Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community

• Published in: Biotechnology and bioengineering Vol. 117; no. 7; pp. 2008 - 2022
• Main Authors: Wen, Zhiqiang, Ledesma‐Amaro, Rodrigo, Lu, Minrui, Jiang, Yu, Gao, Shuliang, Jin, Mingjie, Yang, Sheng
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2020
• Subjects: adaptive laboratory evolution; Agmatine; Agmatine deiminase; Aldehyde dehydrogenase; Aldehydes; Bioprocessing; Butanol; cell wall lyases; Cell walls; consolidated bioprocessing; Consortia; Evolutionary genetics; Fermentation; Genes; Lignocellulose; low pH resistance; Metabolic engineering; Microbial activity; Microorganisms; pH control; pH effects; Sugar; low pH resistance; consolidated bioprocessing; agmatine deiminase; adaptive laboratory evolution; cell wall lyases
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.27333

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. The mutualism of Clostridium cellulovorans and Clostridium beijerinckii on lignocelluloses by nutrition‐detoxify cooperation yet relies on pH‐stat control. Here, combined evolutionary engineering and genetic manipulation improved the ability of C. cellulovorans to tolerate and produce butanol at a pH of 5.5. The engineered twin‐clostridium consortium produced 3.94 g/L butanol directly from alkali‐extracted deshelled corn cobs without pH control. This work explores a proof of concept on how to improve the robustness of a synthetic microbial community for bioproduction.