Engineering and manufacturing of probiotic E. Coli to treat metabolic disorder

The fields of synthetic biology and microbiome research developed greatly over the last decade. The convergence of those two disciplines is now enabling the development of new therapeutic strategies, using engineered microbes that operate from within the gut as living medicines. Inborn errors of metabolism represent candidate diseases for these therapeutics, particularly those disorders where a toxic metabolite causing a syndrome is also present in the intestinal lumen. Phenylketonuria (PKU), a rare inherited disease caused by a defect in phenylalanine hydroxylase (PAH) activity, is one such disease and is characterized by the accumulation of systemic phenylalanine (Phe) that can lead to severe neurological deficits unless patients are placed on a strict low-Phe diet. As an alternative treatment, Escherichia coli Nissle (EcN), a well-characterized probiotic, was genetically modified to efficiently import and degrade Phe (SYNB1618). The coupled expression of a Phe transporter with a Phe ammonia lyase (PAL) allows rapid conversion of Phe into trans-cinnamic acid (TCA) in vitro, which is then further metabolized by the host to hippuric acid (HA) and excreted in the urine. Experiments conducted in the enu2-/- PKU mouse model showed that the oral administration of SYNB1618 is able to significantly reduce blood Phe levels triggered by subcutaneous Phe injection. Decreases in circulating Phe levels were associated with proportional increases in urinary HA, confirming that Phe metabolism was caused by the engineered pathway in SYNB1618. Subsequent studies have shown that SYNB1618 is similarly operative in a non-human primate model, providing a translational link to inform future human clinical studies. Consistent with preclinical studies, recent Phase 1/2a clinical data demonstrate that oral administration of SYNB1618 resulted in significant dose-dependent production of biomarkers specifically associated with SYNB1618 activity, demonstrating proof-of-mechanism of this cell therapy

Engineering of probiotic E.coli to treat metabolic disorders

The fields of synthetic biology and microbiome research developed greatly over the last decade. The convergence of those two disciplines is now enabling the development of new therapeutic strategies, using engineered microbes that operate from within the gut as living medicines. Inborn errors of metabolism represent candidate diseases for these therapeutics, particularly those disorders where a toxic metabolite causing a syndrome is also present in the intestinal lumen. Phenylketonuria (PKU), a rare inherited disease caused by a defect in phenylalanine hydroxylase (PAH) activity, is one such disease and is characterized by the accumulation of systemic phenylalanine (Phe) that can lead to severe neurological deficits unless patients are placed on a strict low-Phe diet. As an alternative treatment, Escherichia coli Nissle (EcN), a well-characterized probiotic, was genetically modified to efficiently import and degrade Phe (SYN-PKU). The coupled expression of a Phe transporter with a Phe ammonia lyase (PAL) allows rapid conversion of Phe into trans-cinnamic acid (TCA) in vitro, which is then further metabolized by the host to hippuric acid (HA) and excreted in the urine. Experiments conducted in the enu2-/- PKU mouse model showed that the oral administration of SYN-PKU is able to significantly reduce blood Phe levels triggered by subcutaneous Phe injection. Decreases in circulating Phe levels were associated with proportional increases in urinary HA, confirming that Phe metabolism was caused by the engineered pathway in SYN-PKU. Subsequent studies have shown that SYN-PKU is similarly operative in a non-human primate model, providing a translational link to inform future human clinical studies. In addition to SYN-PKU, a second EcN strain was genetically engineered to rapidly import and degrade branched-chain amino acids (BCAAs) for the treatment of maple syrup urine disease (SYN-MSUD). MSUD, similar to PKU, is a rare genetic disorder caused by a defect in branched-chain ketoacid dehydrogenase activity leading to the toxic accumulation of BCAAs, particularly leucine, and their ketoacid derivatives. The controlled expression in SYN-MSUD of two BCAA transporters, a leucine dehydrogenase, a ketoacid decarboxylase and an alcohol dehydrogenase, result in the efficient degradation of BCAAs into branched-chain alcohols. In a mouse model of MSUD, the oral delivery of SYN-MSUD suppressed the increase in blood BCAAs level induced by a high-protein challenge and prevented the associated moribund phenotype, as measured by locomotor activity. In conclusion, the therapeutic effects observed with SYN-PKU and SYN-MSUD in pre-clinical studies support the further evaluation of engineered microbes as promising approaches for serious inborn errors of metabolism

Engineering a highly enantioselective HRP by directed evolution

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009.Cataloged from PDF version of thesis.Includes bibliographical references.There is an ever-growing demand for enantiopure chemical compounds, particularly new pharmaceuticals. Enzymes, as natural biocatalysts, possess many appealing properties as robust asymmetric catalysts for synthetic chemistry. However, their enantioselectivity toward most synthetically useful, non-natural substrates is typically low. Therefore, improving enzymatic enantioselectivity toward a given substrate is a practically important but arduous task. Here we report a highly efficient selection method for enhanced enzymatic enantioselectivity based on yeast surface display and fluorescenceactivated cell sorting (FACS). By exploiting the aforementioned method, in just three rounds of directed evolution we both greatly increased (up to 30-fold) and also reversed (up to 70-fold) the enantioselectivity of the commercially useful enzyme, horseradish peroxidase (HRP), toward a chiral phenol. In doing so, we discovered that mutations close to the active site not only preserve HRP catalytic activity but impact its enantioselectivity far greater than distal mutations. We thus examined how a single mutation near the active site (Argl78Glu) greatly enhances (by 25-fold) the enantioselectivity of yeast surface-bound HRP. Using kinetic analysis of enzymatic oxidation of various substrate analogs and molecular modeling of enzyme-substrate complexes, this enantioselectivity enhancement was attributed to changes in the transition state energy due to electrostatic repulsion between the carboxylates of the enzyme's Glu- 178 and the substrate's D enantiomer. In addition, the effect of yeast surface immobilization and influence of a fluorescent dye on controlling the enantioselectivity of the discovered HRP variants was investigated. Soluble variants were also shown to have marked improvements in enantioselectivity, which were rationalized by computational docking studies.by Eugene Antipov.Ph.D

How a Single-Point Mutation in Horseradish Peroxidase Markedly Enhances Enantioselectivity

The effect of all possible mutations at position 178 on the enantioselectivity of yeast surface-bound horseradish peroxidase (HRP) toward chiral phenols has been investigated. In contrast to their wild-type predecessor, most HRP mutants are enantioselective, with the Arg178Glu variant exhibiting the greatest, 25-fold, (S)/(R) preference. Using kinetic analysis of enzymatic oxidation of various substrate analogues and molecular modeling of enzyme−substrate complexes, this enantioselectivity enhancement is attributed to changes in the transition state energy due to electrostatic repulsion between the carboxylates of the enzyme’s Glu178 and the substrate’s (R)-enantiomer.National Institutes of Health (U.S.) (Grant R01-GM66712

DIMACS Series in Discrete Mathematics and Theoretical Computer Science In vitro Selection for a Max 1s DNA Genetic Algorithm

Abstract. Genetic algorithms, DNA computing, and in vitro evolution are brie y discussed. Elements of these are combined into laboratory procedures, and preliminary results are shown. The traditional test problem for genetic algorithms called the MAX 1s problem is addressed. Preliminary experimental results indicate successful laboratory \separation by tness " of DNA encoded candidate solutions. 1

A design for DNA computation of the OneMax problem

Elements of evolutionary computation and molecular biology are combined to design a DNA evolutionary computation. The traditional test problem for evolutionary computation, OneMax problem is addressed. The key feature is the physical separation of DNA strands consistent with OneMax "fitness.&quot

DNA Computing Implementing Genetic Algorithms

Genetic algorithms, DNA computing, and in vitro evolution are briefly discussed. Elements of these are combined into laboratory procedures, and preliminary results are shown. The traditional test problem for genetic algorithms called the MAX 1s problem is addressed

In vitro Selection for a Max 1s DNA Genetic Algorithm

. Genetic algorithms, DNA computing, and in vitro evolution are brie#y discussed. Elements of these are combined into laboratory procedures, and preliminary results are shown. The traditional test problem for genetic algorithms called the MAX 1s problem is addressed. Preliminary experimental results indicate successful laboratory #separation by #tness" of DNA encoded candidate solutions. 1. Introduction Evolution is a concept of obtaining adaptation through the interplay of selection and diversity. Analogies from evolution have been used in both computing and molecular biology. These two areas are called respectively #evolutionary computation " and #in vitro evolution." From the beginning of DNA based computing to the present there have been calls #11, 22, 28# to consider carrying out evolutionary computations using genetic materials in vitro. In this paper we identify elements of evolutionary computations and in vitro evolution that we recommend combining to address three simp..

In Vitro Selection for a OneMax DNA Evolutionary Computation

Aspects of Evolutionary Computation, DNA computing, and in vitro evolution are combined in proposed laboratory procedures. Preliminary experimental results are shown. The traditional test problem for Evolutionary Computation known as the OneMax problem is addressed. The preliminary experimental results indicate successful laboratory "separation by fitness" of DNA encoded candidate solutions