Directed evolution of the 3-hydroxypropionic acid production pathway by engineering aldehyde dehydrogenase using a synthetic selection device
Introduction
3-Hydroxypropionic acid (3-HP) is an important platform chemical that can be used as a feedstock for production of polymer and other chemicals, including acrylic acid and acrylamide (Jiang et al., 2009, Kumar et al., 2013). 3-HP can be synthesized using a variety of chemical methods, but it is not suitable for industrial production owing to the high cost of the precursors and processes, and environmental incompatibility (Della Pina et al., 2011); therefore, the biological synthesis of 3-HP has attracted much attention.
A number of efforts have been made to synthesize 3-HP via biological methods (Borodina et al., 2015, Lan et al., 2015, Li et al., 2016, Rathnasingh et al., 2012, Sabet-Azad et al., 2015, Song et al., 2016). In particular, the production of 3-HP from glycerol in recombinant microorganisms as shown in Fig. 1A has been revealed to be efficient by development of high-productivity strains and a strain with the highest productivity of 1.8 g/L/h has been developed so far (Chu et al., 2015, Jung et al., 2014, Kim et al., 2014, Ko et al., 2012, Lim et al., 2016, Sankaranarayanan et al., 2014). However, further challenges remain because of the problems with enzymes in the 3-HP biosynthetic pathway (Kumar et al., 2013, Sankaranarayanan et al., 2014, Zhou et al., 2015). One of the important issues is the insufficient activity of aldehyde dehydrogenase (ALDH); alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH) has been the most successful for production of 3-HP from glycerol (Chu et al., 2015; Lim et al., 2016; Park et al., 2017; Saxena et al., 2009; Son et al., 2017). The low activity of KGSADH causes accumulation of toxic 3-hydroxypropionaldehyde (3-HPA), which not only inhibits cell growth but also reduces enzyme activity, resulting in detrimental effects on 3-HP production (Barbirato et al., 1996a, Barbirato et al., 1996b, Celińska, 2010, Kumar et al., 2013).
Directed evolutionary strategy with a well-designed library and an efficient screening or selection method has been the most successful in engineering enzymes (Belsare et al., 2017, Packer and Liu, 2015, Woolston et al., 2013). The low probability of incidence of a positive mutant in a randomly generated library made it necessary to examine a large number of clones, which generally causes an experimental burden of repeating assays (Dietrich et al., 2010). However, advances in analysis of protein structures and functions have help to construct designed libraries, which has increased the probability to isolate positive clones (Di Lorenzo et al., 2007, Jennewein et al., 2006, Jeong et al., 2012, Park et al., 2017, Yamanishi et al., 2012). Nevertheless, efficient screening or selection methods are still required to apply the directed evolution strategy, as the size of the library is usually too large to perform individual analysis even though it was constructed based on structural and functional information of the target proteins. This indicates that development of efficient methods to isolate positive mutants from libraries is the most critical part for directed evolution of enzymes, and therefore various high-throughput screening or selection methods have been proposed and developed according to the target properties of enzymes (Dietrich et al., 2010, Woolston et al., 2013). In addition, to engineer an enzyme in the metabolic pathway, the method should be able to associate the enzyme activity with the production of target metabolites rather than the reaction that the enzyme is involved, as the ultimate goal of modifying the enzyme in the biosynthetic pathway is to improve metabolite production.
For these reasons, genetically encoded biosensors composed of biomolecules recognizing metabolites have been used (Dietrich et al., 2010, Fowler et al., 2010, Jang et al., 2015, Liu et al., 2015, Michener and Smolke, 2012, Selvamani et al., 2017, Yang et al., 2013). One of the strategies is the use of transcription factor (TF)-promoter pairs that respond to small molecule ligands and consequently regulate the transcriptional outputs (Canton et al., 2008, Dietrich et al., 2013, Voigt, 2006). The TF-based biosensors have contributed to a number of recent metabolic engineering approaches (Liu et al., 2015, Liu et al., 2017, Mahr and Frunzke, 2016). In this case, a cognate promoter of transcription factor with reporter gene or selection marker can convert metabolite accumulation into a fluorescence or growth-coupled response, and consequently high-throughput screening becomes available. However, the majority of the metabolite responsive TF-promoters are heterologously expressed in metabolite-producing strains, thus optimized redesign of the genetic devices is required to minimize disturbances, including growth inhibition caused by metabolic burden, which may affect productivity in the host (Mahr and Frunzke, 2016).
Recently, 3-HP-responsive transcription factors (LysR) have been reported to exist in Pseudomonas denitrificans (Zhou et al., 2015). LysR is a transcription activator and binds to the cognate promoter with 3-HP as an effector to activate the transcription of downstream gene. This inducible gene expression system can be used to develop an artificial genetic circuit for screening 3-HP-producing microbes. In this study, a synthetic selection device with 3-HP-responsive transcription factor (LysR) and tetA as a selection marker was developed for high-throughput screening. By selecting C4-LysR between two types of the LysR transcription factors and modulating the expression level of C4-LysR, the device could be redesigned to have the appropriate operational range and dynamic range. Following determination of the appropriate selection pressure, it was applied to directed evolution of KGSADH. As a result, a strain with a 25% increase in 3-HP production was selected after only two serial cultures and the mutant KGSADH of the strain revealed a 2.79-fold higher catalytic efficiency compared with the wild-type KGSADH. Therefore, we demonstrated that KGSADH could be effectively engineered for the optimal production of 3-HP using well-designed library and the selection device.
Section snippets
Bacterial strains, plasmids, and reagents
The Escherichia coli strains and plasmids used in this study are presented in Table 1. Mach1-T1R was used for general cloning purposes. Routine cultures for the construction of plasmids and strains were performed in Luria-Bertani (LB) broth or on an LB agar plate containing appropriate antibiotics. Plasmid DNA and genomic DNA were prepared using a AccuPrepR Nano-Plus Plasmid Mini Extraction Kit (Bioneer, Daejeon, Korea) and a GeneAllR Exgene™ Cell SV Kit (GeneAll Biotechnology, Seoul, Korea),
Development of a synthetic 3-HP selection device
Recently, a gene expression system induced by 3-HP was revealed in P. denitrificans (Zhou et al., 2015). In this system, 3-HP induces LysR-type transcriptional regulators to activate the expression of catabolic genes involved in the degradation of 3-HP. We were able to develop a synthetic 3-HP selection device by using the transcription factors and promoters of the catabolic genes (Fig. 1B). The genetic device on a single expression vector contains an expression module for transcription factor
Conclusion
In this study, we report a synthetic 3-HP selection device using a 3-HP-responsive transcription factor derived from P. denitrificans. The device was designed to have desired performance using a synthetic constitutive promoter and 5′-UTR. This was applied to directed evolution of KGSADH, one of the enzymes involved in 3-HP biosynthesis in recombinant E. coli to select for KGSADH with improved activity toward the substrate, 3-HPA. Only two rounds of enrichment culture under selective condition
Acknowledgements
This research was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project (grant number ABC-2015M3A6A2066119) and the National Research Foundation of Korea (NRF) (grant number NRF-2015R1A2A1A10056126), funded by the Ministry of Science and ICT, Korea. This research was also supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (grant number 20174030201600).
Competing interests
The authors declare that they have no competing interests.
References (44)
- et al.
Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine
Metab. Eng.
(2015) - et al.
Using a riboswitch sensor to examine coenzyme B12 metabolism and transport in E. coli
Chem. Biol.
(2010) - et al.
Elevated production of 3-hydroxypropionic acid by metabolic engineering of the glycerol metabolism in Escherichia coli
Metab. Eng.
(2014) - et al.
Enhanced production of 3-hydroxypropionic acid from glycerol by modulation of glycerol metabolism in recombinant Escherichia coli
Bioresour. Technol.
(2014) - et al.
Aldehyde dehydrogenase activity is important to the production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae
Process Biochem.
(2012) - et al.
Metabolic engineering of cyanobacteria for photosynthetic 3-hydroxypropionic acid production from CO2 using Synechococcus elongatus PCC 7942
Metab. Eng.
(2015) - et al.
High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch
Metab. Eng.
(2012) - et al.
Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains
J. Biotechnol.
(2012) - et al.
Production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde by recombinant Escherichia coli co-expressing Lactobacillus reuteri propanediol utilization enzymes
Bioresour. Technol.
(2015) - et al.
Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency
Metab. Eng.
(2013)
A novel α-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism
J. Biol. Chem.
3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species
Appl. Environ. Microbiol.
Physiologic mechanisms involved in accumulation of 3- hydroxypropionaldehyde during fermentation of glycerol by Enterobacter agglomerans
Appl. Environ. Microbiol.
A simple combinatorial codon mutagenesis method for targeted protein engineering
ACS Synth. Biol.
Refinement and standardization of synthetic biological parts and devices
Nat. Biotechnol.
Debottlenecking the 1,3-propanediol pathway by metabolic engineering
Biotechnol. Adv.
Metabolic engineering of 3-hydroxypropionic acid biosynthesis in Escherichia coli
Biotechnol. Bioeng.
A green approach to chemical building blocks. The case of 3-hydroxypropanoic acid
Green Chem.
Enhancement of the stability of a prolipase from Rhizopus oryzae toward aldehydes by saturation mutagenesis
Appl. Environ. Microbiol.
High-throughput metabolic engineering: advances in small-molecule screening and selection
Annu. Rev. Biochem.
Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis
ACS Synth. Biol.
Riboselector:riboswitch-based synthetic selection device to expedite evolution of metabolite-producing microorganisms
Methods Enzymol.
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2022, Electrochimica ActaCitation Excerpt :Regulating both native and recombinant metabolic pathways is one of the most important tasks for microbial production in the future [1]. Recent molecular/synthetic biology researches have advanced genetic tools to control the recombinant pathways for over-expression and active-regulation, such as synthetic genetic circuits [2,3] and product-responsive transcription factors [4]. These studies have improved the biocatalytic activity effectively by reducing the activation energy barrier.
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These authors contributed equally to this work.