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Background: Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation., Results: This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively., Conclusion: This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives., (© 2024. The Author(s).)

Biological production of citramalate has garnered attention due to its wide application for food additives and pharmaceuticals, although improvement of yield is known to be challenging. When glucose is used as the sole carbon source, carbon loss through decarboxylation steps for providing acetyl-CoA from pyruvate is inevitable. To avoid this, we engineered a strain to co-utilize glucose and cost-effective acetate while preventing carbon loss for enhancing citramalate production. The production pathway diverged to independently supply the precursors required for the synthesis of citramalate from glucose and acetate, respectively. Moreover, the phosphotransferase system was inactivated and the acetate assimilation pathway and the substrate ratio were optimized to enable the simultaneous and efficient utilization of both carbon sources. This yielded results (5.0 g/L, 0.87 mol/mol) surpassing the yield and titer of the control strain utilizing glucose as the sole carbon source in flask cultures, demonstrating an economically efficient strain redesign strategy for synthesizing various products.

Background: Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using Escherichia coli overexpressing citramalate synthase (cimA gene). Deletion of gltA encoding citrate synthase and leuC encoding 3-isopropylmalate dehydratase were critical to achieving high citramalate yields. Acetate is an undesirable by-product potentially formed from pyruvate and acetyl-CoA, the precursors of citramalate during aerobic growth of E. coli. This study investigated strategies to minimize acetate and maximize citramalate production in E. coli mutants expressing the cimA gene., Results: Key knockouts that minimized acetate formation included acetate kinase (ackA), phosphotransacetylase (pta), and in particular pyruvate oxidase (poxB). Deletion of glucose 6-phosphate dehydrogenase (zwf) and ATP synthase (atpFH) aimed at improving glycolytic flux negatively impacted cell growth and citramalate accumulation in shake flasks. In a repetitive fed-batch process, E. coli gltA leuC ackA-pta poxB overexpressing cimA generated 54.1 g/L citramalate with a yield of 0.64 g/g glucose (78% of theoretical maximum yield), and only 1.4 g/L acetate in 87 h., Conclusions: This study identified the gene deletions critical to reducing acetate accumulation during aerobic growth and citramalate production in metabolically engineered E. coli strains. The citramalate yield and final titer relative to acetate at the end of the fed-batch process are the highest reported to date (a mass ratio of citramalate to acetate of nearly 40) without being detrimental to citramalate productivity, significantly improving a potential process for the production of this five-carbon chemical.

Abstract Background The filamentous fungus Aspergillus niger is frequently used for industrial production of fermentative products such as enzymes, proteins and biochemicals. Notable examples of industrially produced A. niger fermentation products are glucoamylase and citric acid. Most notably, the industrial production of citric acid achieves high titers, yield and productivities, a feat that has prompted researchers to propose A. niger to serve as heterologous production host for the industrial production of itaconic acid (IA), a promising sustainable chemical building-block for the fabrication of various synthetic resins, coatings, and polymers. Heterologous production of IA in A. niger has resulted in unexpected levels of metabolic rewiring that has led us to the identification of IA biodegradation pathway in A. niger. In this study we have attempted to identify the final product of the IA biodegradation pathway and analyzed the effect of metabolic rewiring on the bioproduction of 9 industrially relevant organic acids. Results IA biodegradation manifests in diminishing titers of IA and the occurrence of an unidentified compound in the HPLC profile. Based on published results on the IA biodegradation pathway, we hypothesized that the final product of IA biodegradation in A. niger may be citramalic acid (CM). Based on detailed HPLC analysis, we concluded that the unidentified compound is indeed CM. Furthermore, by transcriptome analysis we explored the effect of metabolic rewiring on the production of 9 industrially relevant organic acids by transcriptome analysis of IA producing and WT A. niger strains. Interestingly, this analysis led to the identification of a previously unknown biosynthetic cluster that is proposed to be involved in the biosynthesis of CM. Upon overexpression of the putative citramalate synthase and a genomically clustered organic acid transporter, we have observed CM bioproduction by A. niger. Conclusion In this study, we have shown that the end product of IA biodegradation pathway in A. niger is CM. Knock-out of the IA biodegradation pathway results in the cessation of CM production. Furthermore, in this study we have identified a citramalate biosynthesis pathway, which upon overexpression drives citramalate bioproduction in A. niger.