Source: AUBURN UNIVERSITY submitted to NRP
BIOMANUFACTURING OF VALUABLE (R)-3-HYDROXYBUTYRATE FROM LIGNOCELLULOSIC FEEDSTOCKS THROUGH MICROBIAL ENGINEERING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1031640
Grant No.
2024-67022-41530
Cumulative Award Amt.
$300,000.00
Proposal No.
2022-10776
Multistate No.
(N/A)
Project Start Date
Nov 15, 2023
Project End Date
Nov 14, 2025
Grant Year
2024
Program Code
[A1531]- Biorefining and Biomanufacturing
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
(N/A)
Non Technical Summary
(R)-3-hydroxybutyrate (R-3HB) is a versatile chemical compound used to make biodegradable plastics, vitamins, antibiotics, and more. Its production is in high demand due to its unique properties. However, creating R-3HB with the required chiral purity is a complex challenge. In this project, we aim to demonstrate the feasibility of making chirally pure R-3HB from plant waste materials, like wood and crop leftovers, by modifying Clostridium pathways. We'll create and test two different pathways to produce R-3HB in the Clostridium host. Then, we'll combine these approaches and make further genetic changes to boost R-3HB production in the bacterium. If successful, this research could pave the way for an efficient and sustainable process to create R-3HB from agricultural waste, supporting the growth of a bio-based economy. This project aligns with efforts to promote a sustainable bioeconomy and strengthen agriculture. It will serve as a valuable reference for scientists working on improving microbial processes for valuable products. Ultimately, this transformative research has the potential to benefit the environment, the economy, and society, promoting agricultural sustainability and advancing our understanding of environmental responsibility.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5114010202060%
5114010104040%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 2020 - Engineering;
Goals / Objectives
In this project, we attempt to demonstrate the feasibility of producing high-value chirally pure (R)-3-hydroxybutyrate (R-3HB) from lignocellulosic materials through metabolic engineering of Clostridium and bioprocess development. To achieve the goal, the following research tasks have been proposed: First, we will build and evaluate two synthetic pathways for R-3HB production in Clostridum. Further, we will combine both pathways and conduct further genome engineering to enhance R-3HB production in the engineered strain using lignocellulosic materials. With further systematic development after this Seed Grant, we aim to ultimately establish an enabling bioprocess for efficient R-3HB production from agri-industrial waste streams, thus supporting the development of a sustainable bioeconomy.
Project Methods
Objective 1: Develop an R-3HB production pathway in the Clostridium host. Firstly, we will evaluate 'Pathway I' by introducing specific genes into the bacterium and measuring R-3HB production. If successful, this pathway could yield high-purity R-3HB. We'll also optimize the genetic code to match Clostridium's codon usage to enhance production. Additionally, we'll introduce specific genetic mutations to further boost R-3HB output.Objective 2: Establish an alternative pathway for R-3HB production. In addition, we will introduce 'Pathway II' into Clostridium to explore its potential for R-3HB production. This pathway involves overexpressing specific genes and converting intermediates to R-3HB. After introducing this pathway, we'll evaluate its effectiveness for producing chirally pure R-3HB.Objective 3: Enhance R-3HB production level and selectivity through further genome engineering. To improve R-3HB production, we will integrate the genes responsible for R-3HB production into Clostridium chromosome, making the bioprocess more stable and sustainable. We'll also delete the key gene in the natural pathway to redirect carbon flow towards R-3HB production. These genetic modifications will help us achieve higher selectivity and yield in R-3HB production.

Progress 11/15/23 to 11/14/24

Outputs
Target Audience:The target audience comprises academic researchers engaged in producing biofuels and biochemicals through metabolic engineering, synthetic biology, and bioprocess engineering. Additionally, it includes industry professionals and the agricultural sector seeking to scale up biomanufacturing for biofuels and biochemicals. Policymakers and funding bodies dedicated to fostering the bioeconomy and environmental sustainability are also key groups, as well as members of the general public who support renewable energy initiatives and prioritize environmental stewardship. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Postdoc has been trained in cellular metabolism, genome engineering, and fermentation of Clostridium strains. 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 conduct systematic genome engineering to enhance R-3HB production by knocking out competitive carbon utilization pathways and boosting cofactor regeneration. 2) We will optimize fermentation processes for R-3HB production using lignocellulosic feedstock.

Impacts
What was accomplished under these goals? 1) To investigate the enzymatic properties involved in the catalytic synthesis of 3-HB and to facilitate the enhancement of enzyme activity and stability, genes from heterologous sources were assembled into a plasmid vector. These genes were first induced and expressed in E. coli, and SDS-PAGE analysis post-purification confirmed that only two genes produced significant amounts of soluble proteins at the expected sizes. Further optimization is needed for other genes, including adding fusion expression tags, co-expressing chaperone proteins, and refining protein induction conditions. 2) Multiple synthetic pathways for 3-HB synthesis were constructed in our Clostridium host strain. Additionally, expression vectors for these pathway genes were designed to modulate copy number. To date, we have successfully established a 3-HB production pathway, achieving a maximum titer of approximately 1 g/L after 24 hours of fermentation. We also assessed the tolerance of our Clostridium host to 3-HB, revealing that the strain can endure high levels of 3-HB, positioning it as a promising candidate for efficient 3-HB production.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Ma, Y., Guo, N., Wang, S., Wang, Y., et al., Metabolically engineer Clostridium saccharoperbutylacetonicum for comprehensive conversion of acid whey into valuable biofuels and biochemicals. Bioresource Technology 2024, 400, 130640
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Y. Ma, Y., Guo, N., Li, X., Jiang, Z., Zhang, D., Guo, L., Wang, Y. Development of an Efficient Recombinant Protein Expression System in Clostridium based on the Bacteriophage T7 System. 2024 AIChE Annual Meeting. San Diego, CA. October 27-31, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: L. Murphy, Y. Wang, J. Wang. Understanding mutant strategies of Clostridium tyrobutyricum using a systems identification-based framework for genome-scale metabolic model analysis. 2024 AIChE Annual Meeting. San Diego, CA. October 27-31, 2024.