Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The target audience includes academic researchers focused on producing biofuels and biochemicals through metabolic engineering, synthetic biology, and bioprocess engineering. Additionally, it includes industry professionals (including the ones from paper and pulping industries) and agriculture sectors interested in realizing the biomanufacturing of biofuels and biochemicals in the commercial scale. Policymakers and funding bodies committed to the bioeconomy and environmental sustainability, along with members of the general public who support renewable energy and are concerned with environmental sustainability, are also key target groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One Post-doc has been trained for cellular metabolism, genome engineering, and fermentation of Clostridium strains. One PhD student has been trained, who worked on the characterization of PMS. How have the results been disseminated to communities of interest?We have published one peer-reviewed journal article. We have presented the relevant research results at conferences. The PDs have also disseminated the relevant results through invited presentations and other teaching activities. What do you plan to do during the next reporting period to accomplish the goals?1) We will further troubleshoot the constructed synthetic pathway to enable R-1,3-BD production. 2) We will carry out systematic genome engineering work to further enhance the production of R-1,3-BD. 3) We will optimize the fermentation for R-1,3-BD production using PMS as feedstock.
Impacts
What was accomplished under these goals?
We have collected representative PMS samples from three main types of PMS based on their sources: Kraft mill primary sludge, recycled mill sludge, and tissue mill sludge. A comparison of the chemical compositions of three different PMS has been carried out and determined that tissue mill sludge would best suit this project with the following compositions: cellulose-68.1%, hemicellulose-13.5%, extractives-9.1%, lignin <0.1%, and ash 5.8%. In this project, we will use tissue mill sludge due to its higher cellulose content and much lower ash and lignin contents. In order to enable R-13-BD production, initially, we carried out the following steps in our Clostridium host: 1) Overexpressed the optimized ctfA(V8)-ctfB(V5); 2) Overexpressed the optimized phaB gene by both plasmid-based overexpression and chromosomal integration. However, we were not able to observe R-13-BD production. Further, it has been reported that the phaB gene from Cupriavidus necator (CnphaB) was heterologously expressed in E. coli BL21(DE3) with characterized enzyme activity. Following this, the recombinant E. coli strain expressing CnphaB was constructed, and through induced expression and protein purification, the necessary enzymes for fermentation were obtained. In Clostridium host, acetoacetyl-CoA requires sequential catalysis by PhaB and AdhE2 enzymes to convert into the target product R-1,3-BD. Previous work in our lab successfully created the C. tyrobutyricum Δcat1::adhE2 strain, where adhE2 was effectively expressed with robust enzyme activity, leading to a high-yielding butanol-producing strain. Based on these findings, the experiment conducted mixed fermentation with the exogenously expressed and purified PhaB enzyme and the C. tyrobutyricum Δcat1::adhE2 strain to further verify the feasibility of using acetoacetyl-CoA as a precursor for synthesizing the target product R-1,3-BD. Following various conditions explored regarding pH, enzyme, and cell concentrations, the mixed fermentation has been completed. Chromatographic analysis of the fermentation products is underway, with the chiral purity and yield of R-1,3-BD to be determined after further analysis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Y. Ma, N. Guo, X. Li, Z. Jiang, D. Zhang, L. Guo, Y. Wang. 2023. Development of an efficient recombinant protein expression system in Clostridium saccharoperbutylacetonicum based on the bacteriophage T7 system. ACS Synthetic Biology. 12(10):3092-3105.
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