(157ao) Aerobic Ester Production through Escherichia coli-Clostridium Co-Culture

Butanol toxicity and oxygen sensitivity are two of the major obstacles faced in traditional acetone-butanol-ethanol (ABE) fermentation of Clostridium. Butanol toxicity could be resolved by converting butanol into butyl butyrate and extracting butyl butyrate simultaneously. Introducing an aerobe or facultative anaerobe into Clostridium culture could enable the aerobic fermentation of Clostridium and hence avoid the costly anaerobic pretreatment.

In this study, we developed a synthetic co-culture system consisting of an engineered E. coli strain and a wild-type Clostridium strain to allow the production of butyl butyrate at aerobic condition. In this co-culture system, facultative anaerobic E. coli consumes oxygen in the peripheral environment to facilitate the growth of Clostridium. After oxygen is depleted, butanol and butyrate are produced by Clostridium. Meanwhile, butyl butyrate is produced from butanol and butyrate by the engineered E. coli and extracted from the aqueous cell culture by the hydrophobic extractant.

To realize the conversion of butanol and butyrate into butyl butyrate, two modules were designed to be co-expressed in E. coli, including a butyryl-CoA-generating module and an ester-producing module. The ester-producing module was constructed by expressing an alcohol acyltransferase from strawberry (SAAT). After testing a variety of enzymes that can convert butyrate into butyryl-CoA, acetoacetyl-CoA: acetate CoA transferase (CtfAB) from C. acetobutylicum was found to be the most efficient one. Mono-culturing the CtfAB- and SAAT-expressing E. coli with butanol and butyrate fed led to the production of 380 mg/L butyl butyrate within 24 hours, which is the highest titer of butyl butyrate that has been produced from E. coli. Moreover, 110 mg/L butyl butyrate was produced within 36 hours when the CtfAB- and SAAT-expressing E. coli was aerobically co-cultured with Clostridium using glucose as the sole carbon source. To our best knowledge, this is the first time that butyl butyrate was aerobically produced by an E. coli-Clostridium co-culture. In addition, the butyl butyrate titer achieved here is more than twice of what was obtained from the anaerobic mono-culture of an engineered Clostridium strain.

As a system harnessing both the genetic tractability of E. coli and superior acid- and alcohol-producing ability of Clostridium, this co-culture system is of great potential to improve the conventional Clostridium fermentation and produce high value-added chemicals.