Compared to ethanol, butanol has gained more and more interest as a substitute of gasoline because of its higher energy content, lower volatility and less corrosion. Butanol can be produced by anaerobic microorganisms such as Clostridium acetobutylicum and C. beijerinckii in acetone-butanol-ethanol fermentation (ABE fermentation), which however suffers from a low butanol yield (~20% w/w), low titer (<15 g/L) and low productivity because of butanol toxicity and the production of other byproducts including acetone, ethanol, acetate and butyrate. Conventional ABE fermentation involves complicated metabolic pathways, including acidogenesis, solventogenesis and spore-forming life cycle, which are difficult to control or manipulate. Despite extensive efforts in metabolic engineering of solventogenic Clostridia, little progress has been made in improving butanol yield, titer, and productivity in ABE fermentation. In this work, we aim at developing a novel solventogenic Clostridia strain by cloning and expressing genes in the butanol biosynthesis pathway into C. tyrobutyricum, a butyric acid bacterium that normally produces butyric acid with acetic acid, carbon dioxide and hydrogen as byproducts from glucose and xylose. C. tyrobutyricum has high tolerance to butyric acid (>50 g/L) and butanol (>20 g/L). It's relatively simple metabolic pathway and high metabolic flux to butyryl-CoA (the precursor for butanol biosynthesis) render it an ideal host for butanol production.
In this study, several butanol producing mutants of C. tyrobutyricum were obtained by overexpressing aldehyde-alcohol dehydrogenase (adhE2) under the control of native thiolase promoter. A butanol titer of 1.1 g/L was obtained in batch fermentations carried out in serum bottles with wild-type C. tyrobutyricum overexpressing adhE2 (WT-ad). Higher butanol titers were achieved in fermentations with ptb (phosphotransbutyrylase) and ack (acetate kinase) knockout mutants. The ack knockout mutant (ACK-ad) produced the highest butanol (~10 g/L) with a high yield (0.26-0.33 g/g glucose consumed). Butyric acid was the major byproduct along with some acetic acid and ethanol in the fermentation. Compared to WT-ad, the ack knockout mutant (ACK-ad) produced more C4 products (butanol and butyrate) and less C2 products (acetate and ethanol) with a high C4/C2 ratio of 10.6 (mol/mol), whereas the ptb knockout mutant (PTB-ad) produced more C2 and less C4 products with a low C4/C2 ratio of 1.4. It is clear that ack and ptb genes play important roles in controlling the metabolic flux distribution in C. tyrobutyricum. These results suggest that knockout of genes in the butyrate biosynthesis pathway in the mutant ACK-ad can further increase the butanol yield to approaching the theoretical value of 0.41 g/g glucose.
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