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Abstract 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.

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. Highlights: Synechocystis sp. PCC 6803 produced 6.35 g/L of citramalate with a space–time yield of 1.59 g/L/day from carbon dioxide. The rate of citramalate production by Synechocystis. sp. PCC 6803 was improved from 0.068 to 1.59 g/L/day, a ~ 23-fold increase. The switch between growth and production can be controlled using process parameters in place of complex genetic circuits and chemical inducers. Overall carbon fixation rates of Synechocystis sp. PCC 6803 photobioreactor cultures were increased from 0.12 g/L/day to 0.77 g/L/day. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • An efficient raffinose utilization host was generated using ALE on a solid medium. • The evolved strain exhibited better raffinose catabolism than the parental strain. • Growth, raffinose consumption rates were 0.40 h−1 and 1.2 g/g dcw /h, respectively. • Causative mutations and their mechanisms were investigated. • An engineered strain produced 6.2 g/L of citramalate from 20 g/L of raffinose. [ABSTRACT FROM AUTHOR]

Abstract 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.

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.

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. [ABSTRACT FROM AUTHOR]

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.

Citramalic acid (citramalate) is important for the chemical synthesis of methylmethacrylate, a bulk monomer used for manufacturing biodegradable polymers. Escherichia coli strains have been engineered for the biological production of citramalate since a rare mutant of citramalate synthase was invented as a mesophilic catalyzer to form the chemical. However, acidic byproducts are routinely excreted by E. coli host cells during fermentation, which could significantly reduce the carbon conversion efficiency of biosynthetic chemicals, including citramalate. Herein, an unusual mutant called S17–3 was reported, and this mutant demonstrated superb recovery efficacy from glucose to citramalate and secreted a very low level of fermentation byproducts during the fermentative production of citramalate. However, S17–3 tends to produce a large amount of colonic acid (CA). Genetically manipulating the regulators involved in CA synthesis revealed that the deletion of rcsD could significantly improve the citramalate titer. Fed-batch fermentation under optimized conditions led to a maximum titer of 46.2 g/L citramalate accumulated in the broth, with a conversion efficiency of 0.80 g citramalate per gram glucose and a close-to-the-theoretical mole transversion efficiency (mol/mol) of 0.97. This study facilitates the development of a practical bioproduction of citramalate using E. coli strains. [Display omitted] • BL21 (DE3) and S17-3 were engineered and compared for the production of citramalate. • rcsD deletion enhanced the citramalate yield in recombinant E. coli S17-3. • The highest carbon conversion ratio ever reported was obtained for the production of citramalate from glucose. [ABSTRACT FROM AUTHOR]

The volatile profile of ‘Redchief Delicious’ apple (Malus ×domestica Borkh.) fruit was evaluated at 18 time points from 3 weeks before to 8 weeks after onset of autocatalytic ethylene production to capture the dynamics associated with development from mature green to senescent fruit. Minor amounts of ester production began several days before the onset of ethylene production. Ester production rose rapidly as internal ethylene levels increased beyond 22 nmol·L−1 (0.5 µL·L−1). Peak ester production roughly coincided with maximum ethylene synthesis, declining thereafter. Ester production was further evaluated according to the acid- (alkanoate) and alcohol- (alkyl) derived portions of the ester. The maximum rate of production for a given ester tended to occur later in development as the chain length of the alcohol-derived portion declined. The production rate for many esters paralleled the rate of emanation of their respective alcohol substrates, suggesting that availability of the alcohols limits ester production more than availability of the acid substrates. Combining production rates with sensory descriptors and human sensitivity to individual volatiles permitted approximations of aroma sensations likely engendered by the fruit throughout ripening. Overripe and alcoholic sensations are predicted to increase 2 weeks after the initiation of ripening in response to an increase in the production of ethyl esters. Acetate esters predominated, comprising 50% to 80% of esters throughout maturation and ripening, indicating that the substrate acetyl-CoA may be at saturating levels for alcohol acyl transferase (AAT) at the final step of ester formation. Acetate feeding did not enhance ester production, although label from 13C-acetate was extensively incorporated into esters. The data are consistent with the action of multiple AAT isozymes differing in activity and substrate preference. Incorporation of labeled 13C-acetate into precursors of esters, alcohols, and acids, reflected ester biosynthesis via 1- and 2-carbon chain elongation pathways in ripening ‘Redchief Delicious’ apple fruit.

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.

Abstract 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 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.

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 FROM AUTHOR]

Aims: The legumes most often used in temperate pastures such as Trifolium subterraneum have relatively high external P requirements for maximum growth. We investigated root traits associated with P acquisition in current and novel pasture legumes, as well as temperate grasses which have lower P requirements. Methods: Thirteen legume species, two pasture grasses, and three high carboxylate-exuding crop species ( Lupinus albus, L. angustifolius, Cicer arietinum) were grown in a glasshouse for six weeks. Rhizosphere carboxylates and root morphological traits were measured. Results: Ornithopus spp. had rhizosphere carboxylates in similar quantities to the Lupinus spp. (> 40 μmol g root dry mass) . Trifolium subterraneum lines had relatively large average root diameter, reduced specific root length and very short average root hair length resulting in specific root hair cylinder volumes (RHCVs) only 14-20 % of the grasses. However, O. sativus, O. compressus and Biserrula pelecinus had specific RHCVs more comparable to the grasses. Conclusions: Novel pasture legume species with root morphology more comparable to that of grasses than T. subterraneum were identified. Of these, Ornithopus spp. were notable as they also had high rhizosphere carboxylates relative to root dry mass. [ABSTRACT FROM AUTHOR]