Improvement of lignocellulose bioconversion in Clostridium cellulolyticum and identification of active lignocellulose degraders in temperate grassland

Abstract

As the most abundant sustainable carbon resource on the earth, lignocellulose is considered as one of the most promising feedstocks to produce biofuels, which can mitigate the environmental issues brought by burning of fossil fuels. However, the lignocellulosic biofuels face grand challenges on multiple fronts, including low-cost technology for utilization of cellulose and highly efficient conversion from cellulose to biofuels by high-yield microorganisms. Therefore, these challenges are calling for engineering designs to increase efficiency and reduce the costs. As a model mesophilic clostridial species for studying lignocellulose degradation, Clostridium cellulolyticum can perform one-step bioconversion of lignocellulose to biofuels and is considered as a potential candidate for future industrial biofuels productions. However, the efficiency of lignocellulose bioconversion in C. cellulolyticum is not high enough, which impedes its further application in industries. Thus, the major aim of this dissertation is to engineer the C. cellulolyticum by CRISPR-Cas9 editing method to improve its lignocellulose bioconversion efficiency. In addition to being feedstock for biofuels productions, lignocellulose is the primary carbon input in the natural soil and the microbial decomposition of the lignocellulose is an important global carbon sink. With current global warming, both photosynthesis rate by plants and the carbon decomposition rate by microorganisms can be enhanced but may not equally. As a result, whether warming can cause a positive feedback for C exchange between the terrestrial and atmosphere is unclear. Thus, another major aim of this dissertation is to identify the active lignocellulose bacteria and understand their lignocellulose degradation mechanisms responding to warming. In C. cellulolyticum, a unique extracellular multi-enzyme complex named cellulosome plays the most important role in degrading the cellulose. The cellulosome has great commercial values and can be used for consolidated bio-saccharification. However, the function for a cellulosomal component named X2 in C. cellulolyticum was still unclear, which limited our understanding for the cellulose degradation mechanisms by the cellulosome and its future commercial application. To have a better understanding of the in-vivo biological function of X2 modules, we employed CRISPR-Cas9 editing to create dual X2 modules mutant (△X2-NC) by deleting the conserved motif (NGNT) of X2 modules. Compared to the wild type strain, the degradation efficiency and saccharification ability in the △X2-NC were decreased. Further, the in vivo adhesion assay and the in vitro enzymatic assay found that the biological function of the X2 module was associated with the binding affinity between the cells and its cellulose substrate. This study provides new perspectives on engineering cellulolytic bacteria or modification of commercial cellulases for industrial application. Major cellulosomal components are encoded by a 26 kb cip-cel gene operon named cip-cel. Two major large transcripts were detected when C. cellulolyticum was grown on cellulose. However, the abundance of 3’- transcript is much lower than the 5’- transcript. To increase the expression of the 3’- transcript of the cip-cel operon, we employed CRISPR-Cas9 editing system to insert a synthetic promoter (P4) and an endogenous promoter (P2) within cip-cel operon in Clostridium cellulolyticum. Both engineered strains increased the transcript abundance of downstream polycistronic genes and enhanced in vitro cellulolytic activities of isolated cellulosomes. Compared to the control strain, both engineered strains could degrade more cellulose and demonstrated a greater growth rate and a higher cell biomass yield. Our strategy, editing regulatory elements of catabolic gene clusters, provides new perspectives on improving cellulose bioconversion in microbes. Earlier studies have found that the accumulation of cellobiose could inhibit both cell growth and cellulase productions in the cellulolytic clostridia bacteria, such as Clostridium thermocellum and C. cellulolyticum, which would further decrease the efficiency of cellulose bioconversion. To overcome it, two strategies were applied to release the carbon catabolite repression caused by cellobiose. First, an exogenous β-glucosidase gene from C.cellulovorans was integrated into the upstream of the lactate hydrogenase gene (ccel_2485) of C. cellulolyticum genome for enhancing the enzymatic bioconversion of cellobiose to glucose. We found that the engineered strain could degrade 12% more cellulose than the WT at the final time point, accompanied with 25% more ethanol production. Second, the regulator for carbon catabolite repression (CCR) was inactivated in C. cellulolyticum. However, the mutant could not utilize the cellulose anymore, indicating that inactivation of CCR regulator is not an effective strategy for releasing the repression of cellobiose. Together, the integration of the exogenous β-glucosidase gene in the genome provides new perspectives on improving cellulose bioconversion in C. cellulolyticum, and also provides a new potential site in the genome of C. cellulolyticum for future integration and engineering. Finally, microbial decomposition of soil organic carbon (SOC), which are mainly derived from lignocellulose, has a strong impact on future atmospheric greenhouse gas concentrations, which serve as important feedbacks to climate warming. However, the underlying decomposition mechanisms remain poorly understood. In order to understand the microbial mechanisms of lignocellulose decomposition and how the active microbes respond to current global warming, we identified active taxa responsible for carbon (C) degradation in temperate grassland subjected to experimental warming. Using a stable-isotope probing incubation experiment with 13C-labeled straw to simulate grassland litter, a total of 56 active amplicon sequence variants (ASVs) were detected only in the warmed samples. Many ASVs belonged to fast-growing bacteria such as α-Proteobacteria, Bacillales, Actinobacteria, and Bacteroidetes, which were further verified by our observation that their relative abundances were increased (p < 0.050) by warming over consecutive seven years. Interestingly, warming increased the phylogenetic diversity of active bacterial communities and β-diversity among active bacterial communities. The carbon-degrading potentials of the active bacterial communities were also stimulated by warming. In summary, these results should provide essential support to future field and global scale simulations and enable more accurate predictions of feedbacks between climate change and carbon cycling. Overall, this dissertation provides valuable insights into engineering C. cellulolyticum for improving its lignocellulose bioconversion efficiency. Our strategies can be applied for engineering other clostridial cellulolytic bacteria, such as C.cellulovorans and C.thermocellum, to improve their lignocellulose bioconversion efficiency. Additionally, the newly identified active lignocellulose degraders in temperate grassland may also provide new insights into finding new industrial potential strain to produce lignocellulosic biofuels.