Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose
Introduction
Benzoic acid (BA) is an industrially important and platform aromatic compound, which finds a broad array of applications. For example, BA can be used as a precursor to synthesize many important commodity chemicals such as terephthalic acid via the Henkel reaction (Ogata et al., 1957), phenol via decarbonation mechanism (Buijs, 1999), and epsilon-caprolactam via the Snia Viscosa process (Hirabayashi et al., 2004). Due to the anti-microbial property, BA and its various salts (e.g., potassium, sodium, and calcium) have also been extensively utilized as preservatives in the food, cosmetics, and pharmaceutical industries (Maki and Takeda, 2000).
Currently, BA is commercially produced from toluene oxidation by chemical processes, which are operated at high temperature and pressure with the use of heavy metal catalysts such as cobalt or manganese naphthenate (Maki and Takeda, 2000). Also, toluene is derived from non-renewable petroleum resource, which makes the overall process not sustainable. For these reasons, there has been much interest to develop an efficient and eco-friendly method to produce BA from renewable feedstocks. In this regard, a biological approach employing microbial cell factories for BA production from renewable biomass holds significant promise. Apart from the environmental benefits associated with the mild bioprocesses, biologically obtained BA is considered to be “natural”, which is a major advantage for its applications in food, cosmetics, and pharmaceuticals.
Natural BA biosynthesis has been mainly found in plants, where BA serves as an essential moiety in the synthesis of a variety of plant natural products (Widhalm and Dudareva, 2015; Wildermuth, 2006). To date, three metabolic pathways via a common precursor l-phenylalanine (l-PHE) for BA production have been identified in plants (Widhalm and Dudareva, 2015), which will be discussed in detail later. BA biosynthesis by native microorganisms has also been reported, but only in one bacterium named Streptomyces maritimus, where BA is synthesized via a plant-like β-oxidation pathway as an intermediate during enterocin biosynthesis (Xiang and Moore, 2003). This wild-type S. maritimus was employed for BA fermentation in a complex medium containing glucose, cellobiose, or starch as a carbon source, leading to the production of 257, 337, and 460 mg/L of BA, respectively (Noda et al., 2012). However, the use of wild-type producer without a full understanding of the underlying biosynthetic mechanism and complex regulation hindered further improvement of titer.
Microbial BA production using a heterologous host has been rarely attempted except for the following two studies published very recently. In the first study, Escherichia coli strains harboring a seven- or nine-step biotransformation pathway were constructed, which enabled BA production (16.2 or 17.8 g/L) from styrene or l-PHE, respectively, using concentrated suspensions of resting (non-growing) cells. Furthermore, BA production (8.6 g/L) from the simple carbon source glycerol was achieved by a coupled fermentation-biotransformation process, in which l-PHE was first produced from glycerol by fermentation with an E. coli l-PHE overproducer, followed by its conversion to BA using the above biotransformation process (Zhou et al., 2020). Despite the relatively high BA titers, the total reaction volume was rather small (~10 mL), and the coupled fermentation-biotransformation approach involved separate cultivation of individual strains and several rounds of cell harvest and suspension. While we were preparing our manuscript, the results of the second study on de novo production of BA were reported. This nice work on metabolic engineering of Pseudomonas taiwanensis strain employing a β-oxidation pathway resulted in production of 232.0 and 366.4 mg/L of BA from glucose and glycerol, respectively (Otto et al., 2020). Although this pathway employed is the same as one of the five pathways we examined in this study, we decided to report the results of our works that led to the production of BA to a higher titer.
In the present study, we report the development of metabolically engineered E. coli strains harboring a plant-like β-oxidation pathway or a synthetically designed pathway for the production of BA directly from glucose. First, three different natural BA biosynthetic pathways from plants and a newly designed synthetic BA pathway were systematically evaluated for BA production by in silico flux response analyses. Next, the selected β-oxidation pathway and the synthetic pathway were separately established in E. coli to enable de novo BA production from glucose. To increase BA production, various sources for key biosynthetic enzymes and different plasmid configurations for expressing the genes involved in each metabolic pathway were examined. Moreover, improved BA production was achieved by applying several metabolic engineering strategies including the increase of precursor supply, elimination of competitive pathways, transporter engineering, and byproduct reduction. Ultimately, the best-performing engineered strains harboring each metabolic pathway were examined in fed-batch fermentations to demonstrate the potential for BA production from glucose.
Section snippets
Strains and media
The bacterial strains used in this study are listed in Table 1. The molecular cloning and plasmid maintenance experiments were performed in E. coli DH5α. BA production was investigated using E. coli W3110 or NST74 (Tribe, 1987) and its derivatives as host strains. In the course of plasmid construction and strain development, E. coli cells were routinely grown in lysogeny broth (LB) broth or on LB agar plates (1.5% w/v) supplemented with appropriate antibiotics when necessary: 100 mg/L of
Design and evaluation of different metabolic pathways for BA biosynthesis from glucose
As mentioned earlier, BA biosynthesis has been known in plants. The three identified plant metabolic pathways leading to BA formation are derived from a common precursor l-PHE (Widhalm and Dudareva, 2015). Also common to the three pathways, the first committed step is the deamination of l-PHE to CA by phenylalanine ammonia lyase (PAL). Then, CA is converted to BA by the removal of two carbons from its three-carbon side chain, wherein different strategies are employed by the three pathways.
Conclusions
In this paper, we report the development of two different metabolically engineered E. coli strains capable of producing BA directly from glucose. Prior to strain development, three distinct natural BA biosynthetic pathways derived from plants and a newly designed synthetic BA pathway were systematically evaluated for their performance in BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately
CRediT authorship contribution statement
Zi Wei Luo: designed the research, conducted the experiments and analyzed the data, wrote the manuscript. All authors read and approved the final manuscript. Sang Yup Lee: designed the research, wrote the manuscript. All authors read and approved the final manuscript.
Declaration of competing interest
The authors declare no competing interests.
Acknowledgement
We thank Changdai Gu and Gi Bae Kim for their help in performing the in silico flux simulation experiments. This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries [NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557] from the Ministry of Science and ICT through the National Research Foundation of Korea.
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2023, Biotechnology AdvancesCitation Excerpt :In addition to traditional products, cinnamic acid and its derivatives (Rodrigues et al., 2017), flavonoids (Santos et al., 2011), stilbenoids (represented by resveratrol) (Thapa et al., 2019), coumarin and its derivatives (Lin et al., 2013; Ren et al., 2018; Yang et al., 2015), alkaloids (Fossati et al., 2014; Galanie et al., 2015), hydroquinone glycoside compounds (represented by arbutin) (Shen et al., 2017), and phenylpropane derivatives (represented by vanillin) (Ni et al., 2015; Plaggenborg et al., 2006) can also be produced using this pathway in microbes. In recent years, the importance of the shikimate pathway has been highlighted by the production of benzylamine (an intermediate of the high-energy propellant CL-20) (Pandey et al., 2021), hydroxytyrosol (Yao et al., 2020), 3,4-dihydroxy-l-phenylalanine (L-DOPA) (Tang et al., 2021), benzoic acid (Luo and Lee, 2020), 4-hydroxystyrene (Gargatte et al., 2021), and aromatic polyesters (Yang et al., 2018). Thus, the shikimate pathway is a general route for the biosynthesis of aromatic and hydroaromatic compounds, and its product spectrum includes amino acids, organic acids, polymers, and fuels.