23 research outputs found
Engineered Vesicles for Perchlorate Degradation
This study demonstrates the use of engineered vesicles to reduce perchlorate. Specifically, cell-free extracts containing perchlorate reductase and chlorite dismutase enzymes were encapsulated in a triblock copolymer vesicle functionalized with the outer membrane porin OmpF. The porin allows for perchlorate transport into the vesicles, inside which the encapsulated enzymes transform perchlorate to chloride. Perchlorate reduction was quantified using a methyl viologen colorimetric technique. The vesicle solutions had perchlorate-reducing activities ranging from 35-45 units per liter. This work shows that vesicles can provide a mechanism to utilize environmentally-relevant biological enzymes. When incorporated into a vesicle, the enzymes could be used outside of environmental conditions where they would normally be expressed by natural bacteria
Understanding the role of histidine in the GHSxG acyltransferase active site motif: evidence for histidine stabilization of the malonyl-enzyme intermediate.
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Towards Biochemical Conversion of CO2 to Higher Value Chemicals Using Enzyme Design and Engineered Polyketide Synthases
Polyketide synthases produce a remarkable number of diverse products. Many medicines have been produced either directly from polyketide products found in nature, or with limited modification via organic semisynthesis. However, we remain in the early days of engineering the biosynthesis of polyketide synthases. Despite being an active area of research for over 20 years, no commercial application of an engineered polyketide synthase exists. Rapidly advancing technologies like next generation sequencing, DNA synthesis, liquid handling automation and mass spectrometry give us tools to do things faster, and in new ways. In the work presented in this doctoral dissertation I will describe how I have applied some of these new tools to advance our understanding of polyketide synthase biochemistry, as well develop catalysts for new small molecules using engineered polyketide synthases.Chapter 1 begins with a discussion of the current state of the art and challenges associated with engineering these enzymes as well as offering opinions about routes forward to narrow the considerable gap between the promise and reality of engineered PKSs.Chapter 2 presents my work applying mass spectrometry to study the formolase enzyme, which is part of an engineered carbon fixation pathway. This work further characterized the product profile of the formolase and describes alternative routes of carbon assimilation from the pathway. Once this pathway is further evolved, it could supply reduced carbon to engineered polyketide synthase proteins. Chapter 3 explores the role of the histidine in the GHSxS active site motif of acyl transferase domains. The role of this residue in the literature was unclear, with some reports suggesting that this histidine could even serve as an alternative nucleophile. This study showed that removal of this histidine increases hydrolysis, suggesting it is important for the stabilization of acyl-enzyme intermediates. Chapter 4 probes the mechanism for gem-dimethylation in complex polyketide biosynthesis. Specifically, this report shows that methylation can precede condensation in polyketide biosynthesis, contrary to the canonical understanding. Chapter 5 describes the development of the yersiniabactin PKS as a gem-dimethylation catalyst. Specifically, the engineering of this enzyme towards production of several useful gem-dimethylated compounds, as well as the parallel engineering of a thioesterase domain to release gem-dimethylated intermediates is described. The final chapter will summarize this work, as well as suggesting future efforts to further enable the engineering of polyketide assembly lines
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Understanding the role of histidine in the GHSxG acyltransferase active site motif: evidence for histidine stabilization of the malonyl-enzyme intermediate.
Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. The ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate
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Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.
Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS' first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to "debug" PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry
PaR-PaR Laboratory Automation Platform
Labor-intensive multistep biological tasks, such as the
construction
and cloning of DNA molecules, are prime candidates for laboratory
automation. Flexible and biology-friendly operation of robotic equipment
is key to its successful integration in biological laboratories, and
the efforts required to operate a robot must be much smaller than
the alternative manual lab work. To achieve these goals, a simple
high-level biology-friendly robot programming language is needed.
We have developed and experimentally validated such a language: Programming
a Robot (PaR-PaR). The syntax and compiler for the language are based
on computer science principles and a deep understanding of biological
workflows. PaR-PaR allows researchers to use liquid-handling robots
effectively, enabling experiments that would not have been considered
previously. After minimal training, a biologist can independently
write complicated protocols for a robot within an hour. Adoption of
PaR-PaR as a standard cross-platform language would enable hand-written
or software-generated robotic protocols to be shared across laboratories
Understanding the Role of Histidine in the GHSxG Acyltransferase Active Site Motif: Evidence for Histidine Stabilization of the Malonyl-Enzyme Intermediate
<div><p>Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. The ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.</p></div
Acylation and transacylation activities of WT, H640A, S641A, and H640A+S641A.
<p>A) Transacylation activity as observed by high-resolution LC/QTOFMS. Data for the variants (WT, H640A, S641A, H640A+S641A) incubated with malonyl-CoA and a wildtype negative control without malonyl-CoA are shown (WT – Mal-CoA). B) Formation of malonyl-AT complex for wildtype (monoisotopic peptide m/z  =  1000.9903, z  =  4) and H640A (monoisotopic peptide m/z  =  984.4849, z  =  4) as observed by high-resolution LC/QTOFMS. Data for wildtype and H640A (WT, H640A) incubated with malonyl-CoA as well as a wildtype negative control without malonyl-CoA (WT – Mal-CoA) are shown. The mass for each chromatogram is shown in parenthesis to the right. Additional details on chromatogram preparation in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109421#pone.0109421.s001" target="_blank">Methods S1</a>.</p
Hydrolysis rates for yersiniabactin AT mutants at a concentration of 35 µM malonyl-CoA.
<p>n.d.  =  below limit of detection; error bars are the standard deviation of 3 replicates.</p><p>Hydrolysis rates for yersiniabactin AT mutants at a concentration of 35 µM malonyl-CoA.</p