransformation. However, a persistent problem with the experimental system is the relatively poor consumption of ethanol that points to need for further engineering of the system. Collectively, pathway-based NADH regeneration is possible with ethanol utilisation, with biotransformation efficiency dependent on mode of biotransformation (resting cell versus growing cell) and growth medium used.