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<b><span style="font-size:14.0pt; line-height:200%">PhD Dissertation Defense of Yixi Wang</span></b><span style="font-size:11.0pt; line-height:200%"></span></p>
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<b>DATE:</b> Friday, December 3, 2021<span style="font-size:11.0pt; line-height:200%"></span></p>
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<b>TIME:</b> 2pm<span style="font-size:11.0pt; line-height:200%"></span></p>
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<b>COMMITTEE CHAIR:</b> Prof. Patrick Cirino<span style="font-size:11.0pt; line-height:200%"></span></p>
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<b>LOCATION:</b> <span style="color:black">Zoom link: </span><span style="font-size:11.0pt"></span></p>
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<span style="font-size:11.0pt"><a href="https://urldefense.com/v3/__https://uh-edu-cougarnet.zoom.us/j/91729008849?pwd=b1NLTXYySVpTT1lhejdkZW1wSmRNdz09__;!!LkSTlj0I!F1kA9oWNvQlrl-EOUREVt8q86ocGNBSylf9NDzg1y14wmPacGCf8owQMf_uJZfUJCcZajU8mG8015bJy8VWXogzsMHI$">https://uh-edu-cougarnet.zoom.us/j/91729008849?pwd=b1NLTXYySVpTT1lhejdkZW1wSmRNdz09</a></span></p>
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<b> </b></p>
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<b>TITLE: </b>Engineering <i>Escherichia coli </i>for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates<b></b></p>
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<b> </b><span style="font-size:11.0pt"></span></p>
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<b><span style="font-size:13.5pt"> </span></b></p>
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<b><span style="font-size:13.5pt"> </span></b></p>
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Short-chain alkanes are abundant carbon sources useful for the production of fuels and<span style="letter-spacing:.05pt">
</span>chemicals, but their efficient utilization is met with many technical and economic hurdles<span style="letter-spacing:.05pt">
</span>relating to their low energy density, high cost of transportation, and the catalytic challenges<span style="letter-spacing:.05pt">
</span>associated with selective and controlled functionalization of these hydrocarbons. While<span style="letter-spacing:.05pt">
</span>biological routes to alkane functionalization are promising, those requiring oxygen still suffer<span style="letter-spacing:.05pt">
</span>from<span style="letter-spacing:-.15pt"> </span>energy<span style="letter-spacing:-.15pt">
</span>and<span style="letter-spacing:-.15pt"> </span>carbon<span style="letter-spacing:-.15pt">
</span>inefficiencies<span style="letter-spacing:-.15pt"> </span>due<span style="letter-spacing:-.15pt">
</span>to<span style="letter-spacing:-.05pt"> </span>aerobic<span style="letter-spacing:-.2pt">
</span>respiration.<span style="letter-spacing:-.4pt"> </span>Therefore,<span style="letter-spacing:-.1pt">
</span>oxygen-independent<span style="letter-spacing:-2.85pt"> </span>alkane<span style="letter-spacing:-.1pt">
</span>utilization<span style="letter-spacing:-.05pt"> </span>has<span style="letter-spacing:-.05pt">
</span>the potential<span style="letter-spacing:-.05pt"> </span>to<span style="letter-spacing:-.05pt">
</span>offer<span style="letter-spacing:-.1pt"> </span>greater biocatalytic<span style="letter-spacing:-.1pt">
</span>efficiency.</p>
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In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs<span style="letter-spacing:.05pt">
</span>most commonly through the addition of fumarate to a sub-terminal carbon, producing an<span style="letter-spacing:.05pt">
</span>alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a<span style="letter-spacing:.05pt">
</span>catalytic<span style="letter-spacing:-.2pt"> </span>glycyl<span style="letter-spacing:-.1pt">
</span>radical<span style="letter-spacing:-.1pt"> </span>and<span style="letter-spacing:-.1pt">
</span>require<span style="letter-spacing:-.05pt"> </span>a<span style="letter-spacing:-.15pt">
</span>partner,<span style="letter-spacing:-.1pt"> </span>activating<span style="letter-spacing:-.1pt">
</span>enzyme<span style="letter-spacing:-.15pt"> </span>for<span style="letter-spacing:-.15pt">
</span>hydrogen<span style="letter-spacing:-.1pt"> </span>abstraction.<span style="letter-spacing:-.35pt">
</span>While<span style="letter-spacing:-2.85pt"> </span>many genes encoding putative alkylsuccinate synthases have been identified, primarily from<span style="letter-spacing:.05pt">
</span>nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported<span style="letter-spacing:-2.85pt">
</span>to<span style="letter-spacing:-.05pt"> </span>be<span style="letter-spacing:-.05pt">
</span>functionally expressed in<span style="letter-spacing:-.05pt"> </span>a<span style="letter-spacing:-.05pt">
</span>heterologous host organism.</p>
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This thesis described the functional expression of the (1-methylalkyl)succinate synthase<span style="letter-spacing:.05pt">
</span>(Mas) system from <i>Azoarcus sp</i>. strain HxN1 in recombinant <i>Escherichia coli</i>. In detail, an<span style="letter-spacing:.05pt">
</span>anaerobic system was established, different media, buffer and host strains were compared to<span style="letter-spacing:.05pt">
</span>determine suitable culturing conditions for Mas expression. The anaerobic biosynthesis of the<span style="letter-spacing:.05pt">
</span>expected products of fumarate addition to hexane, butane, and propane was confirmed and<span style="letter-spacing:.05pt">
</span>quantified by mass spectrometry. Maximum production of (1-methylpentyl)succinate was<span style="letter-spacing:.05pt">
</span>observed when <i>masC</i>, <i>masD</i>, <i>masE</i>, <i>masB</i>, and <i>masG
</i>are all present on the expression plasmid;<span style="letter-spacing:.05pt">
</span>omitting<span style="letter-spacing:-.1pt"> </span><i>masC<span style="letter-spacing:-.05pt">
</span></i>reduces<span style="letter-spacing:-.05pt"> </span>production<span style="letter-spacing:-.1pt">
</span>by<span style="letter-spacing:-.05pt"> </span>66%,<span style="letter-spacing:-.05pt">
</span>while<span style="letter-spacing:-.05pt"> </span>omitting<span style="letter-spacing:-.05pt">
</span>any<span style="letter-spacing:-.05pt"> </span>other<span style="letter-spacing:-.1pt">
</span>gene<span style="letter-spacing:-.15pt"> </span>eliminates<span style="letter-spacing:-.05pt">
</span>production.</p>
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</span>
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Meanwhile, deleting <i>iscR </i>(encoding the repressor of the <i>E. coli </i>iron-sulfur cluster operon)<span style="letter-spacing:.05pt">
</span>improves product titer, as did performing the biotransformation at reduced temperature (18<sup>o</sup>C),<span style="letter-spacing:-2.85pt">
</span>both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this<span style="letter-spacing:.05pt">
</span>enzyme system. Protein engineering was investigated and protein purification was prepared for<span style="letter-spacing:-2.85pt">
</span>further<span style="letter-spacing:-.1pt"> </span>enzyme<span style="letter-spacing:-.05pt">
</span>assay works.</p>
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Functional, heterologous expression of alkylsuccinate synthases which catalyze anaerobic<span style="letter-spacing:.05pt">
</span><span style="letter-spacing:-.05pt">alkane activation has proven </span>difficult. As the first case of functionally expressing alkylsuccinate<span style="letter-spacing:-2.85pt">
</span>synthase<span style="letter-spacing:-.1pt"> </span>in<span style="letter-spacing:-.05pt">
</span><i>E. coli</i>,<span style="letter-spacing:-.05pt"> </span>our<span style="letter-spacing:-.05pt">
</span>work<span style="letter-spacing:-.05pt"> </span>should prove<span style="letter-spacing:-.1pt">
</span>useful for<span style="letter-spacing:-.1pt"> </span>further<span style="letter-spacing:.05pt">
</span>enzymatic alkane<span style="letter-spacing:-.1pt"> </span>utilization.</p>
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