Browsing by Subject "Protein engineering"

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Biosynthetic routes to short-chain carboxylic acids and their hydroxy derivatives
    (2013-07) Dhande, Yogesh Khemchandra
    The current drive for sustainable routes to industrial chemicals and energy due to depleting fossil reserves has motivated research in the field of biotechnology. Biomass is abundant on earth and photosynthesis, if utilized properly, can lead the way to a sustainable carbon cycle. The recent advances in metabolic engineering, DNA sequencing, and protein engineering are driving research to achieve this goal. In this work, the native leucine and isoleucine biosynthetic pathways in Escherichia coli were expanded for the synthesis of pentanoic acid (PA) and 2-methylbutyric acid (2MB) respectively. Several aldehyde dehydrogenases and 2-ketoacid decarboxylases were studied for the conversion of ketoacids into respective carboxylic acids. The optimal combinations of these enzymes enabled production of 2.6 g/L 2MB with IPDC-AldH and 2.6 g/L of PA with IPDC-KDHba in shake-flask fermentations. The extension of these pathways to synthesize value-added chemicals like hydroxyacids was attempted. The cytochrome P450 BM-3 enzyme was engineered to enhance its ability to oxidize unactivated C-H bonds in the short-chain carboxylic acids. Nine mutants were created by rational. The mutant L437K showed 20 to 30-fold higher activity compared to the wild-type. A screening strategy was developed to evolve isobutyrate-hydroxylating activity in P450s based on the valine degradation pathway in Pseudomonas aeruginosa. As a growth-based selection strategy, it will allow screening of a large library. This work expands the library of chemicals that can be produced biologically as a step forward to the sustainable future.
  • Thumbnail Image
    Item
    Enzyme catalyzed perhydrolysis, molecular basis and application
    (2011-10) Yin, Delu (Tyler)
    Enzyme catalyzed perhydrolysis converts a carboxylic acid or ester to a peracid. In the former reaction, the amount of peracid generated is thermodynamically controlled (Keq = 3) – while in the latter, the reaction is kinetically controlled, thus a higher concentration of peracid can be generated. Enzymes that catalyze perhydrolysis of carboxylic acids share high sequence similarity and are thought to use an esterase-like mechanism. Alternatively, carboxylic acids can also use a non-covalent mechanism, such as those used by hydroxynitrile lyases. To test whether carboxylic acid perhydrolases use an esterase-like mechanism, we identify a key covalent intermediate by mass spectrometry that can be attributed to an esterase-like mechanism but not a non-covalent mechanism. We also find that carboxylic acid perhydrolases are good catalysts for hydrolysis of peracetic acid, suggesting that their natural role is to degrade peracids generated as by-products in a living organism. Next, we determine how perhydrolases increase the rate of perhydrolysis. Carboxylic acid perhydrolases increase the rate of perhydrolysis by either increasing the selectivity for hydrogen peroxide or lower the activation barrier towards acylenzyme formation. We measure the selectivity of hydrogen peroxide using wild-type Pseudomonas fluorescens esterase (PFE) and L29P PFE (a model carboxylic acid perhydrolase). The L29P PFE variant is less selective for hydrogen peroxide than the wild-type despite having higher perhydrolysis activity. We measure the rate of acyl-enzyme formation using isotope exchange of acetic acid in H218O/H216O. The L29P PFE variant catalyzes the isotope exchange rate faster than the wild-type. Thus, carboxylic acid perhydrolases favor the formation of acyl-enzyme from carboxylic acids. We find that carboxylic acid perhydrolase (L29P PFE) does not catalyze ester perhydrolysis for accumulating high concentrations of peracetic acid. Instead, wild-type PFE and a new variant, F162L PFE accumulate up to 130 mM of peracetic acid. We measure kinetic parameters and show that hydrolysis of peracetic acid limits maximum accumulation. The F162L PFE variant minimizes hydrolysis of peracetic acid by lower ing the Km and increasing the kcat for ethyl acetate hydrolysis. The kinetic parameters are also used to predict the maximum amount of peracetic acid that can be accumulated. The F162L PFE variant is used to improve the efficiency of lignocellulose pretreatment from a previously published result using wild-type PFE. Enzymatically generated peracetic acid reacts converts lignin into smaller and more soluble lignin pieces. The chemoenzymatic process is further improved by forming peracetic acid in a biphasic layer which allows the reuse of enzyme. The pretreatment reaction conditions were also optimized by increasing the temperature to 60 °C and reducing the reaction time to 6 hours.
  • Thumbnail Image
    Item
    Evolution-guided Engineering of Alpha/Beta Hydrolases
    (2017-06) Jones, Bryan
    This work applies principles from evolution to engineering enzyme properties. Specifically, by examining the phylogeny and evolved sequence diversity in a group of α/β-hydrolase fold enzymes from plants, we are able to engineer proteins with broader chemoselectivity, altered enantioselectivity, and increased stability. A number of ancestral α/β-hydrolases fold proteins were reconstructed in one set of experiments. These were more likely than related modern proteins to have relaxed chemoselectivities and, in one case, was more useful for synthesizing medicinally important molecules. Relative to modern enzymes, ancestral enzymes near functional branch points could catalyze more esterase and hydroxynitrile lyase reactions, as well as a number of other types of reactions: decarboxylation, Michael addition, γ-lactam hydrolysis, and 1,5-diketone hydrolysis. This finding helps to demonstrate the important role that enzyme promiscuity plays in the evolution of new enzymes. Additional experiments and structural analysis on one of these reconstructed ancestral enzymes, the early hydroxynitrile lyase HNL1 found that it is both more thermostable and more promiscuous than its modern relatives, HbHNL and MeHNL. X-ray crystallographic studies revealed, counterintuitively, that larger amino acids in the active site of the ancestor actually increased the size of the substrate binding pocket relative to modern relatives. To take advantage of the promiscuity observed in HNL1, it was used in the asymmetric synthesis of a precursor for the important pharmaceutical propranolol. Another set of experiments altered enantioselectivity by making phylogenetically informed mutations. The active sites from two related hydroxynitrile lyases, HbHNL and AtHNL, were modified to resemble their last common ancestor. This resulted in altered enantioselectivity, and in the case of AtHNL, reversed enantioselectivity. Surprisingly modeling suggested that some of these mutants use a previously undescribed mechanism. This may have been the extinct ancestral mechanism that served as an evolutionary stepping stone that allowed descendant lineages to diverge to either the S-HNL mechanism used by HbHNL, or the R-HNL mechanism used by AtHNL. A final set of experiments used a variety of methods to identify stabilizing mutations in another plant α/β-hydrolase, SABP2. All of the methods were able to identify stabilizing mutations. The most stabilizing mutations were identified by methods that used no structural information. Random mutagenesis identified highly stabilizing mutations, but required screening thousands of mutants. The most efficient approaches were found to be those that used sequence information from either one stable homolog, or the consensus of many homologs, to identify potential stabilizing mutations. Residues that evolution has conserved are often important for stabilizing a protein. We created a software application, Consensus Finder, to automate the process of identifying stabilizing mutations by consensus.
  • Thumbnail Image
    Item
    Improving the unnatural and promiscuous nitroaldolase activity of modern and ancestral HNLs
    (2016-05) Lim, Huey Yee
    Enzymes in superfamilies such as the α/β-hydrolase fold superfamily have similar structures and active sites because they evolved from a common ancestor. Since enzymes within a superfamily catalyze different reactions, the common ancestor may have been a promiscuous catalyst. Our hypothesis is that promiscuous ancestral enzymes may be better starting points to obtain new catalytic activities compared to modern enzymes. To test this hypothesis, I tried to improve the promiscuous and unnatural nitroaldol activity in both a modern and an ancestral hydroxynitrile lyase using semi rational and random approaches. My results showed that the ancestral enzyme was not a better starting point compared to the modern enzyme after the modern enzyme was stabilized by a single mutation.
  • Thumbnail Image
    Item
    Synthetic Biology Approach to New Sustainable Materials
    (2018-03) McClintock, Maria
    Rapid industrialization and an abundance of cheap petroleum fueled the production and development of a great variety of synthetic polymers in the twentieth century. Over the past 60 years, these materials have become a part of the fabric of modern life. They are pervasive; from the polyurethanes in our cars and furniture, to the polypropylene in a state of the art medical implant, we rely on synthetic polymers every day of our lives, to accomplish tasks both trivial and critical. However, production of these chemicals from petroleum feedstocks is unsustainable and damaging to the environment. One potential option for more sustainable production is to use microbial fermentation to generate industrial chemicals. Microbial fermentation offers the opportunity to produce chemicals from biomass, making the compounds produced renewable feedstocks. Furthermore, the conditions used for, and byproducts produced from, microbial fermentation are benign. However, many microbial monomers face challenges in terms of economic viability and utility. With this in mind, my PhD research has focused on developing engineering systems for production of novel and viable monomers, as well as implementation of biological monomers for material applications. Using metabolic engineering, I have implemented the first heterologous pathway for production of dipicolinic acid, an aromatic di-acid that could be used as a biological replacement for isophthalic acid, a major component of the performance polymers Nomex® and polybenzimidazole, as well as a useful additive in many other polymers. By working with collaborators, I have used ancestral reconstruction to improve the production of anhydromevalonolactone, a monomer that can serve as a sustainable alternative to poly(acrylate). Finally, I have worked to establish a new platform for developing zwitterionic materials. In this project, we were able to engineer E. coli to produce N-acetyl-serine, a compound that can be dehydrated to form an acrylate monomer with a protected amine. I then polymerized this monomer with styrene and developed zwitterionic coatings that show improved resistance to cell adhesion. Overall, my work has contributed to the development of new metabolic pathways and material applications of biologically-derived monomers.