Genetic manipulation of Escherichia coli central carbon metabolism for efficient production of fumaric acid
Introduction
Fumaric acid, a four carbon carboxylic acid with a double bond and two carboxylic groups, is an intermediate of tricarboxylic acid (TCA) (Gu et al., 2013). It is also a platform chemical, and widely used as a precursor of numerous products, including pharmaceuticals, fine chemicals, and biodegradable polymers (Zhou et al., 2014). Currently, fumaric acid is produced by chemical isomerization of maleic anhydride, obtained from benzene or butane oxidation (Roa Engel et al., 2008). However, chemical synthesis process faced a number of issues including limited petroleum resources, rising oil prices, and environmental pollution. To mitigate environmental concerns, metabolic engineers and industrial microbiologists have sought to engineer alternate biosynthetic route to efficiently covert renewable feed stock to fumaric acid through genetically-modified organisms (Li et al., 2014, Liu et al., 2017b, Zhang et al., 2015b).
Fumaric acid production by fermentation was primarily conducted with Rhizopus oryzae or Rhizopus arrhizus (Liu et al., 2017c, Xu et al., 2012). According to previous report, 45.31 g/L and 37.52 g/L fumaric acid was obtained from xylose and glucose, respectively, using a new selected strain Rhizopus arrhizus RH 7-13-9# immobilized on a net (Liu et al., 2017a, Liu et al., 2015). In another wok, Rhizopus oryzae ATCC 20344 was used as strain, after optimization of seed culture conditions, uniformly dispersed mycelial clumps with a diameter of ∼0.1 mm were formed, which led to a high yield of fumaric acid as 50.2 g/L from glucose in shake flasks (Zhang et al., 2015a). However, the filamentous fungal fermentation process was difficult to scale up for the industrial applications, due to difficulties in controlling the cell morphology, and oxygen transfer limitations during large-scale production (Xu et al., 2013a, Xu et al., 2017, Xu et al., 2012). With the advantages of well annotate genomic information, facile genetic tools, low nutrient requirements, and rapid growth rate, Escherichia coli has become a prominent host strain for fumaric acid production (Cao et al., 2011).
Improvement of substrate utilization and elimination of byproducts formation were two of the most widely-used strategies for developing bio-based chemical production. Previous studied have demonstrated several useful strategies to improve fumaric acid production in E. coil (Chong et al., 2014, Xu and Koffas, 2010, Xu et al., 2011). For example, pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC) in E. coil was overexpressed to enhance the OAA (oxaloacetic acid) precursors through glycolytic pathway (Gao et al., 2018, Hu et al., 2018, Ma et al., 2013, Xu, 2018). Fumaric acid reductase (FrdABCD) and fumarase (FumABC) were disrupted to reduce the conversion of fumaric acid to the byproducts malic acid and succinic acid (Zhang et al., 2015c). Moreover, lactate dehydrogenase (LDH) and pyruvate formate-lyase (PFL) as the pyruvate-competing pathways for lactic acid formation were also deleted to decrease the waste of carbon source (Salleh et al., 2015). In addition, in order to regulate oxidation and reduction levels by cofactors, the NAD+/NADH and NADP+/NADPH ratios were also improved to enhance the biosynthesis of fumaric acid (Siedler et al., 2011, Zhanga et al., 2009). In this study, we attempted to further improve fumaric acid production by increasing substrate uptake rate and minimizing the carbon loss by preventing the formation of byproduct malic acid and acetic acid. The native glucose transport system was also replaced to minimize the loss of phosphoenolpyruvate (PEP) precursors.
In E. coli, several systems could transport sugar into the cytoplasm coupling consumption of PEP. The PEP dependent glucose-specific phosphotransferase system (PTSG) was the most efficient system for transporting glucose, which could act as the major regulatory point in controlling carbon flux and distribution in the central carbon metabolism(Liang et al., 2015). But PEP was also the direct precursor in OAA and fumaric acid synthesis and participated directly in energy generating reactions. We hypothesized that the modification of the glucose transport system would impact the accumulation of fumaric acid. Meanwhile, previous experimental data showed that malic acid and acetic acid were the main byproducts in the production of fumaric acid using E. coli. Therefore, in previous study, strain E. coli ABCDIA with fumaric acid pathway was further engineered by deleting fumarate reductase (frdABCD), fumarase (fumABC), DNA-binding response regulator (arcA), isocitratelyase repressor (iclR) (Xu et al., 2013b, Zhang et al., 2017). This deletion strain was used as host strain to investigate whether disruption of glucose transport system and modification of byproduct pathways would further improve fumaric acid production. In summary, we significantly improved both the productivity and yield of fumaric acid with these combined strategies. The reported work should be useful to build a sustainable platform for high-yield production of C4 carboxylic acid fumaric acid (Fig. 1).
Section snippets
Strains, plasmids and medium
The strains and plasmids used in this study were listed in Table 1. The fumaric acid producer strain E. coli ABCDIA was used as the original strain (Zhang et al., 2017). The gene encoding PPC and acetyl-CoA synthetase (ACS) were amplified from E. coli K12 genomic DNA. Plasmid pETDUet-1 was used as expression vector. For routine cultures during plasmid construction and strain cultivation, Luria-Bertani (LB) broth or LB agar plate (1.5% w/v, agar) was employed for culturing cell during strain
Effect of ptsG gene deletion on fumaric acid production
PTSG, the phosphoenolpyruvate-dependent sugar phosphotransferase system, is the major sugar translocation system across cell membrane, facilitating uptake of glucose with concomitant consumption of the high energy substrate phosphoenolpyruvate (PEP). To minimize PEP consumption, E. coli native PTSG system (encoded by ptsG gene) was replaced by galactose permease (encoded by galp gene) and glucokinase (encoded by glk gene), which could restore glucose uptake without consumption of PEP, resulted
Conclusion
In this study, a metabolically engineered E. coli strain was constructed to investigate whether the engineering of the ptsG, mdh, acs gene would decrease byproducts and improve fumaric acid production in E. coli. The results showed that the production of fumaric acid was greatly increased by engineering glucose uptake system and manipulation of precursor and byproduct pathway, which meant these modified strategies were beneficial for fumaric acid production process. The reported strategies
Acknowledgements
This research was financially supported by National Key Research Program (2016YFD0400601, 2017YFD0400603, 2017YFB0306900), the Natural Science Foundation of China (21476017), China Petroleum Science and Technology Innovation Fund (2017D-5007-0502), the Hong Kong, Macao and Taiwan Scientific and Technological Cooperation Projects (2015DFT30050), the Amoy Industrial Biotechnology R&D and Pilot Conversion Platform (3502Z20121009).
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