# Engineering Clostridium Tyrobutyricum for High Butanol Production through Induction Expression of Exogenous NADPH-Dependent HBD

## TL;DR

Scientists engineered a bacteria to produce more butanol by controlling when a specific enzyme is active, improving efficiency and reducing unwanted byproducts.

## Contribution

A novel inducible expression system for HBD in Clostridium tyrobutyricum enhances butanol production while reducing acetate and ethanol.

## Key findings

- Inducing hbd(Ck) at 12 h increased butanol yield by 20%, productivity by 87.5%, and selectivity by 52%.
- Optimized induction timing reduced acetate formation and improved redox balance in the engineered strain.
- Scale-up in a bioreactor achieved high butanol titer (21.8 g/L) with improved productivity and yield.

## Abstract

Clostridium tyrobutyricum Δcat1::adhE2 is a promising
cell factory
for butanol production because of its robustness, high butanol tolerance,
and minimal butyrate production. However, excessive acetate and ethanol
production remains a major bottleneck limiting its butanol yield.
Coexpressing an exogenous hbd(Ck) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase
(HBD) from Clostridium kluyveri with adhE2 could increase the C4 carbon flux, resulting in increased
butanol and decreased acetate and ethanol production. However, constitutively
overexpressing hbd(Ck) in Δcat1::adhE2 shows little improvement in
butanol yield, productivity, and selectivity, which might be caused
by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔcat1::adhE2-Pbgal-hbd(Ck) with a dynamic expression of hbd(Ck) controlled by an inducible promoter was developed. In
serum bottle fermentation at 37 °C, when the hbd(Ck) expression was induced at 12 h or in the early
exponential phase, butanol production increased ∼20% in yield
(from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16
to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g
total products) compared to the control strain without expressing
any hbd(Ck), whereas hbd(Ck) expression induced at 0 or 24 h in MΔcat1::adhE2-Pbgal-hbd(Ck) or constitutively in MΔcat1::adhE2-Pcat1-hbd(Ck) showed significantly lower butanol
yield and productivity. At 25 °C, MΔcat1::adhE2-Pbgal-hbd(Ck) with 12 h induction produced the highest butanol
titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity,
and 0.83 g/g product selectivity due to much reduced acetate formation.
Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased
productivity to 0.39 g/L·h while also achieving high butanol
titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The
optimized induction timing resulted in a balanced NAD­(P)H pool, effectively
channeling substrates toward butanol biosynthesis. It was concluded
that the timing for hbd(Ck) expression
was critical as it affected glucose catabolism, cell growth, redox
balance, and carbon flux distribution. These findings underscore the
potential of dynamic metabolic regulation to overcome bottlenecks
in biobutanol production, providing a scalable and economically viable
bioprocess for industrial application.