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

Abstract

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.

Metabolic and Process Engineering of Clostridium beijerinckii for Butyl Acetate Production in One Step

“…C. beijerinckii NCIMB 8052 was cultivated as previously described in an anaerobic chamber (AW400SG, Electro-Tech, Co., Ltd., U.K.) for 12–24 h at 37 °C in liquid or solidified yeast extract–tryptone–glucose (YTG) medium (5 g/L of yeast extract, 5 g/L of tryptone, and 10 g/L of glucose) before fermentation or tolerance studies. All stock cultures were maintained in 20% (v/v) glycerol and frozen at −80 °C.…”

Current Progress in Production of Building-Block Organic Acids by Consolidated Bioprocessing of Lignocellulose

“…This makes it difficult to compare the effectiveness of these different strategies. As regards their ability to lower pH limit allowing growth of a microorganism, no study reported so far was able to go beyond 0.5 pH unit, irrespective from the strategy (strain evolution/engineering) used [198,210,[216][217][218]. Results seem somehow more promising as regards to the ability of these studies to improve tolerance towards an organic acid.…”

“…Enhancement of acid tolerance of a number of microorganisms has been obtained through different approaches, such as chemical mutagenesis [198], genome shuffling [210,211], adaptive laboratory evolution (ALE) [190,212], multiplex automated genome engineering (MAGE) [213], rational metabolic engineering [214,215], or combination of these strategies [198,216] (Table 3). Improved acid tolerance has been differently evaluated and quantified, for instance through survival at extreme pH (e.g., pH 2.5-3.0), or ability to grow/survive at more acidic pH than the wild-type strain, or increased growth rate and/or biomass production at moderately acidic pH or in presence of challenging concentration of organic acid.…”

“…In addition, the role of small non-coding RNAs (sRNAs) and RNA chaperones in microbial response to a number of stresses has been receiving increasing attention [227]. pH limit for growth lowered from ≈6.0 to 5.5 [216] CM + ALE Fibrobacter succinogenes -pH limit for growth lowered from 6.10 to 5.65 [198] * Acid tolerant strains with improved organic acid (i.e., lactic acid) production.…”

Challenges and Future Perspectives of Promising Biotechnologies for Lignocellulosic Biorefinery

“…To further improve the adaptation between members of the co-culturing system, genetic engineering and adaptive evolution are adopted. For example, Wen et al [108] designed a co-culture system to produce butanol from lignocellulose by C. cellulovorans and C. beijerinckii. Butanol fermentation preferred low pH by C. beijerinckii, while C. cellulovorans cannot grow well at a pH below 6.4.…”