Across our research areas, we engineer living cells to support new chemistry,
to introduce the new chemistry within proteins to expand their function,
and to rely on the new chemistry for controlled use of cells in new application contexts.
to introduce the new chemistry within proteins to expand their function,
and to rely on the new chemistry for controlled use of cells in new application contexts.
Direction 1: Genome engineering for new-to-life biocatalysis
Fundamental science
Microbial whole cells or their lysates have promise to serve as lower cost biocatalysts than purified or secreted enzymes, especially for longer transformation pathways. The ability to perform reactions inside living cells can also leverage natural metabolism as a source of sustainable inputs. However, cellular environments introduce competing pathways that erase important chemical functionality.
Our lab shows how electrophilic functional groups such as aldehydes and nitro compounds can be prone to rapid modification in cellular environments and how we can slow down or eliminate these processes through many targeted gene deletions. We investigate how we can harness these platforms for "new-to-life" biocatalysis that installs chemical functional groups onto valuable building blocks.
Applications
Most chemicals and materials we use in our daily lives are currently sourced from petroleum. To develop alternative routes to obtain these molecules from sustainable feedstocks or to source them from waste streams, we need low cost and efficient catalysts. Our early work has demonstrated how our engineered strains can be useful for upgrading plastic and biomass wastes, for producing core motifs of pharmaceutical small molecule classes, and for semi-synthesis of non-standard amino acids.
Synergy across research directions
We have used aldehyde and nitro group stabilizing strains for semi-synthesis of non-standard amino acids (Direction 2) and to extend the method of synthetic auxotrophy to aldehydes (Direction 3).
Relevant publications:
Robust cellular transformations of PET deconstruction products by import of glycol esters
Reductive enzyme cascades for valorization of polyethylene terephthalate deconstruction products
Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells
Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells
Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products
(Review) Innovations toward the valorization of plastic waste
Microbial whole cells or their lysates have promise to serve as lower cost biocatalysts than purified or secreted enzymes, especially for longer transformation pathways. The ability to perform reactions inside living cells can also leverage natural metabolism as a source of sustainable inputs. However, cellular environments introduce competing pathways that erase important chemical functionality.
Our lab shows how electrophilic functional groups such as aldehydes and nitro compounds can be prone to rapid modification in cellular environments and how we can slow down or eliminate these processes through many targeted gene deletions. We investigate how we can harness these platforms for "new-to-life" biocatalysis that installs chemical functional groups onto valuable building blocks.
Applications
Most chemicals and materials we use in our daily lives are currently sourced from petroleum. To develop alternative routes to obtain these molecules from sustainable feedstocks or to source them from waste streams, we need low cost and efficient catalysts. Our early work has demonstrated how our engineered strains can be useful for upgrading plastic and biomass wastes, for producing core motifs of pharmaceutical small molecule classes, and for semi-synthesis of non-standard amino acids.
Synergy across research directions
We have used aldehyde and nitro group stabilizing strains for semi-synthesis of non-standard amino acids (Direction 2) and to extend the method of synthetic auxotrophy to aldehydes (Direction 3).
Relevant publications:
Robust cellular transformations of PET deconstruction products by import of glycol esters
Reductive enzyme cascades for valorization of polyethylene terephthalate deconstruction products
Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells
Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells
Reductive amination cascades in cell-free and resting whole cell formats for valorization of lignin deconstruction products
(Review) Innovations toward the valorization of plastic waste
Direction 2: Combining biosynthesis & incorporation of non-standard amino acids
Fundamental science
Proteins are incredible polymeric nano-machines and the molecular workhorses of cells. Despite their diversity of structure and function, their abilities are sometimes limited by the chemistry of the twenty standard amino acids used for protein synthesis. In my group, we program cells to create non-standard amino acids (nsAAs), either through de novo biosynthetic pathways that begin from simple carbon sources or using semi-synthesis routes that begin from inexpensive precursors. Our key questions and contributions focus on critically examining when complete or partial biosynthesis of nsAAs is most valuable compared to supplementing a chemically synthesized nsAA.
Applications
One context where de novo biosynthesis of an nsAA is essential is when the organism requires autonomous access to the novel sidechain chemistry. We are working towards the development of more efficacious live bacterial vaccines through the synthesis and incorporation of an immunogenic amino acid within designer antigens in mucosal tissue within animal models. However, for centralized manufacturing of proteins in bioreactors, de novo biosynthesis might not be the most efficient strategy. Accordingly, we have developed a polyspecific semi-synthesis pathway to access many different kinds of L-phenylalanine derivatives from supplemented aryl aldehydes. This system could be transformative in the kinds of nsAAs it allows labs to create and test, often at ~100-fold lower cost than the chemically synthesized nsAA.
Synergy across research directions
Our semi-synthesis pathway relies on stabilization of the aryl aldehyde precursor through gene deletions (Direction 1), and our work on immunogenic amino acids features nitro groups that are better stabilized through gene deletions (Direction 1). When the labor of nsAA biosynthesis and incorporation is split across two organisms, and when the target protein is an essential gene, the result is an engineered reliance of one strain on another (Direction 3).
Relevant publications:
Combined biosynthesis and site-specific incorporation of phenylalanine derivatives from aryl aldehydes or carboxylic acids in engineered bacteria
A one-pot biocatalytic cascade to access diverse L-phenylalanine derivatives from aldehydes or carboxylic acids
Discovery of L-threonine transaldolases for enhanced biosynthesis of beta hydroxylated amino acids
A platform for distributed production of synthetic nitrated proteins in live bacteria
(Review) Advances in engineering microbial biosynthesis of aromatic compounds and related compounds
Proteins are incredible polymeric nano-machines and the molecular workhorses of cells. Despite their diversity of structure and function, their abilities are sometimes limited by the chemistry of the twenty standard amino acids used for protein synthesis. In my group, we program cells to create non-standard amino acids (nsAAs), either through de novo biosynthetic pathways that begin from simple carbon sources or using semi-synthesis routes that begin from inexpensive precursors. Our key questions and contributions focus on critically examining when complete or partial biosynthesis of nsAAs is most valuable compared to supplementing a chemically synthesized nsAA.
Applications
One context where de novo biosynthesis of an nsAA is essential is when the organism requires autonomous access to the novel sidechain chemistry. We are working towards the development of more efficacious live bacterial vaccines through the synthesis and incorporation of an immunogenic amino acid within designer antigens in mucosal tissue within animal models. However, for centralized manufacturing of proteins in bioreactors, de novo biosynthesis might not be the most efficient strategy. Accordingly, we have developed a polyspecific semi-synthesis pathway to access many different kinds of L-phenylalanine derivatives from supplemented aryl aldehydes. This system could be transformative in the kinds of nsAAs it allows labs to create and test, often at ~100-fold lower cost than the chemically synthesized nsAA.
Synergy across research directions
Our semi-synthesis pathway relies on stabilization of the aryl aldehyde precursor through gene deletions (Direction 1), and our work on immunogenic amino acids features nitro groups that are better stabilized through gene deletions (Direction 1). When the labor of nsAA biosynthesis and incorporation is split across two organisms, and when the target protein is an essential gene, the result is an engineered reliance of one strain on another (Direction 3).
Relevant publications:
Combined biosynthesis and site-specific incorporation of phenylalanine derivatives from aryl aldehydes or carboxylic acids in engineered bacteria
A one-pot biocatalytic cascade to access diverse L-phenylalanine derivatives from aldehydes or carboxylic acids
Discovery of L-threonine transaldolases for enhanced biosynthesis of beta hydroxylated amino acids
A platform for distributed production of synthetic nitrated proteins in live bacteria
(Review) Advances in engineering microbial biosynthesis of aromatic compounds and related compounds
Direction 3: Biological & ecological containment towards environmental contexts
Fundamental science
Synthetic biologists have created many tools to make engineered organisms more functional in settings outside of traditional reactors. Furthermore, we are actively expanding the chemical repertoire of engineered microbes. To responsibly harness these new opportunities for engineered microbes, we need to better develop and characterize techniques for intrinsic biocontainment. Our lab focuses on synthetic auxotrophy, where an organism is engineered to depend on a synthetic nutrient for growth. By engineering a second organism to produce the synthetic nutrient, we have transitioned the system from a chemical dependence to a biological or ecological dependence. We are also interested in degradation of essential proteins or toxic proteins for further control of microbial survival.
Applications
We are actively working towards the design of controlled rhizobacteria such as Bacillus subtilis or Pseudomonas putida for applications in new contexts. More will be shared publicly soon.
Synergy across research directions
Our efforts on nsAA biosynthesis create more chemistries we can engineer reliance upon for ecological containment (Direction 2), and the strains that rely on nsAAs for their survival can double as selection platforms to advance new-to-life biocatalysis (Direction 1) or biosynthesis pathways (Direction 2) via directed evolution.
Relevant publications:
Combinatorial mutagenesis of N-terminal sequences reveals unexpected and expanded stability determinants of the Escherichia coli N-degron pathway
Engineered orthogonal and obligate bacterial commensalism mediated by a non-standard amino acid
Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells
(Review) Deployment of engineered microbes: Contributions to the bioeconomy and considerations for biosecurity
Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights
Engineering posttranslational proofreading to discriminate nonstandard amino acids
Synthetic biologists have created many tools to make engineered organisms more functional in settings outside of traditional reactors. Furthermore, we are actively expanding the chemical repertoire of engineered microbes. To responsibly harness these new opportunities for engineered microbes, we need to better develop and characterize techniques for intrinsic biocontainment. Our lab focuses on synthetic auxotrophy, where an organism is engineered to depend on a synthetic nutrient for growth. By engineering a second organism to produce the synthetic nutrient, we have transitioned the system from a chemical dependence to a biological or ecological dependence. We are also interested in degradation of essential proteins or toxic proteins for further control of microbial survival.
Applications
We are actively working towards the design of controlled rhizobacteria such as Bacillus subtilis or Pseudomonas putida for applications in new contexts. More will be shared publicly soon.
Synergy across research directions
Our efforts on nsAA biosynthesis create more chemistries we can engineer reliance upon for ecological containment (Direction 2), and the strains that rely on nsAAs for their survival can double as selection platforms to advance new-to-life biocatalysis (Direction 1) or biosynthesis pathways (Direction 2) via directed evolution.
Relevant publications:
Combinatorial mutagenesis of N-terminal sequences reveals unexpected and expanded stability determinants of the Escherichia coli N-degron pathway
Engineered orthogonal and obligate bacterial commensalism mediated by a non-standard amino acid
Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells
(Review) Deployment of engineered microbes: Contributions to the bioeconomy and considerations for biosecurity
Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights
Engineering posttranslational proofreading to discriminate nonstandard amino acids
