Trinh Lab    
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Trinh lab seeks to fundamentally understand and engineer complex cellular systems. One of the research thrusts is to develop the platform MODCELL technology that engineer modular chassis cells for rapid development of novel microbial biocatalysts for industrial biocatalysis. The other research thrust is to develop the ViPaRe technology to effectively inactivate rapidly evolving and resistant pathogens. To pursue the goal, Trinh lab is interested in applying and developing both theoretical and experimental tools in interdisciplinary fields of systems and synthetic biology together with metabolic and biochemical engineering, and microbial physiology. Below is a list of research topics that Trinh lab is pursuing.

 
  Topic 1. Understand and Develop Tools To Rewire Cellular Metabolisms  
 

A metabolic network describing a cellular metabolism typically contains hundreds to thousands of reactions catalyzed by functional enzymes to convert substrates into precursor metabolites used for cell synthesis or other metabolites secreted to extracellular environments (Figure 1). These functional enzymes are directly encoded by functional genes that determine cell phenotypes.

By applying the constraint-based metabolic network modeling, we can decompose a complex metabolic network into unique pathways, each of which contains a minimal set of enzymatic reactions supporting cell functions.  Each of these independent pathways can represent physiological states of cell operation. Complete knowledge of these pathways allows the selection of cell phenotypes of interest, which establishes a basis for designing cells with optimized metabolic functionalities. The engineered cells are particularly designed to function only according to the most efficient pathways to produce target metabolites. In addition, the operation of these pathways is always enforced by coupling both cell growth and production of the target metabolite. Through genetic engineering, cells exhibiting only phenotypes of interest are constructed and characterized.

Trinh lab is currently interested in elucidating complex cellular metabolisms and developing metabolic network modeling tools to design modular microbial cell factories that can be assembled from a universal optimal cell chassis and different production modules in a systematic and rapid way. These microbial factories are engineered to produce advanced biofuels and biochemicals from renewable feedstocks, and high-valued products such as fine chemicals, secondary metabolites, and proteins/enzymes.

We are developing the following computational tools:

+ SMET (Systematic Multiple Enzyme Targeting): This tool combines both structural and dynamic modeling to systematically identify target enzymes and their levels of up/down expresion to enhance production of target molecules. See [2].

+ MMF (Minimal Metabolic Functionality): This tool identifies a minimal set of gene deletions to achieve high production of target molecules. See [4], [5], [6], [7], [8].

+ ModCell (Modular Cell): This tool designs optimal modular cells that can couple with exchangeable production modules to build modular microbial cell factories for efficient production of desirable molecules. See [1], [3].

We are applying these tools to construct and characterize several different platforms including bacteria (Escherichia coli, Clostridium thermocellum) and yeasts (Saccharomyces cerevisiae, and Yarrowia lipolytica)

Funding sources: National Science Foundation, Department of Energy.

Related publications:

[1] Trinh, C.T.*, Liu, Y., Conner, D.J., 2015. Rational Design of Efficient Modular Cells (under review).

[2] Flowers, D., Thompson, R. A., Birdwell, D., Wang, T.*, Trinh, C.T.*, 2013. SMET: Systematic Multiple Enzyme Targeting - A Novel Method To Rationally Design Optimal Strains For Target Chemical Overproduction, Biotechnol J 8(5): 605-618. DOI: 10.1002/biot.201200233

[3] Trinh, C.T., 2012. Elucidating and optimizing Escherichia coli metabolism for obligate anaerobic butanol and isobutanol production, Appl Microbiol Biotechnol, 95(4): 1083-1094.

[4] Trinh, C.T., Li, J., Blanch, H.W., Clark, D.S., 2011. Redesigning Escherichia coli metabolism for anaerobic production of isobutanol, Appl Environ Microbiol 77(14): 4894-4904.

[5] Unrean, P., Trinh, C.T., Srienc, F., 2010.  Rational design and construction of an efficient E. coli for production of diapolycopendioic acid.  Metab Eng 12(2):112-122.

[6] Trinh, C.T., Srienc, F., 2009. Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol into Ethanol.  Appl Environ Microbiol 75(21): 6696-6705.

[7] Trinh, C.T., Unrean, P., Srienc, F., 2008. A minimal Escherichia coli cell for most efficient ethanol production from hexoses and pentoses. Appl Environ Microbiol 7: 3634-3643. (Cover Art)

[8] Trinh, C.T., Carlson, R., Wlaschin, A.P., Srienc, F., 2006. Design, construction and performance of the most efficient biomass producing E. coli bacterium. Metab Eng 8: 628-638.

 
   
  Figure 1: Harnessing cellular metabolism as optimal modular microbial cell factories.  
  Topic 2. Understand and Develop Tools to Engineer Cellullar Robusness  
 

One of the challenging tasks to convert lignocellulosic biomass into biofuels is to develop robust solventogenic microorganisms that can tolerate chemical inhibitors present in the fermentation broth.   These inhibitors are produced during pretreatment, enzymatic hydrolysis, and fermentation.  Depending on the biofuel process, inhibitors can contain weak organic acids (e.g., succinic acid, lactic acid, acetic acid, formic acid), furan derivatives (e.g., furfural, hydroxyl methyl furfural), phenolic compounds derived from lignin, and/or organic solvents derived from pretreatments (e.g., ionic liquids) or synthesized by microorganisms as fermentative products (e.g., ethanol and butanol).   These inhibitors cause detrimental effect on biocatalyst performance by decreasing both cell growth and solvent production.  It is difficult to metabolically engineer microorganisms resistant to these chemical inhibitors because the genotype and phenotype links resulting in chemical resistance in microorganisms are very often unknown.

To approach this problem, Trinh lab uses the genomics approach by applying the gene swapping and amplification technique (Figure 2).  This approach is designed to transfer novel phenotypes from not-well-characterized microorganisms exhibiting high resistance to chemical inhibitors to an engineered host.  Trinh lab is also interested in investigating mechanisms of chemical toxicity that affects cell growth and solvent production by analyzing response of cellular metabolism through systems biology approach. 

Funding sources: National Science Foundation, Department of Energy

Related publications:

Trinh, C.T., Huffer, S., Clark, M.E., Blanch, H.W., Clark, D.S., 2010. Elucidating Mechanisms of Solvent Toxicity in ethanologenic Escherichia coli. Biotechnol Bioeng 106(5):721-730. (Spotlight)

Ryu, S., Labbe, N., Trinh, C.T.*, 2015. Simultaneous saccharification and fermentation of cellulose in ionic liquid for efficient production of alpha-ketoglutaric acid, Appl Microbiol Biotechnol, 99(10): 4237-4244.

 
   
 

Figure 2: Gene swapping and amplification technique.

 
  Topic 3. Understand and Develop Tools to Engineer Synthetic Pathways  
 

Developing an efficient and robust whole-cell biocatalyst to produce a target chemical requires the recruitment of heterologous genes to constitute a synthetic metabolic pathway in the microorganism.   Even though advances in the recombinant DNA technology have developed powerful and convenient molecular biology tools to transfer genes among species, it is still a challenging task to optimize the operation of synthetic metabolic pathways in a microorganism due to imbalanced metabolic fluxes not only in the synthetic metabolic pathway but also in other associated pathways.  This imbalance results in the accumulation of intermediates that are toxic to cells, which inhibits cell growth and decreases production of a target metabolite.  

Trinh lab is interested in developing novel methods to design and optimize the performance of synthetic operons that can dynamically control fluxes of synthetic and native metabolic pathways for enhanced product formation in engineered cells.

Funding sources: UT lab startup; UT SEERC; UT LDRD; NSF CBET CB; NSF CBET BBE; NSF MCB SSB

Related publications:

Layton, D., Trinh, C.T.*, 2014. Engineering Modular Ester Fermentative Pathways in Escherichia coli, Metab Eng, 26:77–88.

Trinh, C.T., 2012. Elucidating and optimizing Escherichia coli metabolism for obligate anaerobic butanol and isobutanol production, Appl Microbiol Biotechnol 95(4): 1083-1094.

Trinh, C.T., Li, J., Blanch, H.W., Clark, D.S., 2011. Redesigning Escherichia coli metabolism for anaerobic production of isobutanol, Appl Environ Microbiol 77(14): 4894-4904.

 
  Topic 4.  Develop tools for directed metabolic pathway evolution  
 

An optimized cell designed to couple cell growth and operation of a target pathway can be utilized as an useful host to conduct the in vivo metabolic pathway evolution because cell growth is inhibited without the functioning of the target pathway.  The selection basis for the molecular pathway evolution is growth phenotype which is easy to implement.  In the recent studies, we successfully demonstrated this approach by developing optimized E. coli cells that can convert mixed sugars and biodiesel waste, glycerol into bioethanol.

We are exploring this approach in different optimized cells to improve titers, yields, and productivities for production of biofuels, biochemicals, and secondary metabolites.  With this approach, the metabolic pathway evolution can be implemented by replacing the enzyme of a reaction within the target pathway with a potential candidate from a library of mutated enzymes or from different microorganisms that have similar or related functions.  Only cells containing complementary enzymes can support cell growth.  The candidate can be selected by using advanced cell-culturing techniques such as a cytostat, turbidostat, and/or CO2-stat (Figure 3).  With these selection methods, only host cells containing enzymes with high activities are enriched in the bioreactor while others are washed out.  We anticipate that the molecular pathway evolution is complementary to the rational approach to optimize the performance of a synthetic metabolic pathway. Toward this end, we are also interested in better understanding the fundamental mechanism of the molecular pathway evolution resulting in desirable phenotypes by using systems-biology tools and hence discovering novel genotype-phenotype links to implement inverse metabolic engineering.

Funding sources: NSF CBET CB; NSF CBET BBE

Related publications:

Trinh, C.T., Srienc, F., 2009. Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol into Ethanol.  Appl Environ Microbiol 75(21): 6696-6705.

 
   
  Figure 3: Schematic setup of a turbidostat/CO2_stat.  

 

  Topic 5.  Understand and Develop Tools to Fight Rapidly Evolving and Resistant Pathogens  
 

In the war against virulent pathogens, defensive measures must be developed quickly and be readily modifiable to counter rapid and unpredictable evolution of resistance.  The current paradigm relies on small molecule discovery technologies that are unable to scale to production fast enough to counter pathogens that can rapidly adapt to neutralize existing treatments. To address the challenge, we seek to develop the ViPaRe (Virulent Pathogen Resistance) technology capable of inactivating rapidly evolving and resistant infectious pathogens.

 

 

 

 

  Updated on 09.13.2017