Presentation Information
[2Biocat-08]Rational Enzyme Design for the Biosynthesis of Non-natural Compounds
○Yutaro Mori1, Tomokazu Shirai2, Chiaki Ogino1 (1. Kobe University (Japan), 2. RIKEN (Japan))
Keywords:
Protein Engineering,Biorefinery,Enzyme Design
To replace fossil resource–derived chemical compounds with bio-based alternatives, it is essential to develop biotechnological methods aimed at achieving the bioproduction of non-natural compounds that have not yet been synthesized by microorganisms. It has long been generally recognized that enzymes possess substrate specificity, enabling them to catalyze reactions only with their intended substrates. However, in addition to such “specialist” enzymes, there are also many “generalist” enzymes in nature that exhibit broad substrate specificity. Some of these enzymes display reactivity toward previously unknown substrates, and by harnessing such enzyme promiscuity, several recent studies have successfully achieved the biosynthesis of non-natural compounds. Furthermore, the rational design of enzyme variants using in silico computational methods provides a powerful means to further advance these efforts. In this presentation, we present recent advances in the biosynthesis of natural and non-natural compounds, including 1,3-butadiene and other unsaturated hydrocarbons.
We designed a novel pathway by combining a cis,cis-muconic acid (ccMA)-producing pathway with engineered ferulic acid decarboxylase (FDC). FDC is a prenylated flavin mononucleotide (prFMN)-dependent enzyme that catalyzes the decarboxylation of α,β-unsaturated carboxylic acids to form terminal alkenes. Based on its substrate promiscuity, we selected ccMA as a precursor for butadiene production.
To enable efficient conversion of ccMA to butadiene, we performed rational enzyme design using in silico docking simulations. By introducing hydrophilic residues into the substrate-binding pocket, we improved interactions with the dicarboxylic substrate. As a result, several FDC mutants showed enhanced activity. The T395Q mutant exhibited a 109-fold increase, and the Y394H:T395Q double mutant achieved a 1,002-fold improvement compared to the wild-type enzyme. This approach allowed efficient identification of high-performance variants from a relatively small mutant library.By integrating the optimized FDC mutants with a ccMA-producing E. coli strain, we successfully achieved direct biosynthesis of 1,3-butadiene from glucose. Fermentation optimization revealed that oxygen levels critically affect both precursor production and enzyme activity. By controlling dissolved oxygen and applying fed-batch cultivation, we achieved a final titer of 2.1 g/L of 1,3-butadiene.However, accumulation of intermediates such as ccMA and pentadienoic acid (PA) indicated that the second decarboxylation step was rate-limiting. To overcome this, we developed a second FDC mutant optimized for PA conversion. Combining two specialized FDC variants significantly improved pathway efficiency, resulting in up to a several-fold increase in butadiene production and complete conversion of intermediates.
This study demonstrates the potential of integrating rational enzyme design with metabolic engineering to enable the biosynthesis of non-natural compounds and advance sustainable biomanufacturing. In addition to 1,3-butadiene described in this study, we have successfully produced other non-natural compounds using engineered protein variants. As the biosynthesis of previously inaccessible yet important compounds continue to be realized through such approaches, the development of a sustainable bioeconomy is expected to accelerate.
We designed a novel pathway by combining a cis,cis-muconic acid (ccMA)-producing pathway with engineered ferulic acid decarboxylase (FDC). FDC is a prenylated flavin mononucleotide (prFMN)-dependent enzyme that catalyzes the decarboxylation of α,β-unsaturated carboxylic acids to form terminal alkenes. Based on its substrate promiscuity, we selected ccMA as a precursor for butadiene production.
To enable efficient conversion of ccMA to butadiene, we performed rational enzyme design using in silico docking simulations. By introducing hydrophilic residues into the substrate-binding pocket, we improved interactions with the dicarboxylic substrate. As a result, several FDC mutants showed enhanced activity. The T395Q mutant exhibited a 109-fold increase, and the Y394H:T395Q double mutant achieved a 1,002-fold improvement compared to the wild-type enzyme. This approach allowed efficient identification of high-performance variants from a relatively small mutant library.By integrating the optimized FDC mutants with a ccMA-producing E. coli strain, we successfully achieved direct biosynthesis of 1,3-butadiene from glucose. Fermentation optimization revealed that oxygen levels critically affect both precursor production and enzyme activity. By controlling dissolved oxygen and applying fed-batch cultivation, we achieved a final titer of 2.1 g/L of 1,3-butadiene.However, accumulation of intermediates such as ccMA and pentadienoic acid (PA) indicated that the second decarboxylation step was rate-limiting. To overcome this, we developed a second FDC mutant optimized for PA conversion. Combining two specialized FDC variants significantly improved pathway efficiency, resulting in up to a several-fold increase in butadiene production and complete conversion of intermediates.
This study demonstrates the potential of integrating rational enzyme design with metabolic engineering to enable the biosynthesis of non-natural compounds and advance sustainable biomanufacturing. In addition to 1,3-butadiene described in this study, we have successfully produced other non-natural compounds using engineered protein variants. As the biosynthesis of previously inaccessible yet important compounds continue to be realized through such approaches, the development of a sustainable bioeconomy is expected to accelerate.
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