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Background: Cyanobacteria have long been suggested as an industrial chassis for the conversion of carbon dioxide to products as part of a circular bioeconomy. The slow growth, carbon fixation rates, and limits of carbon partitioning between biomass and product in cyanobacteria must be overcome to fully realise this industrial potential. Typically, flux towards heterologous pathways is limited by the availability of core metabolites. Citramalate is produced in a single enzymatic step through the condensation of the central metabolites pyruvate and acetyl-CoA; improvements in citramalate productivity can, therefore, be used as a measure of overcoming this limitation. Furthermore, citramalate is a useful biomaterial precursor and provides a route to renewable methyl methacrylate and poly(methyl methacrylate), which is often traded as Perspex or Plexiglas., Results: Here, we describe a phenomenon where the concerted optimisation of process parameters significantly increased citramalate production in Synechocystis sp. PCC 6803. Design of experiment principles were used to determine the optima for each parameter and the interplay between multiple parameters. This approach facilitated a ~ 23-fold increase in citramalate titre from initial unoptimised experiments. The process of scale-up from batch cultures to 0.5, 2, and 5 L photobioreactors is described. At the 2-L scale, citramalate titres from carbon dioxide reached 6.35 g/L with space-time yields of 1.59 g/L/day whilst 5-L PBRs yielded 3.96 ± 0.23 g/L with a productivity of 0.99 ± 0.06 g/L/day. We believe the decrease in productivity from 2-L to 5-L scale was likely due to the increased pathlength and shading for light delivery reducing incident light per cell. However, changes in productivity and growth characteristics are not uncommon when scaling up biotechnology processes and have numerous potential causes., Conclusions: This work demonstrates that the use of a process parameter control regime can ameliorate precursor limitation and enhance citramalate production. Since pyruvate and/or acetyl-CoA give rise to numerous products of biotechnological interest, the workflow presented here could be employed to optimise flux towards other heterologous pathways. Understanding the factors controlling and thus increasing carbon partitioning to product will help progress cyanobacteria as part of a carbon-neutral circular bioeconomy. This is the first study using design of experiment to optimise overall carbon fixation rate and carbon partitioning to product, with the goal of improving the performance of a cyanobacterium as a host for biological carbon capture., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: N.S.S. is a board member and cofounder of C3 Biotechnologies Ltd, and shareholder of C3 Biotechnologies Ltd., (© 2024. The Author(s).)

Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently. Vibrio sp. dhg was selected as the starting strain for the Adaptive Laboratory Evolution (ALE) strategy to leverage its significantly higher metabolic efficiency. We conducted ALE on a solid minimal medium supplemented with raffinose to prevent the enrichment of undesired phenotypes due to the shared effect of extracellular raffinose hydrolysis among multiple strains. As a result, we generated the VRA10 strain that efficiently utilizes raffinose without leaving behind degraded di- or monosaccharides, achieving a notable growth rate (0.40 h -1 ) and raffinose consumption rate (1.2 g/g dcw /h). Whole genome sequencing and reverse engineering identified that a missense mutation in the melB gene (encoding a melibiose/raffinose:sodium symporter) and the deletion of the two galR genes (encoding transcriptional repressors for galactose catabolism) facilitated rapid raffinose utilization. The further engineered strain produced 6.2 g/L of citramalate from 20 g/L of raffinose. This study will pave the way for the efficient utilization of diverse raffinose-rich byproducts and the expansion of alternative carbon streams in biorefinery applications., Competing Interests: Declaration of interest The authors declare no competing interests., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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.

Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis , may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene ( cimA ) variant for expression in I. orientalis . We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated- cimA strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple ( Malus × domestica ) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase ( IPMS ). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. 13 C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in "Jonagold" fruit ( MdCMS_1 and MdCMS_2 ), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine 387 (MdCMS_1) or glutamate 387 (MdCMS_2). Glutamate 387 was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate 387 produced a very low proportion of 2-methylbutanol- and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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 ., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25  g liter -1 citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids. IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules., (Copyright © 2019 Webb et al.)

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.

The volatile ester and alcohol profiles of ripening apple fruit from 184 germplasm lines in the USDA Malus Germplasm Repository at the New York Agricultural Experiment Station in Geneva, NY, USA, were evaluated. Cluster analysis suggested biochemical relationships exist between several ester classes. A strong linkage was revealed between 2-methylbutanoate, propanoate, and butanoate esters, suggesting the influence of the recently proposed "citramalic acid pathway" in apple fruit. Those lines with a high content of esters formed from 2-methylbutan-1-ol and 2-methylbutanoate (2MB) relative to straight-chain (SC) esters (high 2MB/SC ratio) exhibited a marked increase in isoleucine and citramalic acid during ripening, but those lines with a low content did not. Thus, the data were consistent with the existence of the hypothesized citramalic acid pathway and suggest that the Geneva Malus Germplasm Repository, appropriately used, could be helpful in expanding our understanding of mechanisms for fruit volatile synthesis and other aspects of secondary metabolism.