Synthesis method for itaconate driven by thioesterase

Abstract

This invention provides a method for the synthesis of itaconic acid driven by thioesterase. Through experiments illustrated in the examples, this invention provides a novel method for the synthesis of itaconic acid driven by thioester hydrolysis.

Description

[Technical Field]

[0001] This invention relates to a method for synthesizing itaconic acid driven by thioesterase. [Previous Technology]

[0002] Recently, recycled materials derived from biomass feedstocks have received increasing attention. Itaconic acid, a C5 dicarboxylic acid with a bifunctional group and an unsaturated double bond, has numerous applications in polymer synthesis. Itaconic acid has been recognized by the US Department of Energy (DOE) as one of the top 12 valuable bio-based chemical additives. Furthermore, itaconic acid plays a crucial role in the sustainability of renewable alternatives to various petroleum-based chemicals, such as acrylic acid and methacrylic acid, thus reducing reliance on oil-based energy and long-term environmental damage. Itaconic acid has the following chemical formula: [formula missing], and is an essential precursor for many products (e.g., polypropylene fibers, rubber, synthetic diamonds, and lenses), widely used in the chemical industry. Traditionally, itaconic acid was isolated from *Aspergillus terreus*. However, Aspergillus oryzae grows slowly and cannot form itaconic acid during its sporulation period. Therefore, the industry urgently needs a novel method for producing itaconic acid.

[0003] Itaconic acid is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Its production has long relied on the decarboxylation of the TCA cycle intermediate cis-aconitate to form a methylene-linked dicarboxylic acid, losing a carbon in the process. While fungi have achieved success in the original producer, the complex life cycle of fungi and their desire for additional properties such as photosynthetic biosynthesis have led to significant efforts to engineer fungal pathways in recombinant hosts. The insolubility of fungal cis-aconitate decarboxylases and the necessity of TCA cycle disruption often pose challenges to the development and operational costs of recombinant strains. The inevitable loss of cis-aconitate due to the reversible interconversion of citric acid and isocitrate further complicates securing precursor supply; previous technologies have addressed this issue by replacing natural aconitase with artificially engineered dehydrating enzymes. The itaconic acid detoxification pathway has evolved in certain infectious bacteria (such as *Yersinia pestis*, *Pseudomonas aeruginosa*, and *Burkholderia xenovorans*) to counteract the growth inhibition caused by itaconic acid's inhibition of the glyoxylate pathway and methylcitrate cycle. During infection, the natural defense mechanism of mammalian macrophages functions through the synthesis and release of itaconic acid, thereby allosterically deactivating isocitrate and methylcitrate lyases and preventing pathogens from uptakeing acetyl-CoA or propionate-CoA under glucose-restricted conditions. Itaconic acid degradation begins with the activation of dicarboxylic acid via coenzyme A of itaconic acid-CoA transferase (ICT). The obtained itaconyl-CoA was then converted to (S)-citramalyl-CoA via itaconyl-CoA isomerase and mesaconyl-CoA hydratase active modules. Finally, (S)-citramalyl-CoA was cleaved into acetyl-CoA and pyruvate, which occurred under the action of (S)-citramalyl-CoA lyase (Ccl), an enzyme with homologs common in the 3-hydroxypropionic acid cycle.

[0004] Escherichia coli (scientific name: Escherichia coli, usually abbreviated as E. coli) is a bacterium found in the intestines of humans and animals, mainly residing in the large intestine, accounting for about 0.1% of intestinal bacteria. Escherichia coli is a short, motile, non-spore-forming Gram-negative rod-shaped bacterium with blunt ends.

[0005] Although researchers in this field have discovered that Escherichia coli can be used to produce itaconic acid, in order to accumulate precursors for itaconic acid production, it is necessary to inhibit the important gene icd in the citrate cycle (which encodes isocitrate dehydrogenase) in E. coli strains. Inhibition of the icd gene results in auxotrophic strains of α-ketoglutaric acid (α-KG) and glutamic acid. However, this interference often leads to glutamic acid deficiency, resulting in slow cell growth, which prevents this strain from growing and producing itaconic acid in basal culture medium.

[0006] To address this long-standing problem, conventional technology involves adding large amounts of yeast extract or glutamic acid to maintain growth, thereby providing the essential amino acid (i.e., glutamic acid) that the itaconic acid-producing strain that inhibits the ICD gene cannot synthesize on its own, thus helping the strain to grow.

[0007] In order to provide a different route for the synthesis of itaconic acid from the traditional one and hopefully overcome the above problems, those skilled in the art urgently need to develop a novel method for the synthesis of itaconic acid to benefit the vast number of people who have this need. [Summary of the Invention]

[0008] In view of the above, the object of the present invention is to provide a method for synthesizing itaconic acid driven by thioesterase, comprising condensing pyruvate and acetyl-CoA through a reverse degradation pathway to form (S)-citramalyl-CoA, then dehydrating and isomerizing the (S)-citramalyl-CoA to itaconic acid, and then hydrolyzing the itaconic acid to itaconic acid driven by thioesterase.

[0009] In one embodiment of the present invention, the thioesterase is derived from Escherichia coli.

[0010] In one embodiment of the present invention, the Escherichia coli is Escherichia coli strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta).

[0011] In one embodiment of the present invention, the Escherichia coli strain JCL299 is co-transformed by pIA1 and ASKA plastids.

[0012] In one embodiment of the present invention, the thioesterase is selected from the group consisting of: FadM, YdiI, YbgC, YbfF, YpfH, TesA, TesB, YbaC, YjfP, BioH, YbdB, FrsA, phenylacetyl-CoA thioesterase (PaaI), PaaY, YeiG, and YqiA.

[0013] In one embodiment of the present invention, the yield of itaconic acid is 10-40 mg / L within 24 hours.

[0014] In one embodiment of the present invention, the PaaI achieves an itaconic acid yield of 150 mg / L within 48 hours.

[0015] In one embodiment of the present invention, the Escherichia coli is cultured using a microaerobic culture condition in which the volume ratio of the culture container to the culture is 6.25:1.

[0016] In one embodiment of the present invention, the PaaI has a specific activity of 0.15 to 0.19 U / mg against itaconic acid coenzyme A.

[0017] In one embodiment of the present invention, the itaconic acid yield reaches 1 g / L within 120 hours.

[0018] This invention creates a novel itaconic acid synthesis pathway that directly uses the central metabolite pyruvate and acetyl-CoA as precursors, and can be synthesized through three reactions (see Figure 1), which has extremely high potential for subsequent optimization and development.

[0019] In summary, this invention provides a novel itaconic acid synthesis method driven by thioester hydrolysis, which generates a thermodynamic driving force and a kinetic trap through a thioesterification reaction, thus redirecting the originally reversible pathway towards the production of the target itaconic acid. This invention replaces the enzyme (itaconyl-CoA transferase, Ict) in the final step of the original pathway with a thioesterase, which has never been reported in previous studies.

[0020] By identifying phenylacetyl-CoA thioesterase (PaaI) with itaconic acid-CoA hydrolytic activity, the proposed itaconic acid pathway exhibited the same theoretical maximum glucose yield and the same redox balance as the natural fungal pathway via the TCA cycle. However, through the reverse degradation pathway, an additional one mole of ATP is generated for every mole of itaconic acid synthesized, which is a beneficial characteristic in microbial biosynthesis. The abundance of pyruvate and acetyl-CoA and the good performance of bacterial enzymes should make this pathway have good potential for development in other unique hosts, such as cyanobacteria or methanogens.

[0021] Currently, the synthetic itaconic acid pathway appears to be primarily limited by the low hydrolysis efficiency of itaconic acid-CoA. Therefore, designing thioesterases to obtain better itaconic acid-CoA activity while maintaining high specificity will be the next important step in further improving itaconic acid produced using this pathway.

[0022] The embodiments of the present invention will be further described below. The examples listed below are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Implementation Method

[0024] Definition

[0025] The values ​​used in this paper are approximate values. All experimental data are expressed within 20%, preferably within 10%, and optimally within 5%.

[0026] Unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the claims described below) shall be understood to include both singular and plural forms.

[0027] The present invention will be further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0028] According to the present invention, by setting an irreversible thioester hydrolysis step to replace the original coenzyme A transfer, the reversal of the itaconic acid catabolism pathway is demonstrated, thereby producing itaconic acid (Figure 1). Since no alternative biochemical pathway for itaconic acid biosynthesis has been proposed and constructed, the present invention is interested in the design of naturally occurring itaconic acid degradation and sets out to reverse its reaction sequence through engineering means (Figure 1).

[0029] According to the present invention, inspired by the reversal of the β-oxidation pathway through fermentation of NADH equilibrium to generate 1-butanol, the present invention attempts to create a synthetic driving force by utilizing thermodynamically favorable thioester hydrolysis to propel carbon flow toward itaconic acid synthesis. Utilizing acetyl transfer potential to drive biochemical reactions is common in metabolic processes. Thioesters have lower resonance stability efficiency, resulting in a greater energy drop during hydrolysis compared to similar oxyesters. By replacing the final coenzyme A transfer step (ΔrG'0 = 3.4 KJ / mol) with coenzyme A hydrolysis (ΔrG'0 = -25 KJ / mol) using a thioesterase (Figure 1), the intrinsically reversible itaconic acid degradation pathway (overall ΔG'0 = -0.8 KJ / mol) is transformed into a thermodynamically favorable pathway (overall ΔG'0 = -29.2 KJ / mol). It is noteworthy that when the acceptor concentration is set at 1 mM, the Gibbs free energy of itaconitum-CoA hydrolysis decreases even more significantly. From a physiological perspective, this level is more suitable for describing intracellular metabolites. Acyl-CoA thioesterases belong to the large family of thioester hydrolases and catalyze the cleavage of thioester bonds, metabolic intermediates found in many essential pathways such as fatty acid and steroid biosynthesis, β-oxidation, and mevalonate metabolism. Their wide range of involvement in reactions gives acyl-CoA thioesterases a broad acceptor range, encompassing, but not limited to, saturated / unsaturated fatty acyl-CoA, methyl-branched acyl-CoA, aromatic acyl-CoA, and carboxylic acyl-CoA. The diversity of thioesterase functions and structures also presents challenges to classification. Currently, acetylglucosamine thioesterases are broadly classified based on their sequence homology, domains, and catalytic mechanisms. Type I belongs to the α / β-hydrolase protein family, while type II possesses a characteristic "hotdog" fold motif. Studies have shown that due to the lack of a highly conserved substrate-binding pocket, type II acetylglucosamine thioesterases typically exhibit broader substrate heterogeneity, enabling them to participate in a variety of reactions with different functions.

[0030] According to the present invention, the first non-natural itaconic acid production pathway driven by thioester hydrolysis is designed. Identifying suitable thioesterases with itaconic acid-CoA activity is a key factor in reversing the itaconic acid degradation pathway for biosynthesis. The low preference of the target thioesterase for the pathway precursors acetyl-CoA and (S)-limonene-CoA is also important for minimizing unwanted subject competition and preventing precursor loss. Although the achieved titer and yield are still lower compared to prior art itaconic acid production pathways, the pathway proposed in this invention has higher energy efficiency, which could become an attractive feature once sufficiently enhanced. Compared to the natural fungal pathway which produces 1 mole of ATP per mole of glucose converted to itaconic acid, the reverse degradation pathway produces 2 moles of ATP due to its unique pathway characteristics (see Figure 7 for detailed reaction equilibrium). Figure 7 shows the detailed stoichiometry and reaction equilibrium of the two itaconic acid formation pathways. PEP represents phosphoenolpyruvate; OAA represents oxaloacetate. The maximum theoretical yield and NADH balance remain unchanged between the two pathways. Although still in the early stages of development, this invention presents a proof-of-concept design for a thermodynamically favorable synthetic itaconic acid pathway. The engineering principles and novel itaconic acid pathway proposed in this invention should be readily transferable to other production systems and recombinant hosts.

[0031] The reagents and chemicals used in the following examples are as follows. Unless otherwise stated, all chemicals and reagents were purchased from Thermo Scientific (Pittsburgh, PA) or Sigma-Aldrich (Saint Louis, MO). Taq DNA ligase, Phusion high-fidelity DNA polymerase, and T5 exonuclease were purchased from New England Biolabs (Ipswich, MA). Oligonucleotides were purchased from IDT (San Diego, CA). KOD DNA polymerase was purchased from EMD Chemicals (San Diego, CA).

[0032] The bacterial strains and DNA manipulation procedures are as follows. *Escherichia coli* BW25113 was designated as the wild type. All plastids were amplified using XL-1 Blue (Stratagene, La Jolla, CA). The construction of strains JCL16 (BW25113, with lacI q provided on F') and JCL299 is described in previous studies (Atsumi, S., Cann, AF, Connor, MR, Shen, CR, Smith, KM, Brynildsen, MP, Chou, KJ, Hanai, T., Liao, JC, 2008a. Metabolic engineering of *Escherichia coli* for 1-butanol production. Metabolic engineering. 10, 305-311.). All plastids were created using purified PCR fragments via Gibson isothermal DNA assembly. A list of plastids used is shown in Table 1. Except for the Escherichia coli thioesterase gene, which was derived from the ASKA (Escherichia coli K12 ORF complete library) plasmid library, the genes encoding Ict, Ich, Ccl, and thioesterase homologs were amplified from the corresponding commercially available genomic DNA: Pseudomonas aeruginosa (ATCC 47085D), Yersinia pseudotuberculosis (DSM 8992), Burkholderia xenovorans LB400 (DSM 17367), Salmonella enterica subsp. Enterica (DSM17058), Rhodobacter capsulatus (ATCC BAA-309), and Rhodobacter sphaeroides (DSM 5864). Phenylacetyl-CoA thioesterase (PaaI)* D16 and H52 saturated variants were created using primers containing mutant DNA sequences. Table 1 strain genotype Reference source BW25113 rrnB T14 ∆lacZWJ16hsdR514 ∆araBADAH33 ∆rhaBAD LD78 Datsenko, K. A., Wanner, B. L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences. 97,6640-6645. XL-1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F’proAB lacI q Z∆M15 Tn10(Tet R )] Stratagene JCL 16 BW25113 / F’ [traD36,proAB + ,lacI q Z∆M15(Tet r )] Atsumi, S., Cann, A. F., Connor, M. R., Shen, C. R., Smith, K. M., Brynildsen, M. P., Chou, K. J., Hanai, T., Liao, J. C., 2008a. Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic engineering. 10,305-311. JCL 299 JCL 16 ΔldhAΔadhEΔfrdBCΔpta Atsumi, S., Cann, AF, Connor, MR, Shen, CR, Smith, KM, Brynildsen, MP, Chou, KJ, Hanai, T., Liao, JC, 2008a. Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic engineering. 10, 305-311. JCY 298 JCL299 ΔgltA This invention JCY 299 JCL299 ΔyciA This invention JCY 399 JCL299 ΔyciAΔgltA This invention plastid genotype Reference source pSW26 P T5 :: His6X-PaIch; pUC ori; Kan R This invention pSW27 P T5 :: His6X-PaIct; pUC ori; Kan R This invention pSW28 P T5 :: His6X-PaCcl; pUC ori; Kan R This invention pSW29 P T5 :: His6X-BxIch; pUC ori; Kan R This invention pSW30 P T5 :: His6X-BxCcl; pUC ori; Kan R This invention pSW31 P T5 :: His6X-StIct; pUC ori; Kan R This invention pSW32 P T5 :: His6X-YpIct; pUC ori; Kan R This invention pSW33 P T5 :: His6X-YpIch; pUC ori; Kan R This invention pSW34 P T5 :: His6X-YpCcl; pUC ori; Kan R This invention pSW39 P T5 :: His6X-RcCcl; pUC ori; Kan R This invention pSW41 P L lacO1::PaIch-YpCcl-YpIct; ColE1 ori; Amp R This invention pIA1 P L lacO1::PaIch-YpCcl; p15A ori; Spec R This invention pIA16 P T5 :: RsMcl2; pUC ori; Kan R This invention pIA26 P L lacO1::PaIch-YpCcl-acs; p15A ori; Spec R This invention pASKA-TE a P T5-lac :: His6X-TEa ; pBR322 ori; CM R This invention pASKA-paaI* b P T5-lac :: His6X-paaI* b ; pBR322 ori; CM R This invention a TE represents the 17 different E. coli genes contained in the ASKA library plasmids (as shown below): fadM, ydiI, yciA, ybgC, ybfF, ypfH, tesA, tesB, ybaC, yjfP, bioH, ybdB, frsA, paaI, paaY, yeiG, yqiA. b PaaI* D16 and H52 saturated mutants were selected and colonized onto the same skeleton as the wild type. c The abbreviations indicate the gene origin: Pa, Pseudomonas aeruginosa; Bx, Burkholderia xenovorans; St, Salmonella enterica serovar typhimurium; Yp, Yersinia pseudotuberculosis; Rc, Rhodobacter capsulatus; Rs, Rhodobacter sphaeroides. d Genes without source abbreviations originate from E. coli.

[0033] The culture medium and process for itaconic acid production are as follows. For all live itaconic acid production and screening experiments, a single colony was selected from an LB (Luria Broth) plate and inoculated into a test tube containing 2 mL of LB medium containing appropriate antibiotics (kanamycin 50 μg / mL, spectinomycin 50 μg / mL, and chloramphenicol 50 μg / mL). The following day, unless otherwise specified, the overnight culture grown at 37°C was inoculated at 1% (v / v) into 20 mL terrific broth (TB) supplemented with 20 g / L glucose and appropriate antibiotics in 125 mL screw-cap flasks (PYREX 4985-125) containing 12 g trypsin, 24 g yeast extract, 2.31 g KH2PO4, 12.54 g K2HPO4, and 4 mL glycerol per liter of water. The culture was allowed to grow at 37°C on a rotary shaker (250 rpm) until an OD600 of 0.4–0.6 was reached, followed by induction with 0.1 mM IPTG and switching to 30°C. Samples were collected and centrifuged throughout the production process to recover the supernatant. For itaconic acid production using YpIct, unless otherwise specified, 5 g / L succinic acid was fed during induction.

[0034] Except for modifications summarized below, the same culture media and conditions as described above were used in all figures. For Figure 2B, production culture was performed using 250 mL baffle flasks (PYREX 4444-250). For Figure 4, the inoculated culture (1% v / v, overnight) was first grown in 500 mL baffle flasks (PYREX 4444-500) at 37°C on a rotary shaker (250 rpm) until an OD600 of 0.4–0.6 was reached. Once the exponential phase was reached, the culture was induced with 0.1 mM IPTG and the temperature was switched to 30°C. After induction at 30°C for 4 hours, the induced culture was aliquoted into the different culture containers shown in the diagram for further itaconic acid production. The specific container product numbers are as follows: Container 1 is a PYREX 250 mL Delong conical flask (with baffle) (4444-250) with a KIMBLE KIM-KAP polypropylene cap (73660-38); Containers 2, 3, and 4 are PYREX 250 / 125 mL narrow-mouth conical flasks with phenolic screw caps (4985-250 / 125); Containers 5, 6, and 7 are BD vacuum blood collection tubes (BD 367820). In the cases shown in Figures 3B and 6A–6B, glucose was added to the production culture every 12 hours (10 g / L for the first 48 hours, then 5 g / L), and the pH of the culture was adjusted to 7.2 using 10 M NaOH or 5 M KOH:NH4OH (1:2, v / v, Figure 6B). Figure 6B shows long-term production using concentrated cells. The inoculated culture was first grown to the exponential phase at 37°C, and then induced for 4 hours at 30°C using the same containers and conditions described in Figure 4. Next, the induced cells were rotated down and resuspended in 20 mL of fresh TB 2% glucose medium to an initial OD600 of approximately 8, and then allowed to culture for several days in 125 mL screw-cap flasks (PYREX 4985-125) at 30°C and 200 rpm.

[0035] The protein purification procedure is as follows. Protein purification was performed using the Zymo Research (Irvine, CA) His-Spin Protein Mini-prep Purification kit. XL-1 strain containing the target enzyme was cultured overnight and inoculated into 20 mL of fresh LB containing appropriate antibiotics. The culture was incubated at 37°C until the exponential phase was reached, then induced with 0.1 mM IPTG. The induced culture was then switched to 30°C for protein expression. After 18 to 24 hours, the culture was obtained by centrifugation, and the resulting precipitate was resuspended in 1 mL of His-binding buffer provided in the kit. The resuspended culture was mixed with 1 mL of 0.1 mm glass beads (Biospec) and homogenized using a MiniBeadBeater-16, model 607 (Biospec). The soluble fraction was collected by centrifugation at 4°C. The supernatant was washed and eluted with buffer according to the Zymo procedure via a His-binding column. The purified protein was mixed with glycerol and stored at -80°C. Protein concentration was determined using Bradford reagent with BSA as a standard.

[0036] Regarding the in vitro production of itaconic acid using purified enzymes. The reaction mixture contained 100 mM pyruvate, 2.5 mM acetyl-CoA, 100 mM succinate, 5 mM DTT, 5 mM MgCl2, and 0.5 µM of various purified enzymes prepared in 0.1 M MOPS-KOH buffer (pH 7.0). The reaction was terminated at different time points by adding 1 M HCl. Protein debris was removed by centrifugation, and the sample was then neutralized to pH 7 by adding 10 M NaOH for HPLC analysis.

[0037] The preparation process for the CoA intermediates is as follows. Since commercially available itaconic acid-co-A (itaconyl-CoA) and (S)-citramalyl-CoA ((S)-citramalyl-CoA) are unavailable, purified YpIct and PaIch were used for the enzymatic synthesis of these two coenzyme A intermediates. To synthesize itaconic acid-co-A (reaction A), the reaction mixture contained 1 or 2 mM acetyl-co-A, 10 mM itaconic acid, and 0.025 g / L YpIct, dissolved in 0.1 M MOPS-KOH buffer (pH 7). After incubation at 30°C for 30 minutes (or at different time points as shown in Figures 5A-5C), the reaction was terminated by adding 10M HCl to a pH of approximately 1. Protein debris was removed by centrifugation at 4°C, and the pH was then neutralized to 7 with 10M NaOH. To synthesize (S)-limonene-CoA (reaction B), reaction A was terminated and neutralized, followed by further mixing with 0.025 g / L PaIch to convert itacone-CoA to (S)-limonene-CoA. It was later found that adding YpIct and PaIch simultaneously to reaction A also yielded excellent (S)-limonene-CoA, and was therefore chosen as the final preparation method. The reaction was terminated and neutralized using the same procedure as described above. The resulting sample was passed through a Nanosep column (Pall Life Sciences) and analyzed using HPLC for different CoA intermediates. Chromatographic peaks corresponding to itacone-CoA and (S)-limonene-CoA were identified by examining the corresponding increase and / or decrease in peak area during the target reaction. The concentration of synthesized itacone-CoA was estimated using the acetyl-CoA absorbance / concentration standard curve and the corresponding decrease in the acetyl-CoA peak in reaction A. Similarly, the concentration of synthesized (S)-limonene-CoA was estimated using the itaconic A standard curve and the corresponding decrease in the itaconic A peak in reaction B. Notably, a certain degree of spontaneous dissociation of CoA from both itaconic and acetyl-CoA was observed and subtracted during the calculation of the itaconic A standard curve and PaaI kinetic assay.

[0038] The kinetic analysis procedure is as follows. The activity of *E. coli* PaaI for different coenzyme A intermediates was determined by monitoring the decrease in the corresponding aceto-CoA peak using HPLC. The reaction mixture A or B obtained from itaconic A or (S)-limonene-CoA preparations was mixed with 0.1 M KCl and purified PaaI (0.02–0.04 g / L). The initial aceto-CoA acceptor concentrations were as follows: 1 mM aceto-CoA (commercial), 0.5 mM itaconic A (estimated based on preparation), or 0.8 mM (S)-limonene-CoA (estimated based on preparation). The reaction was initiated by adding PaaI and carried out at 30°C. At different time points, the reaction was terminated by adding 10 M HCl to a pH of approximately 1. Protein debris was removed by centrifugation at 4°C, and the pH was then neutralized to 7 using 10 M NaOH. The obtained samples were analyzed using HPLC.

[0039] The activity of YpCcl for pyruvate and acetyl-CoA was also determined by monitoring the decrease in the acetyl-CoA peak using HPLC. The reaction mixture contained 10 mM pyruvate, 1 mM acetyl-CoA, and purified YpCcl (0.025 g / L) dissolved in 0.1 M MOPS-KOH buffer (pH 7). The reaction was terminated and analysis was performed using the same procedure as described above.

[0040] The quantitative procedure for metabolites is as follows. Itaconic acid, glucose, and other organic acids were quantitatively analyzed using an Agilent 1260 HPLC equipped with an autosampler and an Agilent Hi-Plex H column (5 mM H₂SO₄, 0.6 mL / min, column temperature 50°C). Glucose was measured using a refractive index detector, and organic acids were detected at 210 nm using a photodiode array detector. For the identification and analysis of free coenzyme A and coenzyme A ester, a Shimadzu Prominence-i LC-2030C 3D Plus HPLC equipped with a GL Sciences C18 column (50 mM KH₂PO₄, pH 5.3 and methanol (9:1, v / v), 1 mL / min, column temperature 40°C) was used. Detection was performed at 259 nm, and concentrations were determined by extrapolation from a standard curve.

[0041] Regarding the use of YpCcl and PaIch for limiting step analysis. The reaction mixture contained 100 mM pyruvate, 2 mM acetyl-CoA, 5 mM DTT, and 5 mM MgCl2, dissolved in 0.1 M MOPS-KOH (pH 7). Purified YpCcl and PaIch were added in a ratio of 1 or 3 to a concentration of 1 or 3 µM. The reaction was allowed to proceed at 30°C for 30 minutes, and then terminated using the same procedure described in the kinetic analysis. The samples were analyzed by HPLC to obtain the concentrations of itaconic acid-CoA and (S)-limonene-CoA. Example 1. Assessment of itaconic acid production by catabolic enzymes

[0042] To examine the inherent possibility of itaconic acid accumulation via its degradation pathway, this embodiment first investigated enzyme candidates from naturally occurring itaconic acid-degrading microorganisms. The encoding genes for citrate-CoA lyase (Ccl), itaconic-CoA hydratase (Ich), and itaconic-CoA transferase (Ict) from *Yersinia pseudotuberculosis* (YP), *Pseudomonas aeruginosa* (Pa), and *Burkholderia xenovorans* (Bx), along with several other homologs identified by protein sequence alignment and bioprospecting, were overexpressed in *Escherichia coli*. Ict from *Salmonella typhimurium* was selected as an alternative due to its similar pathogenicity and 76% protein identity with YpIct. Malyl-CoA lyase from *Rhodobacter capsulatus* (RcCcl) naturally catalyzes the reversible condensation of glyoxylate with either acetyl-CoA or amylate-CoA, and its potential non-specific activity toward pyruvate was also tested. Thermodynamic calculations using the eQuilibrator for each reaction show that the overall Gibbs free energy change of the itaconic acid catabolism pathway is very close to neutral, with no natural impetus in the synthetic direction (see Figure 1). Even if all three enzymes function effectively, observing itaconic acid formation intracellularly can be challenging without a kinetic trap, especially given the strong competition between pyruvate and acetyl-CoA for other essential reactions. This pathway is highly efficient for itaconic acid detoxification, requiring only a catalytic amount of coenzyme A ester to degrade high concentrations of itaconic acid. In the case of YpIct, which exhibits loose coenzyme A ester specificity, itaconic acid can be effectively activated by acetyl-CoA, propionyl-CoA, and butyric acid, in addition to the natural co-acceptor succinate-CoA. The ability to utilize acetyl-CoA, the final product of itaconic acid detoxification, as a coenzyme A donor for the next round of itaconic acid activation avoids the need for a fresh succinate-CoA supply and further enhances the efficiency of the degradation pathway.

[0043] To assess the feasibility of producing itaconic acid via degradative enzymes, in vitro biosynthesis of itaconic acid was first performed using purified enzymes to ensure the functional performance of each enzyme and to allow the precursors pyruvate and acetyl-CoA to reach saturation concentrations. Then, different combinations of bioprospective enzyme homologs were examined sequentially based on itaconic acid formation efficiency.

[0044] Figures 2A and 2B show the performance of itaconic acid as evaluated by catabolic enzymes. (Figure 2A) In vitro validation and comparison of different candidate enzymes found in the itaconic acid degradation pathway. Purified enzymes were used in various combinations for in vitro itaconic acid synthesis. (Figure 2B) In vivo production of itaconic acid using YpCcl, PaIch, and YpIct. Strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) was transformed by plasmid pSW41 and produced itaconic acid for 48 hours with or without succinic acid. Error bars represent the standard deviation of three replicates. PA stands for Pseudomonas aeruginosa; YP stands for Yersinia pseudotuberculosis; BX stands for Burkholderia xenovorans; ST stands for Salmonella enterica serovar typhimurium; RC stands for Rhodobacter capsulatus; Ccl stands for citramalyl-CoA lyase; Ich stands for itaconyl-CoA hydratase; Ict stands for itaconyl-CoA transferase.

[0045] As shown in Figure 2A, YpCcl, PaIch, and YpIct showed the highest in vitro itaconic acid accumulation, reaching nearly 10 mg / L within 30 minutes. Other homolog combinations resulted in a 10-80% reduction in itaconic acid formation levels, with RcCcl showing the worst performance, indicating that despite its high structural similarity to the natural acceptor glyoxylic acid, it has a low acceptor preference for pyruvate. Next, to further examine the potential for itaconic acid accumulation based on intracellular pyruvate and acetyl-CoA concentrations, this embodiment selected the best-performing enzyme into the operon (PaIch-YpCcl-YpIct, plastid pSW41) and attempted to synthesize itaconic acid in vivo by overexpressing this catabolistic pathway in the fermentation-deleted strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta). Studies have shown that eliminating fermentation can improve the production of biochemical products derived from pyruvate and / or acetyl-CoA, such as isobutanol, 1-butanol, and 2,3-butanediol. In this example, the use of JCL299 increased itaconic acid formation levels (~8 mg / L) by approximately four times compared to yields achieved using wild-type *E. coli*. As shown in Figures 2A and 2B, similar levels of itaconic acid production were observed in vitro and in vivo with the addition of succinate as an essential co-acceptor for itaconic A transferase. This observation indicates that intracellular pyruvate and acetyl-CoA concentrations are sufficient to drive carbon flux into itaconic acid via a highly reversible pathway. However, the low accumulation of itaconic acid in both in vivo and in vitro conditions appears to stem from the reversibility of the pathway and a lack of driving force. Therefore, a synthetic driver is needed to further drive carbon flux into the target itaconic acid. Example 2. Screening for thioesterases to promote itaconic acid production.

[0046] In previous studies, NADH accumulation and pathway recombination via an ATP-dependent response have successfully directed carbon flux to the production of 1-butanol via a highly reversible β-oxidation pathway. However, the itaconic acid degradation pathway has not shown any cofactor or redox dependence that could be directly used as a driving force, nor has it exhibited auxotrophic characteristics. Since metabolic pathways found in nature (such as fatty acid biosynthesis) are generally driven by acetyltransferase potential, replacing coenzyme A transferase with thioesterase should provide an efficient kinetic trap to irreversibly push carbon into itaconic acid. Since no thioesterase with itaconic coenzyme A activity has been found, this embodiment bioprospects endogenous thioesterases in *E. coli* because they are readily apparent and have potential for substance contamination, as it is generally necessary to eliminate native thioesterases to minimize the hydrolysis of coenzyme intermediates into acidic byproducts. In addition to thioesterases involved in fatty acid biosynthesis, *E. coli* also possesses a variety of thioesterases with undefined physiological functions. Seventeen enzymes with thioesterase activity (FadM, YdiI, YciA, YbgC, YbfF, YpfH, TesA, TesB, YbaC, YjfP, BioH, YbdB, FrsA, PaaI, PaaY, YeiG, YqiA) and (3S)-malyl-CoA thioesterase (Mcl2) from Rhodobacter sphaeroides, which has potential activity against itaconic acid, were selected. Strains JCL299 containing plastid pIA1 (PLlac01::PaIch-YpCcl) and different thioesterases overexpressing from the pBR302 backbone were then cultured under microaerobic conditions, and the formation of itaconic acid was compared.

[0047] Figures 3A and 3B show the screening of thioesterases driving itaconic acid biosynthesis. (Figure 3A) Strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) was co-transformed with plastids pIA1 and pSW32 (containing YpIct as a negative control) or pIA1 containing the corresponding thioesterase gene and ASKA plastids. Samples were taken after 24 hours of culture. (Figure 3B) Confirmation and comparison of two optimal positive thioesterases for itaconic acid production after 48 hours under optimized culture conditions (glucose supplementation and pH adjustment every 12 hours). The same transformed strain as in Figure 3A was used. Error bars represent the standard deviation of three replicates.

[0048] As shown in Figure 3A, almost all thioesterase candidates exhibited some degree of hydrolytic activity against itaconic acid coenzyme A, reflected in itaconic acid secretion levels of 10 to 40 mg / L over 24 hours. YpIct, which exhibits an overexpression of a skeletal system similar to thioesterases, was used as a negative control for comparison.

[0049] Thioesterases that produce high levels of itaconic acid (>15 mg / L) have been identified as YciA, YbgC, YjfP, and PaaI. Interestingly, the three enzymes other than YjfP belong to type II thioesterases and have been shown to have broad acceptor activity. Among the two thioesterases with the highest itaconic acid production, YciA has been reported to have broad acceptor selectivity, accepting short chains (such as acetyl-CoA and isobutyryl-CoA) and long chains (such as n-decanoyl-CoA and oleoyl-CoA), aliphatic acyl-CoA, and phenyl acyl-CoA). On the other hand, PaaI, found in the phenylacetic acid degradation pathway, is generally considered to be the hydrolysate of phenylacetyl-CoA and its monohydroxy and dihydroxy ring analogs, rather than aliphatic acetylated CoA thioesters. To further confirm the superior performance of PaaI, itaconic acid production was repeatedly carried out using the thioesterase PaaI and YciA under optimized culture conditions and pH adjustment, with the coenzyme A transferase YpIct as a control (Figure 3B). Notably, the redox-neutral nature of the reverse itaconic acid degradation pathway places oxygen demand in a challenging position. Lower oxygen supply is generally ideal for maintaining higher levels of acetylated CoA and pyruvate in the fermentation-deleted strain (JCL299). However, reducing power recovery is required in JCL299, making respiration indispensable.

[0050] Figure 4 shows the preliminary assessment of the ideal aeration level by changing the culture vessel and liquid volume. The left figure shows the culture apparatus, with methods labeled (1) to (7), corresponding to the x-axis on the graph. Transformed plasmids pIA1 and pASKA-paaI strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) were used. Samples were taken after 24 hours of culture. Error bars represent the standard deviation of three replicates.

[0051] As shown in Figure 4, a preliminary assessment of the ideal aeration level was conducted by changing the culture container and liquid volume. The results showed that microaerobic culture (20 mL of culture in a 125 mL unbaffled screw-cap flask) achieved the optimal itaconic acid yield. On the other hand, extreme oxygen supply (complete aeration in a baffled flask and minimal aeration in a sealed tube with a large culture volume) both resulted in a significant reduction in itaconic acid accumulation. Finally, by implementing optimal aeration conditions and pH adjustment, the amount of itaconic acid formed using PaaI was greatly increased to 150 mg / L within 48 hours (Figure 3B), which is approximately 4 times and 15 times higher than that achieved by YciA and the transferase YpIct, respectively. Example 3. Kinetic analysis of the effect of thioesterase PaaI on coenzyme A intermediate in the itaconic acid pathway

[0052] As previously reported, many type II thioesterases exhibit nonspecific activity against a variety of aceto-CoA receptors. Since the itaconic acid degradation pathway involves three different aceto-CoA intermediates—acetyl-CoA, (S)-limonene-CoA, and itaconic acid-CoA—this embodiment characterizes the thioesterase activity of the positive candidate PaaI against each CoA receptor. Because (S)-limonene-CoA and itaconic acid-CoA are not commercially available, these two CoA compounds were first prepared in vitro via itaconic acid degradation.

[0053] Figures 5A-5C show the HPLC preparation and analysis of coenzyme A thioesters. (Figure 5A) Itaconic acid and acetyl-CoA were incubated with YpIct for 30 minutes to form itaconic-CoA. (Figure 5B) After the formation of itaconic-CoA in the reaction shown in Figure 5A, PaIch was added to the reaction mixture and incubated for 30 minutes to convert itaconic-CoA to (S)-limonene-CoA. Simultaneous incubation of acetyl-CoA and itaconic acid with YpIct and PaIch can convert acetyl-CoA to (S)-limonene-CoA with a conversion rate as high as that shown in the sequential reaction, and therefore it was selected as the final synthesis method. (Figure 5C) Thioesterase PaaI was incubated with itaconic-CoA prepared in the reaction shown in Figure 5A.

[0054] As shown in the HPLC chromatograms (Figures 5A-5C), itaconic acid and acetyl-CoA were cultured with coenzyme A transferase YpIct or hydratase PaIch, and different new peaks were formed using a C18 column with coenzyme A retention capacity. In this example, acetyl-CoA was used as the coenzyme A donor because it is comparable in efficiency to succinyl-CoA and works in conjunction with YpIct. When only YpIct was present, the new peak at approximately 15.5 minutes gradually expanded over time, from 0 to 30 minutes, while the acetyl-CoA peak decreased with a similar aspect ratio. PaIch was added after the 15.5-minute peak, resulting in the appearance of a peak at 7.6 minutes, while the area of ​​the 15.5-minute peak decreased. Based on their respective reaction sequences and peak characteristics, the 15.5-minute peak was identified as itaconic-CoA, and the 7.6-minute peak was identified as (S)-limonene-CoA. By incubating limonene-coenzyme A lyase YpCcl with pyruvate and acetyl-CoA, the (S)-limonene-coenzyme A peak was further confirmed. As acetyl-CoA was consumed, the peak at 7.6 minutes gradually appeared.

[0055] After successful preparation and identification of the coenzyme A intermediate, the kinetics of the thioesterase PaaI were further determined. Contrary to the general observation of a wide acceptor range for type II thioesterases, PaaI exhibited relatively low activity for itaconic A (0.17 U / mg, U: μmol / min) compared to the reported levels of its most active acceptor, hydroxyacetyl-CoA, and even lower activity for acetyl-CoA (0.015 U / mg). Furthermore, no activity was detected against the pathway intermediate (S)-limonene-CoA, which was also reflected in the absence of citramalate accumulation in itaconic acid production broth. According to the kinetic analysis of this example, the (S)-limonene-CoA lyase YpCcl exhibited suboptimal but higher activity for acetyl-CoA compared to the levels observed in PaaI (Table 2). Table 2 enzyme Subject Specific activity (U / mg) PaaI Acetyl coenzyme A 0.015 ± 0.006 (S)-Limonene Coenzyme A Undetected (ND) Itacryl Coenzyme A 0.17 ± 0.02 YpCcl Acetate-CoA + Pyruvate 0.06 ± 0.02

[0056] PaaI's preference for itaconic acid-coenzyme A, relative to other coenzyme A intermediates in this pathway, helps minimize the loss of key precursors due to nonspecific hydrolysis and significantly promotes the superior performance of PaaI in itaconic acid production. It is speculated that *E. coli* PaaI belongs to the same superfamily as *Arthrobacter* 4-hydroxyphenacyl-CoA thioesterase. Structural and activity analyses indicate that it is functionally independent of the benzoic acid-based metabolic pathway and exhibits undetectable or minimal activity towards benzoic acid-coenzyme A and aliphatic acetylated coenzyme A thioesters, respectively. Notably, extremely low activity toward C2- to C6 saturated aliphatic thioesters (e.g., acetyl-CoA, butyric-CoA, and hexyl-CoA) and methylmalonidin-CoA, which is structurally similar to itaconic-CoA in terms of carboxylation and methylation characteristics, was also observed in the PaaI homolog of *Arthrobacter*. This example reveals that *E. coli* PaaI thioesterase possesses relatively good itaconic-CoA activity, which increases our understanding of its acceptor range. Since itaconic-CoA activity can be enhanced without overcoming limited activity toward other CoA intermediates, production of itaconic acid via this CoA-binding pathway should greatly benefit from the specificity of PaaI. Example 4. Acetic acid recovery and minimization

[0057] During the optimization of itaconic acid production, a large amount of acetic acid was observed to be secreted (>10 g / L), indicating that the reverse degradation pathway was insufficient to isolate the high-throughput pyruvate and acetyl-CoA. Small amounts of pyruvate (~1 g / L) were also observed in the production medium. As previously described, this example employed microaerobic culture for itaconic acid biosynthesis to allow the accumulation of precursors in the fermentation-deleted strain JCL299 and NADH oxidation via respiration. Testing of (S)-limonene-CoA lyase (YpCcl), which catalyzes the reversible condensation of pyruvate and acetyl-CoA, showed relatively low activity (0.06 U / mg, Table 2) compared to other endogenous competing reactions. Therefore, the suboptimal reaction rate may have led to acetic acid spillover. Insufficient oxygen supply may further promote the formation of ATP-producing acetic acid, which cannot be eliminated solely by PTA deletion.

[0058] Initially, an attempt was made to increase the condensation rate of YpCcl by preserving acetyl-CoA by eliminating citrate synthase (gltA), which is responsible for entering the TCA cycle. However, itaconic acid levels remained unchanged. Since acetic acid is frequently excreted during E. coli fermentation due to metabolic overflow, the most feasible approach is to recover acetic acid for acetyl-CoA. This also facilitates the conversion of acetyl-CoA lost due to the presence of nonspecific thioesterase activity in the cells back into acetic acid. Indeed, overexpression of acetyl-CoA synthase (Acs) resulted in a 30% increase in itaconic acid production; however, similar amounts of acetic acid secretion were observed. Further literature suggests that the highly efficient heterozygous activity of the native thioesterase YciA for acetyl-CoA generally contributes to the accumulation of acetic acid.

[0059] Figures 6A and 6B show the improvement of itaconic acid production by recovering acetic acid and minimizing its formation. (Figure 6A) Different optimization strategies and their itaconic acid formation after 96 hours. Strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) was used as the base strain, with additional gene knockouts indicated by "Δ" in the table. Cells transformed into the corresponding plastids are marked with "✓". Operon structures are shown next to each plastid name. (Figure 6B) Long-term itaconic acid production using high cell density. Time indicates the time required for cell concentration and resuspending in fresh medium. Error bars represent the standard deviation of three replicates.

[0060] As shown in Figure 6A, deletion of endogenous YciA not only reduced acetic acid formation but also further increased itaconic acid production by 15% compared to the level in strain JCL299. Notably, YciA was the second-best candidate enzyme for better itaconic acid formation compared to other screened endogenous thioesterases. The high activity of YciA for acetyl-CoA is a significant drawback for itaconic acid synthesis using this CoA-binding pathway. Therefore, when designing thioesterases to obtain better itaconic A activity, it is essential to maintain a low preference for acetyl-CoA and other CoA intermediates.

[0061] After the initial metabolic pathway adjustment was completed, condition optimization continued. Through a series of tests involving ventilation, pH, intermittent glucose supplementation, and cell density, an itaconic acid yield of 1 g / L was achieved within 120 hours (as shown in Figure 6B), nearly 100-fold higher than the initial level obtained using coenzyme A transferase. Although the current productivity is still not optimal, this invention successfully demonstrates the effectiveness of setting thermodynamic traps in determining the pathway direction. Compared to the final irreversible thioester hydrolysis step, the first two reactions may now be the limiting factors for itaconic acid formation rate.

[0062] Incorporating irreversible steps into the pathway design can enhance overall thermodynamic favorability and typically yields better biotransformation yields given a functional enzyme. Utilizing this concept, this invention constructs the first non-natural itaconic acid biosynthetic pathway driven by thioester hydrolysis. Itaconic acid is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Itaconic acid production has long relied on the decarboxylation of the TCA cycle intermediate cis-aconitine as the sole biosynthetic pathway. Inspired by nature's itaconic acid detoxification design, this invention provides a novel itaconic acid production pathway that is orthogonal to natural metabolism and requires no auxotrophic elimination.

[0063] Figure 7 shows that, compared to the natural fungal pathway which produces 1 mol of ATP for every 1 mol of glucose converted to itaconic acid, the reverse degradation pathway produces 2 mol of ATP due to its unique pathway characteristics. Detailed stoichiometry and reaction equilibrium of the two itaconic acid formation pathways can be observed, where PEP represents phosphoenolpyruvate; OAA represents oxaloacetate. The maximum theoretical yield and NADH balance remain unchanged between the two pathways. The reverse degradation pathway begins with the condensation of pyruvate and acetyl-CoA to form (S)-citrate-CoA, which then undergoes dehydration isomerization to itaconic-CoA, followed by hydrolysis to itaconic acid. Screening revealed that the highest itaconic acid formation efficiency is achieved when phenylacetyl-CoA thioesterase (PaaI) in *E. coli* binds to the reversible activities of citrate lyase and itaconic-CoA hydratase. PaaI preferentially hydrolyzes itaconic acid coenzyme A instead of acetyl-CoA and (S)-limonene-CoA, thus minimizing the inevitable precursor loss due to enzyme contamination. Through acetic acid recycling, acetyl-CoA protection, and condition optimization, this invention achieves a final itaconic acid yield of 1 g / L using a thioesterase-driven pathway, a significant improvement over previous coenzyme A transferase-based degradation pathways. This invention illustrates the importance of thermodynamic advantage as a principle in pathway engineering design.

[0064] In summary, this invention provides a novel itaconic acid synthesis method driven by thioester hydrolysis, which has not been reported in previous studies. By identifying phenylacetyl-CoA thioesterases with itaconic A hydrolytic activity, this invention successfully reconnects the itaconic acid detoxification pathway of itaconic acid biosynthesis. Compared with the natural fungal pathway via the TCA cycle, the proposed itaconic acid pathway exhibits the same theoretical maximum glucose yield and the same redox balance. However, through the reverse degradation pathway, an additional one mole of ATP is generated for every mole of itaconic acid synthesized, which is a beneficial characteristic in microbial biosynthesis. Although current itaconic acid production is lower than conventional production levels, the abundance of pyruvate and acetyl-CoA and the good performance of bacterial enzymes should make this pathway have good development potential in other unique hosts such as cyanobacteria or methanotrophs. Currently, the synthetic itaconic acid pathway appears to be mainly limited by the low efficiency of itaconic A hydrolysis. Therefore, designing thioesterases to obtain better itaconic acid-CoA activity while maintaining high specificity will be the next important step to further improve itaconic acid produced using this pathway.

[0065] The above description is merely illustrative and not restrictive. Any equivalent modifications or alterations made without departing from the spirit and scope of this invention shall be included in the appended claims. [Simplified Explanation of the Diagram]

[0023] Figure 1 shows a schematic diagram of itaconic acid production driven by thioesterases through their degradation pathway. The standard Gibbs free energy change (ΔrG'0) for each step in the itaconic acid synthesis direction was calculated using eQuilibrator and is shown in the gray box. The Gibbs free energy change at a mass concentration of 1 mM, calculated by eQuilibrator, is also shown as ΔrG'm, which may be closer to the intracellular state compared to 1 M under standard conditions. Solid circles indicate the abbreviations for each enzyme. The eQuilibrator calculation conditions were set to pH 7.5 and ionic strength 0.25 M. Figures 2A and 2B show the performance of itaconic acid through catabolic enzymes. (Figure 2A) In vitro validation and comparison of different candidate enzymes found in the itaconic acid degradation pathway. Purified enzymes were used in various combinations for in vitro itaconic acid synthesis. (Figure 2B) In vivo production of itaconic acid using YpCcl, PaIch, and YpIct. Strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) was transformed by plasmid pSW41 and produced for 48 hours with or without succinic acid. Error bars represent the standard deviation of three replicates. PA represents Pseudomonas aeruginosa; YP represents Yersinia pseudotuberculosis; BX represents Burkholderia xenovorans; ST represents Salmonella enterica serovar typhimurium; RC represents Rhodobacter capsulatus; Ccl represents citramalyl-CoA lyase; Ich represents itacone-CoA hydratase; Ict represents itacone-CoA transferase. Figures 3A and 3B show the screening of thioesterases driving itaconic acid biosynthesis. (Figure 3A) Strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) was co-transformed with plastids pIA1 and pSW32 (containing YpIct as a negative control) or pIA1 containing the corresponding thioesterase gene and ASKA plastids. Samples were taken after 24 hours of culture. (Figure 3B) Confirmation and comparison of two optimal positive thioesterases for itaconic acid production at 48 hours under optimized culture conditions (glucose supplementation and pH adjustment every 12 hours). The same transformed strain as in Figure 3A was used. Error bars represent the standard deviation of three replicates. Figure 4 shows the preliminary assessment of ideal aeration levels by changing the culture vessel and liquid volume.The left figure shows the culture apparatus, with method labels (1) to (7), corresponding to the x-axis on the graph. Transformed plasmids pIA1 and pASKA-paaI strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) were used. Samples were taken after 24 hours of culture. Error bars represent the standard deviation of three replicates. Figures 5A-5C show the HPLC preparation and analysis of coenzyme A thioesters. (Figure 5A) Itaconic acid and acetyl-CoA were incubated with YpIct for 30 minutes to form itaconic-CoA. (Figure 5B) After the formation of itaconic-CoA in the reaction shown in Figure 5A, PaIch was added to the reaction mixture and incubated for 30 minutes to convert itaconic-CoA to (S)-limonene-CoA. Simultaneous co-cultivation of acetyl-CoA and itaconic acid with YpIct and PaIch resulted in the conversion of acetyl-CoA to (S)-limonene-CoA, with a conversion rate as high as that shown in the sequential reaction, and was therefore selected as the final synthetic method. (Fig. 5C) Co-cultivation of itaconic acid prepared by the thioesterase PaaI with the reaction shown in Fig. 5A. Figs. 6A and 6B show the increased itaconic acid yield by recovering acetic acid and minimizing acetic acid formation. (Fig. 6A) Different optimization strategies and the itaconic acid formation produced after 96 hours. Strain JCL299 (ΔldhA ΔadhE ΔfrdBC Δpta) was used as the base strain, with additional gene knockouts indicated by "Δ" in the table. Cells transformed into the corresponding plastids are marked with "✓". Operon structures are shown next to each plastid name. (Fig. 6B) Long-term itaconic acid production using high cell density. Time indicates the time required for cell concentration and resuspending in fresh medium. The error bars represent the standard deviation of three replicates. Figure 7 shows the detailed stoichiometry and reaction equilibrium of the two itaconic acid formation pathways. PEP represents phosphoenolpyruvate; OAA represents oxaloacetate.

Claims

1. A method for synthesizing itaconic acid driven by a thioesterase, comprising condensing pyruvate and acetyl-CoA through a reverse degradation pathway to form (S)-citramalyl-CoA, then dehydratisomerizing the (S)-citramalyl-CoA to itaconic acid, and subsequently hydrolyzing the itaconic acid to itaconic acid by a thioesterase-driven process, wherein the reverse degradation pathway utilizes itaconic acid-CoA hydratase (Ich) and (S)-citramalyl-CoA lyase (Ich). The thioesterase is derived from recombinant microorganisms and is selected from the following group: FadM, YdiI, YbgC, YbfF, YpfH, TesA, YbaC, YjfP, BioH, YbdB, FrsA, phenylacetyl-CoA thioesterase (PaaI), PaaY, and YeiG.

2. The method as described in claim 1, wherein the recombinant microorganism is Escherichia coli.

3. The method as described in claim 2, wherein the Escherichia coli is Escherichia coli strain JCL299.

4. The method as described in claim 3, wherein the *E. coli* strain JCL299 is co-transformed with pIA1 plastid and a complete set of *E. coli* K12 ORF Archive (ASKA) plastid, wherein the pIA1 plastid contains the PLlacO1 promoter, a gene encoding itaconic-CoA hydratase (PaIch) from *Pseudomonas aeruginosa*, a gene encoding (S)-citrin-CoA lyase (YpCcl) from *Yersinia pseudotuberculosis*, a p15A origin of replication, and a SpecR resistance gene.

5. The method as described in claim 1, wherein the itaconic acid yield is 10–40 mg / L over 24 hours.

6. The method as described in claim 1, wherein the PaaI produces itaconic acid at a yield of 150 mg / L within 48 hours.

7. The method of claim 2, wherein the Escherichia coli is cultured using a microaerobic culture condition having a culture container to culture volume ratio of 6.25:

1.

8. The method of claim 1, wherein the PaaI has specific activity of 0.15 to 0.19 U / mg against itaconic acid coenzyme A.

9. The method of claim 1, wherein the itaconic acid yield reaches 1 g / L within 120 hours.

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