Research review paperCoenzyme A thioester-mediated carbon chain elongation as a paintbrush to draw colorful chemical compounds
Introduction
CC bond forming reactions are critical steps in synthetic organic chemistry to construct the carbon framework of organic molecules.(Fesko and Gruber-Khadjawi, 2013; Lee et al., 2019) As an alternative, biological reactions carry out carbon chain elongation through the addition of various extender units to a carbon backbone have been exploited for the production of phenylpropanoids (Kallscheuer et al., 2016; Zhou et al., 2019), antibiotics (Go et al., 2015), organic acids (Zhao et al., 2018a; Zhao et al., 2018b) and alcohols(Ohtake et al., 2017; Yu et al., 2018). Furthermore, improving the diversity of biosynthesis will contribute to solving the decreasing energy resources and increasingly severe environmental pollution problems. Cellular anabolism beginning from a small starter molecule (such as CO2, formyl-CoA, acetyl-CoA and malonyl-CoA) and the addition of various carbon backbones at different positions is a promising strategy that leads to numerous chemicals (Cheong et al., 2016). Consequently, bio-based carbon chain elongation strategies are becoming increasingly popular.
In general, coenzyme A (CoA) and its thioesters are possibly the most common intermediate metabolites observed in cellular processes. Approximately 4% of the enzymes in all living cells are CoA dependent enzymes (Harijan, 2017). Hence, CoA thioester-mediated carbon rearrangements are very popular in nature (Cheong et al., 2016; Haapalainen et al., 2006). The structure and high nucleophilicity of CoA allow it to act as a nucleophile and react with carboxyl-containing molecules to generate its thioester derivatives (Gulick, 2009). Subsequently, the α‑carbon of CoA thioesters can be activated into α-carbanion by thiolases (Meriläinen et al., 2009; Meriläinen et al., 2008) and polyketide synthases (PKSs) (Heath and Rock, 2002). The activated α-carbanion could react with another molecule of a CoA thioester to extend the carbon chain. In general, the two thioesters used in the carbon chain elongation are defined as the “primer” and “extender” unit, respectively (Fig. 1) (Cheong et al., 2016). The extender unit supplies the α-carbanion to perform the nucleophilic attack on the acyl group of the primer unit. Consequently, a vast array of chemicals can be synthesized by combining different primers and extender units with Claisen-condensation enzymes (Fig. 1).
As described above, the enzymes that catalyze the Claisen-condensation reaction between the primer and extender units usually act as “gatekeepers” to control the diversity of the products formed. Generally, these enzymes can be simply separated into four categories: Thiolases, PKSs, carboxylases, and the other enzymes. The compatible substrates of these enzymes span a broad range from CO2 and formyl-CoA to bulky acyl/aryl-CoA (Chou et al., 2019; Haapalainen et al., 2006; Musiol-Kroll and Wohlleben, 2018; Palmer and Alper, 2019; Peter et al., 2015). Hence, a diverse range of either natural or non-natural products can be synthesized based on these enzymes. Specifically, when acetyl-CoA, malonyl-CoA and their analogues are used as extender units, iterative Claisen-condensation (ICC) reactions can further extend the diversity of the products by continuously adding them onto the end of the primer units (Kallscheuer et al., 2017). To terminate the ICC reaction, various termination pathways are coupled and to further enhance the diversity of the products formed. Overall, combining different primer and extender units as well as Claisen-condensation enzymes through a variety of ICC pathways and termination pathways can produce a large number of natural or non-natural products (Fig. 1).
Although there are a number of reviews on CC bond formation, few of them have focused on carbon chain elongation using CoA thioesters (Fesko and Gruber-Khadjawi, 2013; Lee et al., 2019). Herein, we thoroughly examine the current status of CoA thioester-mediated carbon chain elongation. We describe the enzymes, pathways and strategies used to produce a diverse range of products. Likewise, we also emphasize the strategies used to improve the titer of the extender units as they relate to cell growth and the final titer of the products (Krivoruchko et al., 2015; Matsumoto et al., 2017; Takamura and Nomura, 1988).
Section snippets
CoA and its thioesters
CoA is an essential and ubiquitous cofactor in all living cells, which plays a crucial role in cellular catabolism and anabolism by controlling the enzymes activity and intermediate metabolism (Gout, 2018; Theodoulou et al., 2014). The stability of CoA and high reactivity of CoA thioesters determine their important functions in metabolic processes. Herein, we describe the biochemical properties and synthetic pathways of CoA and its thioester derivatives.
Enzymes in CoA thioester-mediated carbon chain elongation
Acyl/aryl-CoAs can be separated into two categories, primer and extender units. Based on enzyme-specific Claisen-condensation reactions, the primer and extender units can be used in a tremendous number of combinations. Hence, the enzymes involved in CoA thioester-mediated carbon chain elongation are crucial toward generating a diverse range of products (Fig. 1). Herein, we highlight the catalytic mechanisms and engineering strategies of these enzymes.
Metabolic engineering to improve the diversity and productivity of the products
By protein engineering, novel enzyme mutants were created, which enable generate greater product diversity. Furthermore, the concentration of the extender units, especially acetyl-CoA, malonyl-CoA and some branched extender units, directly determined the titer of target compounds (Krink-Koutsoubelis et al., 2018; Takamura and Nomura, 1988). Herein, we describe the strategies used to enhance the diversity and productivity of the target compounds.
Conclusion and prospects
Overall, CoA thioesters are core compounds in carbon chain elongation reactions. From starting molecules, such as CO2, acetyl-CoA and malonyl-CoA, a vast array of functionalized products can be synthesized based on flexible carbon chain elongation and termination pathways. To further enhance the diversity and productivity, protein and metabolic engineering strategies have been thoroughly explored to modify the substrate scope, enhance the catalytic properties, maintain the CoA balance and
Declaration of Competing Interest
The authors declare no competing financial interests.
Acknowledgements
This work was supported by the National Key R&D Program of China (2019YFA0905502), the National Natural Science Foundation of China (31900066, 21877053), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-24), the Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP) and the Fundamental Research Funds for the Central Universities (JUSRP51705A, JUSRP12056).
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