Extending the shikimate pathway for microbial production of maleate from glycerol in engineered Escherichia coli

Abstract

Maleate is one of the most important unsaturated four‐carbon dicarboxylic acids. It serves as an attractive building block in cosmetic, polymer, and pharmaceutical industries. Currently, industrial production of maleate relies mainly on chemical synthesis using benzene or butane as the starting materials under high temperature, which suffers from strict reaction conditions and low product yield. Here, we propose a novel biosynthetic pathway for maleate production in engineered Escherichia coli. We screened a superior salicylate 5‐hydroxylase that can catalyze hydroxylation of salicylate into gentisate with high conversion rate. Then, introduction of salicylate biosynthetic pathway and gentisate ring cleavage pathway allowed the synthesis of maleate from glycerol. Further optimizations including enhancement of precursors supply, disruption of competing pathways, and construction of a pyruvate recycling system, boosted maleate titer to 2.4 ± 0.1 g/L in shake flask experiments. Subsequent scale‐up biosynthesis of maleate in a 3‐L bioreactor under fed‐batch culture conditions enabled the production of 14.5 g/L of maleate, indicating a 268‐fold improvement compared with the titer generated by the wildtype E. coli strain carrying the entire maleate biosynthetic pathway. This study provided a promising microbial platform for industrial level synthesis of maleate, and demonstrated the highest titer of maleate production in microorganisms so far.

Metabolic Engineering of Escherichia coli for the Biosynthesis of 3-Phenylpropionic Acid and 3-Phenylpropyl Acetate

“…In previous experiments of this study, strains were grown in media containing a mixture of 2.5 g L −1 glucose and 10 g L −1 glycerol, the former supporting cell growth and the latter favoring aromatic compound production. 16,40 However, in this case, the situation that afforded the best yield within 24 h indicated that improving the cell growth in the initial stage of cultivation may result in an improvement in production. Therefore, we changed the carbon source to 10 g L −1 glucose for further enhancement.…”

Recent Progress in Metabolic Engineering of Escherichia coli for the Production of Various C4 and C5-Dicarboxylic Acids

“…Maleate, a structural isomer of fumarate with a cis -form, is projected to reach a market size of $283.3 million by 2027 . Its derivative, maleic anhydride, is utilized extensively for pharmaceuticals, cosmetics, and monomer for polymer synthesis. ,, To avoid the energy-intensive process of chemical synthesis, a novel pathway for maleate production from glucose was engineered in E. coli . In this pathway, chorismate, an intermediate of the shikimate pathway and a precursor for aromatic amino acids such as phenylalanine, tyrosine, and tryptophan, is synthesized using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) derived from glycolysis and the pentose phosphate pathway, respectively.…”

“…Ultimately, these advancements establish highly efficient microbial cell factories. 20,71 Previous studies have indicated that a mixture of dicarboxylate, including 2-methylsuccinate, amounting to 0.65 g/L, was created via the EMC pathway from Methylobacterium extorquens by optimizing the cultural conditions. However, a new method based on the citramalate pathway was suggested for its relatively low productivity.…”

“…20 Furthermore, an alternative approach was implemented within the maleate production pathway, aiming to eliminate the reliance on the relatively inefficient conversion from chorismate to gentisate, which acts as an intermediate. 71 To enable the conversion of chorismate to gentisate, genes for isochorismate synthase (EntC), isochorismate pyruvate lyase (PchB), and salicylate 5-hydroxylase (NagAaGHAb) was introduced. As a result, the engineered strain ultimately produced 14.5 g/L of maleate from glycerol in a 3 L bioreactor over a period of 84 h. 71…”

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli

“…With recent advances in metabolic engineering, 2-PE produced by microbial systems is a potentially sustainable alternative to fossil fuel-based production and isolation from native plants, which has attracted considerable interest (Kong et al, 2020 ; Li et al, 2021 ; Zhan et al, 2022 ; Zhan et al, 2020 ). In consideration of its rapid growth, clear genetic background and highly efficient genome editing, E. coli has been used as a versatile platform strain to produce various biochemicals by applying genetic engineering methods (Guo et al, 2020 ; Guo et al, 2022 ; Lai et al, 2022 ; Sheng et al, 2021 ; Zhang et al, 2022 ), including 2-PE (Guo et al, 2017 ; Kang et al, 2014 ; Wang et al, 2019 ). Although various efforts have been made to improve the synthetic ability of E. coli , the titer, yield and productivity were still less than satisfactory.…”