Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
(N/A)
Non Technical Summary
How to find solutions to achieve the sustainable production of biofuel from nonfood biomass, such as the lignocellulosic material or the other waste streams from food production, is a grand challenge that faces both scientists and policy makers. In the center of this challenge lies the question of how to maximize the economic values that can be extracted from biomass in biofuel production.Lignocellulosic biomass represents the primary feedstock in modern biorefineries for making biofuels. Cellulose (polymer of C6 sugar), hemicellulose (polymer of C5 and C6 sugar), and lignin are the three major components of lignocellulose. In the past two decades, extensive research efforts have focused on the development of processes to utilize the cellulose and the hemicellulose components, while lignin, the aromatic polymer comprising 15-30% dry weight of the lignocellulosic biomass, has traditionally been viewed as a waste product and burned for heat and power. With a full commercialization of lignocellulosic biofuels, the industry would produce estimated 300 million tons of lignin-rich material. The current money equivalent of lignin is $0.18/kg, while the approximate value of lignin used in chemical conversion can be higher than $1.08/kg. Processes that can convert lignin into value-added chemicals would drastically boost the economic values of lignocellulosic biomass and the sustainability of biofuel production.This project takes a 'from wastes to treasure' approach that is built upon a FDA HV1-certified microorganism that was originally isolated from garden soil. The goal is to engineer Pseudomonas putida KT2440 for the bioproduction of value-added chemicals, such as monomers in nylon 6,6 production, from aromatics that can be derived from lignin degradation. The team expects broad impacts from the research outcomes on both improving lignin valorization and enhancing knowledge of microbialmetabolism.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The long-term goal of the research is to establish bioprocesses for economical manufacturing of value-added products from lignin in microbial cell factories. As a stepping-stone, the proposed research will focus on the understanding and engineering of KT2440 for improved metabolic traits and the production of C6 compounds, in particular adipic acid, from lignin-derived aromatics. Three objectives are designed to achieve the proposed goals.Objective 1. Broadening carbon utilization by KT2440. This objective aims to build a robust KT2440 chassis that can efficiently and completely utilize various components of the lignin stream for high-titer and high-yield bioproduction. Two sub-aims are proposed: (1) Expanding the utilization of S lignin derivatives by KT2440. Direct genetic modification will be coupled with Adaptive Lab Evolution (ALE) to expedite the engineering efforts. Studies will be conducted to uncover the causal genetic/biochemical change(s) for phenotypic improvement. (2) Engineering KT2440 for complete co-utilization of mixed carbon source. Due to its intrinsically complex structure, lignin degradation products are chemically heterogeneous, containing aromatics from H (hydroxyphenyl), G (guaiacyl), and S (syringyl) lignin, and sugar, e.g., glucose. We will conduct systems studies to unravel control mechanism(s) over sequential carbon consumption and apply the learned knowledge to the engineering of KT2440 for simultaneous utilization of H/G/S lignin-derived aromatics and glucose. Enhanced sugar metabolism in KT2440 has been reported in the literature and is not covered here. Successful completion of Objective 1 not only benefits the design and implementation of strain engineering strategy in this proposal but also yields knowledge and strains that broadly serve the P. putida research community.Objective 2. Optimizing the direct biosynthesis of adipic acid from lignin-derived aromatics in KT2440. The PD's lab reported the first direct biosynthesis of adipic acid from lignin-derived aromatics using engineered KT2440 strains through the design and implementation of a novel artificial biosynthetic pathway, which exploits the pre-existing carbon skeleton of aromatic starting materials. Objective 2 is built upon this preliminary study and focuses on optimizing the adipate biosynthesis through two sub-aims: (1) Balancing the expression of adipic acid pathway enzymes and (2) Systems host strain engineering. Obtained strain will be examined in the third sub-aim for adipate production from model compounds in lignin degradation and a commonly used lignin stream in bio-valorization studies, alkaline pretreated liquor of corn stover (APL). Systems biology studies will be conducted to guide the design of effective metabolic engineering strategy. Due to resource constraint, elaborate process optimization will be excluded. We will also conduct in-depth lifecycle cost analysis (LCCA) to assess the feasibility of our process, particularly in comparison to a reported two-step process for adipate production from lignin stream.Objective 3. Expanding the product portfolio to include C6 diol and (di)amine. This objective conducts proof-of-concept studies to further extend the adipic acid pathway for direct biosyntheses of three additional industrially important C6 compounds via aldehyde intermediates that are produced by Carboxylic Acid Reductase (CAR)-catalyzed reduction of C6 carboxylates. The effort is facilitated by a growth-coupled method that was recently published by the PD's lab. It allows the high-throughput selection of CAR mutants with improved kinetics on desirable substrates. Sub-aims of this objective target: (1) Structure-guided directed evolution of CAR and (2) Demonstrating the direct biosyntheses of three C6 chemicals in adipate-producing KT2440. Objective 3 builds the foundation for future optimizations of the proposed bioprocesses. It shows the feasibility of extending the product portfolio through adipic acid as a biosynthetic hub.
Project Methods
The project uses a combination of protein engineering, strain engineering, and technoeconomic analysis methods to achieve the research goal.1. General genetic engineering methods include plasmid transformation and genome editing are applied to change the traits of engineered microbes.2. Adaptive laboratory evolution is applied to select mutated microbes with desirable phenotype.3. Omics studies including genomics, transcriptomics, and fluxomics are used to obtain systems-level datasets.4. Genome-scale metabolic modeling is used to simulate and analyze the phenotypical change.5. Both well-established and machine-learning-based bioinformatics are used to analyze omics data to unravel regulatory mechanisms.6. Directed evolution is used improve the kinetic properties of key enzymes.7. Computational modeling is used to select key residues for protein engineering.8. Analytical methods including HPLC, MS, NMR, and UV spectrophotometry are used to analyze the enzyme activities and metabolite production.9. Lifecycle cost analysis (LCCA) is used to gauge the feasibility of the biological process.