Clostridium cellulovorans DSM 743B can produce
butyrate when grown on lignocellulose, but it can hardly synthesize
butanol. In a previous study, C. cellulovorans was
successfully engineered to switch the metabolism from butyryl-CoA
to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC
824; however, its full potential in butanol production is still unexplored.
In the study, a metabolic engineering approach based on a push–pull
strategy was developed to further enhance cellulosic butanol production.
In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA
was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene
(ter), which can irreversibly catalyze crotonyl-CoA
to butyryl-CoA. Then an acid reassimilation pathway uncoupled with
acetone production was introduced to redirect the carbon flow from
butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism
engineering was implemented by inactivating xylR (Clocel_0594) and araR (Clocel_1253), as well as overexpressing xylT (CA_C1345), which is expected to supply additional carbon and reducing power
for CoA and butanol synthesis pathways. The final engineered strain
produced 4.96 g/L of n-butanol from alkali extracted
corn cobs (AECC), increasing by 235-fold compared to that of the wild
type. It serves as a promising butanol producer by consolidated bioprocessing.
Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single‐specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5–5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169‐(pXY1‐Pthl‐augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5‐fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.
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