Engineering and characterization of hybrid carboxylic acid reductases

Kramer, Levi; Le, Xuan Ai; Hankore, Erome Daniel; Wilson, Mark A.; Guo, Jiantao; Niu, Wei

doi:10.1016/j.jbiotec.2019.08.008

Cited by 12 publications

(15 citation statements)

References 28 publications

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“…Recent work showed that retaining the natural T−R interface in bacterial hybrid CAR enzymes leads to higher k cat values . However, a version of NRPS‐4 maintaining the T−R didomain from XtvB (NRPS‐5) results in an approximately 6.5‐fold lower production of both derivatives.…”

Section: Resultsmentioning

confidence: 99%

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains

et al. 2020

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Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2-electron reduction of the enzyme-bound thioester releasing the generated peptides as C-terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde-generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.

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Section: Resultsmentioning

confidence: 99%

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains

et al. 2020

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“…Although successful studies on domain exchange in NRPS systems (43,44) and CAR systems (19,45) have been reported, replacing the native R or PCP-R domains with the ones from NRPS enzymes sharing high sequence similarity resulted in the complete loss of the activity of MmCAR (SI Appendix, Fig. S12), which might be due to an inappropriate domain boundary (SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The efficient reduction of MCFAs by the engineered CAR enzymes will enable the synthesis of versatile aldehyde intermediates with broad applications for further production of, for example, the corresponding alka(e)nes and FA acyl esters. Although CAR enzymes were previously modified by different strategies (39,42,45), this work is unique in comprehensively engineering the CAR enzyme for more selective biosynthesis of MCFOHs. In addition, our engineering strategies may inspire and promote the engineering of other complex multidomain enzymes.…”

Section: Discussionmentioning

confidence: 99%

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast

Zhu

Gradischnig

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme’s substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.

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“…18−20 The modular structure of CAR enables the application of its individual domains in catalysis and the engineering of hybrid enzymes through domain shuffling. 21,22 Due to the broad substrate specificity, CAR can potentially be applied to the syntheses of a wide range of pharmaceutical and industrial chemicals. Meanwhile, CARs often have suboptimal catalytic activities on nonnative substrates, which have prompted large enzyme engineering efforts.…”

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confidence: 99%

“…Carboxylic acid reductase (CAR) catalyzes the NADPH-consuming direct reduction of a carboxylic acid into its corresponding aldehyde through an in situ activation mechanism that is enabled by a post-translationally grafted phosphopantetheinyl group on a serine residue (Figure A). , Over 40 bacterial and fungal CARs have been identified and characterized for in vitro or in vivo syntheses of aldehydes either as end products or as intermediates for subsequent production of alcohols, amines, and hydrocarbons. − Structural and mechanistic studies of CAR reveal a highly organized catalytic process that is orchestrated by sequentially shuttling substrates through an adenylation (A), a phosphopantetheinylation (P), and a reduction (R) domain. − The modular structure of CAR enables the application of its individual domains in catalysis and the engineering of hybrid enzymes through domain shuffling. , Due to the broad substrate specificity, CAR can potentially be applied to the syntheses of a wide range of pharmaceutical and industrial chemicals. Meanwhile, CARs often have suboptimal catalytic activities on nonnative substrates, which have prompted large enzyme engineering efforts. − A high-throughput assay was established to quantify the aldehyde products of CAR-catalyzed reactions .…”

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confidence: 99%

Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy

Kramer

Rodríguez

et al. 2020

ACS Synth. Biol.

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Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. We report the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified Escherichia coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on 2-methoxybenzoate and adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries. Obtained mutants were successfully applied to whole-cell conversions of adipate into 1,6-hexanediol, a C6 monomer commonly used in polymer industry.

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Engineering and characterization of hybrid carboxylic acid reductases

Cited by 12 publications

References 28 publications

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast

Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy

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