Shikimate pathway-dependent catabolic microorganism

Abstract

The invention relates to the field of industrial biotechnology. The invention can be integrated in the biotechnological production of chemicals and fuels as it provides for a more sustainable alternative. In particular, the invention relates to the metabolic engineering of a microorganism for the production of an aromatic compound of interest.

Description

[0001]P135337PC00 Title: Shikimate pathway-dependent catabolic microorganism FIELD OF THE INVENTION The invention relates to the field of industrial biotechnology. The invention can be integrated in the biotechnological production of chemicals and fuels as it provides for a more sustainable alternative. In particular, the invention relates to the metabolic engineering of a microorganism for the production of an aromatic compound of interest. BACKGROUND OF THE INVENTION Aromatic compounds hold significant commercial value in various sectors, such as pharmaceutics, agrochemical industry, food and beverage industry, and cosmetics. Most of these compounds are currently synthesized from petroleum derivatives, raising substantial environmental concerns. Also, aromatic compounds can be produced by microorganisms. However, the yields in natural organisms are low and therefore the production is not efficient enough for industrial biotechnology. The major pathway in microorganisms to produce aromatic compounds is the shikimate pathway. Therefore, the shikimate pathway in microorganisms is a focal point in metabolic engineering for aromatic compound production. It is responsible for the biosynthesis of aromatic amino acids, which can serve as precursors to a variety of valuable aromatic compounds. Traditionally, metabolic engineering strategies focus on overexpressing genes related to the target pathway and eliminating competing pathways to increase product yield. However, this approach sometimes involves deleting essential reactions that render the strain auxotrophic, demanding supplementation with expensive nutrients and thus increasing production costs. Despite advancements in metabolic engineering, numerous challenges persist. The metabolic burden on host organisms, due to overexpression of shikimate pathway genes, can negatively affect cell growth and overall productivity. Additionally, the competing nature of the host’s native metabolism often diminishes the carbon flux towards the target aromatic compounds, thus reducing yields (Zhang et al., 2015). Feedback inhibition in the shikimate pathway poses another major issue. As the intracellular concentration of aromatic compounds increases, the pathway can self-regulate, decreasing its activity and thus reducing production efficiency (Papagianni, 2012). With increasing environmental concerns, a sustainable process for aromatic compound production is necessary. However, existing microbial production methods lack the required efficiency and scalability to replace petrochemical methods. The present invention pertains to a microorganism and a method to develop a microorganism that utilizes the shikimate pathway for growth while redirecting a significant portion of cellular flux towards production of aromatic compounds. In an exemplary embodiment, the catabolic network of Pseudomonas putida is successfully reprogrammed so that the catabolism occurs through the shikimate pathway, enabling the growth-coupled production of aromatic compounds. In another exemplary embodiment, the microorganism was developed to produce 4-hydroxybenzoate (4HB) as an aromatic compound of interest. Extensive phenotypic characterization demonstrated remarkable production yields, achieving up to 89% of the maximum theoretical pathway yield for the high-value 4HB. This yield is, to the best of the inventors’ knowledge, the highest achieved through microbial processes for any aromatic compound. Besides 4HB, the presented microorganism can be used as a platform for the production of a number of other aromatics. A further exemplary embodiment demonstrated equally high production yields for salicylate. The invention allows for sustainable and efficient production of aromatic compounds, addresses challenges associated with low yields and limited pathway utilization in previously developed microorganisms. This invention pertains to an improved method for the microbial production of aromatic compounds, such as 4-hydroxybenzoate (4HB) and salicylate, by engineering a new metabolic architecture in the bacterium Pseudomonas putida KT2440. The genetically modified microorganism offers high yields for the microbial production of an aromatic compound. The invention demonstrates that the shikimate pathway can function as the main catabolic route in a microorganism, and the metabolic engineered microorganism can serve as a useful tool to produce various shikimate pathway-derived compounds. SUMMARY OF THE INVENTION It is an object of the invention to provide a microorganism with an altered metabolic pathway for the production of an aromatic compound of interest. It is a further object of the invention to adapt a microorganism to produce an aromatic compound of interest. In a first aspect, the invention therefore provides a microorganism with an altered metabolic pathway for the production of an aromatic compound of interest, the microorganism comprising: i) functional deletion of pyruvate kinase (pyk) gene; functional deletion of pyruvate kinase A (pykA) gene; functional deletion of phosphogluconate dehydratase (edd) gene; and functional deletion of phosphoenolpyruvate carboxylase (ppc) gene, and ii) overexpression of at least one gene encoding an enzyme performing a reaction derived from the shikimate pathway that can support growth. In a further aspect, the invention provides a microorganism according to theinvention, further comprising one or more of: functional deletion of dehydroshikimate dehydratase (quiC) gene; functional deletion of periplasmic pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase H (pedH) gene; functional deletion of PQQ-dependent alcohol dehydrogenase E (pedE) gene; and functional deletion of phenoxybenzoate dioxygenase subunit alpha (pobA) gene. In a further aspect, the invention provides a microorganism according to the invention wherein the overexpression of at least one gene is selected from: chorismate pyruvate-lyase (ubiC); isochorismate synthase (menF) and isochorismate pyruvate lyase (pchB); and 3-hydroxybenzoate synthase (hyg5); or a combination thereof. In a further aspect, the invention provides a microorganism according to the invention, further comprising functional deletion of tRNA dimethylallyltransferase (miaA) gene. In a further aspect, the invention provides a microorganism according to the invention, further comprising an altered anthranilate synthase (trpE) to reduce activity of trpE, for example by a mutation. In a further aspect, the invention provides a method for adapting a microorganism to produce an aromatic compound of interest, wherein the method comprises: i) functionally deleting pyruvate kinase (pyk) gene; functionally deleting pyruvate kinase A (pykA) gene; functionally deleting phosphogluconate dehydratase (edd) gene; and functionally deleting phosphoenolpyruvate carboxylase (ppc) gene, and ii) overexpressing at least one gene encoding an enzyme performing a reaction derived from the shikimate pathway that can support growth. In a further aspect, the invention provides a method according to the invention, wherein the method further comprises one or more of: functionally deleting dehydroshikimate dehydratase (quiC) gene; functionally deleting PQQ-dependent alcohol dehydrogenase H (pedH) gene; functionally deleting PQQ-dependent alcohol dehydrogenase E (pedE) gene; and functionally deleting phenoxybenzoate dioxygenase subunit alpha (pobA) gene. In a further aspect, the invention provides a method according to the invention, wherein the overexpressing of at least one gene is selected from: chorismate pyruvate-lyase (ubiC); isochorismate synthase (menF) and isochorismate pyruvate lyase (pchB); and 3-hydroxybenzoate synthase (hyg5); or a combination thereof. In a further aspect, the invention provides a method according to the invention, further comprising functionally deleting tRNA dimethylallyltransferase (miaA) gene. In a further aspect, the invention provides a method according to the invention, further comprising altering anthranilate synthase (trpE) to reduce activity of trpE, for example by mutating trpE. In a further aspect, the invention provides a method for production of an aromatic compound of interest by a microorganism, comprising: i) providing a microorganism according to the invention, or a microorganism as produced by the method of the invention, and ii) incubating the microorganism in a growth medium, comprising a carbon source, such as glycerol, glucose, xylose, and / or cellobiose, and thereby producing an aromatic compound of interest, preferably wherein the produced aromatic compound is 3-hydroxybenzoate (3-HB), 4-hydroxybenzoate (4HB), or salicylate. In a further aspect, the invention provides a microorganism according to the invention or to a method according to the invention, wherein the microorganism is a bacterium or a fungus, for example, having an Entner-Doudoroff pathway, preferably a Gram-negative bacterium. In a further aspect the invention provides a microorganism according to the invention, or a method according to the invention, wherein the microorganism is a bacterium selected from: Agrobacterium, Alcaligenes, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Cupriavidus, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Serratia, Shigella, Staphylococcus, Streptomyces, Synnecoccus, and Zymomonas, preferably E. coli, P. putida, C. butyricum, X. campestris, C. glutamicum, and D. radiorans. In a further aspect the invention provides a microorganism according to the invention, or a method according to the invention, wherein the microorganism is Pseudomonas putida. In a further aspect the invention provides a microorganism according to the invention, or a method according to the invention, wherein the microorganism is a fungus selected from the group consisting of Aspergillus, Candida, Chrysosporium, Cryptococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma, Xanthophyllomyces and Yarrowia, preferably S. cerevisiae and A. niger. In a further aspect, the invention provides use of an aromatic compound produced by a method according to the invention, for the production of bioplastics, such as bio-polyesters and bio-polystyrenes, pharmaceuticals, agrochemicals, cosmetics, chemicals, such as dyes, detergents, or biofuel molecules. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Metabolic architecture of the shikimate pathway-dependent catabolism (SDC). Pathways and mutations that were engineered pre- and post-evolution to establish the final SDC strain. Abbreviations: GLY3P, sn-glycerol-3-phosphate; GLYALD, glyceraldehyde; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3- phosphate; 2-PG, 2-phosphoglycerate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; PEP, phosphoenolpyruvate; FBP, fructose bisphosphate, F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; X5P, xylose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabinoheptulosonate-7-phosphate; 3HDS, 3-dehydroshikimate; ANT, anthranilate; 4HB, 4-hydroxybenzoate; PCA, protocatechuate; SUC, succinate; OAA, oxaloacetate; glpR, glycerol regulon repressor; edd, phosphogluconate dehydratase; pedE / H, PQQ (pyrroloquinoline quinone) - dependent alcohol dehydrogenases; garK, glycerate kinase; pyk / A, pyruvate kinase; quiC, dehydroshikimate dehydratase; ubiC, chorismate pyruvate lyase; ppc, phosphoenolpyruvate carboxylase; trpE, anthranilate synthase. Figure 2: An overview of various conversions possible in the shikimate pathway to release pyruvate. Figure 3: In silico assessment of SDC. A) Growth rate predictions using FBA with glycerol as carbon source. Each pyruvate-releasing reaction was set as the sole pyruvate source. All reactions are compared to the growth rate of the wild-type which was set as 100% (µmax= 0.235 h-1). B) Metabolic scheme of the pyruvate-releasing reactions derived from the shikimate pathway. PYK, pyruvate kinase; AGPOP, DGTP:pyruvate 2-O-phosphotransferase; CYSTL, cystathionine b-lyase; SERD_L, L-serine deaminase; ME2, malic enzyme (NADP); OAADC, oxaloacetate decarboxylase; CYSDS, cysteine desulfhydrase; EDA, 2-dehydro-3-deoxy-phosphogluconate aldolase; MCITL2, methylisocitrate lyase; LDH_D, D-lactate dehydrogenase; LDH_D2, D-lactate dehydrogenase (q8); CHRPL, chorismate pyruvate lyase; APATr, B-alanine pyruvate aminotransferase; ALATA_L, L-alanine transaminase; DLYSPYRAT, D-lysinepyruvate aminotransferase; ARUH, L-arginine pyruvate transaminase; AEPTA, 2- aminoethylphosphonate pyruvate transaminase; OCAALD, 4-oxalcitromalate aldolase; L_LACDS, L-lactate dehydrogenase (ubiquinone); DAAD, D-amino acid dehydrogenase; ADCL, 4-aminobenzoate synthase; ANS2, anthranilate synthase 2; 3AIBT2, L-3 aminoisobutyrate transaminase; ANS, anthranilate synthase. Figure 4: Setting up the base strain and biosensor for adaptive laboratory evolution. A) Metabolic scheme of the genetic basis of ΔPYR. B) Growth curve of ΔPYR in glycerol minimal media supplemented with various concentrations of pyruvate. C) Graphical representation of the 4HB-responsive biosensor used in this study. The pobR enzyme from Acinetobacter baylyi ADP1 binds to 4HB upon its presence. This in turn activates PpobA expressing the sfgfp gene. The gene is equipped with an LAA degradation tag, to reduce leakiness of the system D) Relative fluorescence profiles of P. putida ΔglpR equipped with the 4HB-sensor upon exposure to increasing 4HB levels in glycerol minimal medium. Abbreviations: PEP, phosphoenolpyruvate; PYR, pyruvate; AcCoA, acetyl-CoA; OAA, oxaloacetate; CIT, citrate; 4HB, 4-hydroxybenzoate. Data points represent the mean value ± SD from three independent experiments. Figure 5: Isolation of SDC.1 after adaptive laboratory evolution. A) Growth curves of ALE passages in glycerol minimal medium. B) Relative fluorescence profiles (RFU / OD600) plotted against the growth rate (h-1) of the selected mutants after ALE in glycerol minimal medium. Every dot in the graph indicates a single isolated mutant. Figure 6 Characterization of isolated mutants after adaptive laboratory evolution. A) Growth curves of selected mutants after ALE in glycerol minimal medium. B) Relative fluorescence profiles of the selected mutants after ALE in glycerol minimal medium. All strains in the depicted graphs are equipped with the 4HB responsive biosensor, and the evolved mutants were compared to the wild type (KT2440) and the parental ΔPYR strain (KT2440 ΔPYR). Every line indicates a single strain. Figure 7: Exploring the genetic basis of SDC for further optimization. A) Genomic alterations that were discovered in the five strains with the highest relative fluorescence after laboratory evolution. B) Growth curves of the reverse-engineered strains. The miaA and mexT genes were mutated separately and in combination from strain ΔPYR. InFigure 7B the lines of KT2440 ΔPYR (A) and the KT2440 ΔPYR ΔmexT (B) lie on top ofeach other. C) Quantification of 4HB titer and yield in the KT2440 ΔglpR ΔpobA vsΔglpR ΔpobA ΔmiaA and ΔglpR ΔpobA ΔmiaA ΔmexT after 48h of flask cultivation in M9medium supplemented with 100 mM glycerol. D) Quantification of final cell density andconsumed glycerol in the KT2440 ΔglpR ΔpobA vs ΔglpR ΔpobA ΔmiaA and ΔglpR ΔpobAΔmiaA ΔmexT after 48h of flask cultivation in M9 medium supplemented with 100mM glycerol. Abbreviations: queA, S‑adenosylmethionine:tRNA ribosyltransferase‑isomerase; rpoC, DNA‑directed RNA polymerase subunit beta; mexT, Transcriptional regulator MexT; miaA, tRNA dimethylallyltransferase; garK, glycerate kinase; SDC, shikimate pathway-dependent catabolism. Data points represent the mean value ± SD from three independent experiments. Figure 8: Characterization and optimization of SDC. A) Growth curves of the control strains KT2440 ΔglpR and KT2440 ΔglpR ΔpobA compared to the SDC strains. B) Quantification of 4HB yields in the KT2440 ΔpobA vs SDC.2. C) Quantification of glycerate yields in SDC.3 vs SDC.4. D) Quantification of 4HB yields in SDC.3 vs SDC.4 vs SDC.5. Data points and bar graphs represent the mean value ± SD from three independent experiments. *, p <0.1; **, p <0.01; ***, p <0.001; ****, p <0.0001 determined by an unpaired Student’s t-test. Figure 9: Characterization of SDC.6. A) Graphical representation of the genetic modification in SDC6. The pedE and pedH genes were deleted to abolish glycerate formation as a byproduct. The quiC gene was deleted to avoid splitting of the shikimate pathway and the P290S point mutation in trpE was introduced to lower fluxes toward tryptophan biosynthesis. B) Growth curves of the KT2440 ΔglpR, the first isolated SDC.1 and optimized SDC.6 strain. Abbreviations: GLY, glycerol; GLYC, glycerate; E4P, erythrose-4-phosphate; PEP phosphoenolpyruvate; PYR, pyruvate; CHA, chorismate; TRP, tryptophan; 4HB, 4-hydroxybenzoate; EDEMP, Entner-Doudoroff-Embden- Meyerhof-Parnas pathway; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle. Data points represent the mean value ± SD from three independent experiments. Figure 10: A) Biosynthetic pathway of salicylate. Heterologous enzymes menF and pchB convert chorismate to isochorismate and isochorismate to salicylate, respectively. B) Growth curves of strains shikimate pathway-dependent catabolism SDC.4 and SDC.5 carrying pSal on glycerol. C) Salicylate production yield in strains SDC.4_sal and SDC.5_sal. Data points and bar graphs represent the mean value ± SD from three independent experiments. DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS The present invention aims to develop a platform technology for the metabolic engineering of microorganisms, in particular to design microorganisms for the production of aromatic compounds. Using microorganisms to produce aromatic compounds is promising as it contributes to the sustainable and efficient production of aromatic compounds. In the present invention, microorganisms are engineered to steer the metabolic pathway in the cell differently in order to produce an aromatic compound of interest. The present invention can increase the yields and productivity in current microbial production systems. Despite the potential of microbial factories to produce aromatic compounds through sustainable means, metabolic engineering often suffers from low yields and low productivity. This is mainly due to the competition between the native metabolic pathway and the engineered pathway(s) for common precursors and energy. Hence it is an objective of the invention to increase the metabolic flux towards the production of aromatic compounds of interest with high yields. The present invention reduces the demand of petroleum-derived aromatic compounds, thereby having a positive environmental impact. At present, a significant portion of the aromatic compounds produced is derived from petroleum, which not only presents significant environmental concerns but also volatility in cost due to fluctuations in petroleum prices. The engineered microorganism described herein can produce aromatic compounds from renewable feedstocks, offering a more sustainable and potentially cost-effective alternative. The present invention increases metabolic flexibility of microbial production systems. Due to the rigid and inflexible nature of catabolism, traditional metabolic engineering strategies often struggle to efficiently optimize catabolic pathways for biotechnological applications. The development of a new shikimate pathway-dependent catabolism according to the present invention presents a unique solution to this problem by introducing a high degree of metabolic flexibility, enabling the cell to focus more on the production of aromatic compounds of interest. The present invention increases the carbon flux in the shikimate pathway: the shikimate pathway is subject to strict regulation and generally carries low fluxes, making its exploitation for the production of aromatic compounds challenging. By creating a shikimate pathway-dependent catabolism through rational engineering and adaptive laboratory evolution, the present invention seeks to overcome this limitation, driving a higher flux through the shikimate pathway and thereby increasing the production of aromatic compounds. All cells and organisms, including microorganisms, have a catabolic pathway to generate energy and compounds in the cell. In the present invention, the catabolic pathway of a microorganism is reprogrammed so that the catabolism mainly occurs through the shikimate pathway. The shikimate pathway in microorganisms is responsible for the biosynthesis of aromatic amino acids, which can serve as precursors to a variety of valuable aromatic compounds. In the present invention, the shikimate pathway is used for growth of the microorganism while redirecting a significant portion of cellular fluxes towards product formation; so-called growth-coupled production of aromatic compounds. As an exemplary embodiment, the metabolic network of Pseudomonas putida was engineered to produce the aromatic compound 4-hydroxybenzoate (4HB). In a first aspect, the invention therefore provides a microorganism with an altered metabolic pathway for the production of an aromatic compound of interest, wherein the microorganism comprises functional deletions. In a further aspect, the invention provides a method for adapting a microorganism to produce an aromatic compound ofinterest, wherein the method comprises functionally deleting genes from themicroorganism. The functional deletions comprise pyruvate kinase (pyk), pyruvate kinase A (pykA), phosphogluconate dehydratase (edd) and phosphoenolpyruvate carboxylase (ppc). These genes are involved in the metabolic pathway in cells, and by deleting these genes, the cells will have to reroute the metabolic pathway to produce sufficient energy. Despite the considerable diversity among organisms, there is a primary central carbon metabolism shared by almost all of them. This primary metabolism often consists of glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. These metabolic pathways are used to convert carbon sources to energy and produce precursor molecules necessary to synthesize cellular constituents for growth and maintenance of the cell (Noor et al., 2010). As a result, the primary metabolism is often viewed as a rigid network, and any deviation from it can lead to reduced cell viability or even cell death (Stephanopoulos & Vallino, 1991). This is unfortunate as the metabolic pathways can potentially be used to produce a plethora of fuels and chemicals, replacing petroleum-derived processes (Staffas et al., 2013). Therefore, for the sake of the bio-economy, these networks need to be efficiently manipulated to produce chemicals with high titers, rates, and productivity (Nielsen & Keasling, 2016). To achieve this goal, microorganisms need to be radically refactored to ensure a high flux towards the product of interest. However, the introduction of a production pathway often interferes directly with the main metabolism, and fluxes are not easily diverted (Orsi et al., 2022). Moreover, extra complexity is added as many inherent pathways are subjected to tight regulation and therefore carry considerably low fluxes (Nielsen & Keasling, 2016). One of these pathways is the shikimate pathway, the biochemical source of numerous aromatic molecules including aromatic amino acids. While a myriad of valuable molecules can be derived from this pathway, its biotechnological exploitation remains a mounting metabolic engineering challenge (Averesch & Krömer, 2018; Fujiwara et al., 2020; Li et al., 2020). Standard metabolic engineering strategies rely on simple gene overexpressions and flux alterations in the central carbon metabolism. However, these modifications compete directly with cellular fitness and often do not result in economically feasible yields (Braga & Faria, 2020). Therefore, a paradigm shift is essential to fully reshape the rigid carbon metabolism and exert the potential of the shikimate pathway. Pyruvate is a key molecule in central metabolism in all organisms. It is the major source of the citric acid cycle facilitating cellular respiration. In the majority of organisms, pyruvate is predominantly produced via glycolysis. Glycolysis is a metabolic pathway that converts glucose into pyruvate. The most common type of glycolysis is the Embden–Meyerhof–Parnas (EMP) pathway. However, glycolysis also refers to other pathways, such as the Entner–Doudoroff pathway. The Embden–Meyerhof–Parnas (EMP) pathway is a sequence of ten reactions catalyzed by enzymes. In this pathway, one molecule of glucose is converted into two molecules of pyruvate. This results in two ATP molecules and two NADH molecules per every one glucose molecule metabolized. The enzymes that are able to effect the conversions of molecules in this pathway are hexokinase, glucose-6-phosphate isomerase (GPI), phosphofructokinase-1 (PFK-1), fructose-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, phosphopyruvate hydratase, and pyruvate kinase. The Entner-Doudoroff pathway uses glucose as a substrate through a series of enzyme-assisted chemical reactions and catabolizes it into pyruvate. In the process of breaking down glucose, a net yield of one ATP molecule is formed per every one glucose molecule processed, as well as one NADH molecule and one NADPH molecule. This pathway is present in the cells of Gram-negative bacteria, certain Gram-positive bacteria and archaea, as well as in in cyanobacteria, ferns, algae, mosses, and plants. Apart from the main carbon metabolism, pyruvate is produced by several other innate reactions, some derived from the shikimate pathway. In the present invention, the main carbon metabolism routes of the microorganism are blocked by functional deletion of enzymes that are essential in these pathways. Therefore, the microorganism will have to reroute its carbon metabolism to produce pyruvate and energy from carbon sources. In the present invention, the microorganism uses the shikimate pathway as the major pyruvate-releasing pathway in the cell. The engineered microorganism diverts the vast majority of its carbon catabolism flux through the shikimate pathway, which can serve as a useful chassis to produce various aromatic compounds. The shikimate pathway is a metabolic pathway used in bacteria, archaea, fungi, algae, some protozoans, and plants for the biosynthesis of folates and aromatic amino acids (e.g., tryptophan, phenylalanine, and tyrosine). This pathway is not found in animal cells. The shikimate pathway is a seven-step metabolic pathway. Seven enzymes involved in the shikimate pathway are DAHP synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, EPSP synthase, and chorismate synthase. The pathway starts with two substrates, phosphoenol pyruvate and erythrose-4-phosphate, and ends with chorismate, a substrate for the three aromatic amino acids. In addition, the shikimate pathway can produce pyruvate as a source for the citric acid cycle for the production of sufficient energy in the microorganism. In the present invention, the catabolic pathway of a microorganism is reprogrammed so that the catabolism mainly occurs through the shikimate pathway. In order to steer the catabolism through the shikimate pathway, the glycolysis pathway in the cell is disturbed by functional deletions of important genes involved in these pathways. The microorganism comprises functional deletions in pyruvate kinase (pyk), pyruvate kinase A (pykA), phosphogluconate dehydratase (edd) and phosphoenolpyruvate carboxylase (ppc). These genes are involved in the metabolic pathway in cells, and by deleting these genes the cell will have to reroute the metabolic pathway to produce sufficient energy. The gene pyk encodes for pyruvate kinase, an enzyme involved in the last step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP. The gene pykA encodes for the enzyme pyruvate kinase A, which is also called pyruvate kinase 2. This enzyme is present in various species, such as bacteria species. Pyruvate kinase A catalyzes the conversion of PEP to pyruvate and may take over the function of a functionally deleted pyruvate kinase. Therefore, it is important for the present invention to functionally delete both pyruvate kinase and pyruvate kinase A. Functional deletion of pyk and pykA will reduce the conversion of PEP to pyruvate in the cell or microorganism. Therefore, the glycolysis pathway can no longer provide sufficient pyruvate for the cell to grow and survive, and the cell will need to use another pathway to provide sufficient pyruvate. The gene edd encodes a 6-phosphogluconate dehydratase. The reaction facilitated by this enzyme is part of the Entner-Doudoroff pathway together with the sequential pyruvate-releasing step, encoded by eda. The functional deletion of edd will render the whole Entner-Doudoroff pathway inactive. The gene ppc encodes phosphoenolpyruvate carboxylase. This enzyme facilitates the conversion of PEP into oxaloacetate, which subsequently can be converted to pyruvate by the oxaloacetate decarboxylase (PP_1389). Functional deletion of ppc will render the PEP carboxylase pathway inactive. The combination of functional deletions of pyk, pykA, and ppc not only disrupts the flow to pyruvate via glycolysis, but also increases the intracellular PEP pool for the shikimate pathway. In a further aspect of the invention, the microorganism further comprises functional deletion of the gene quiC. The gene quiC encodes 3-dehydroshikimate dehydratase, an enzyme that catalyzes the conversion of 3-dehydroshikimate to 3,4-dihydroxybenzoate. This conversion serves as a point for carbon flow from the shikimate pathway into the citric acid cycle. The deletion of quiC blocks this conversion, thereby channeling the carbon flux more towards to the shikimate-pathway and thereby the production of an aromatic compound of interest, such as 4HB or salicylate. The deletion of pobA prevents the degradation of 4-hydroxybenzoate towards the citric acid cycle and will further optimize the production of an aromatic compound of interest. In a further aspect of the invention, the microorganism further comprises functional deletion of the gene pedH and / or the gene pedE. The gene pedH encodes for dependent alcohol dehydrogenase H, and pedE encodes for dependent alcohol dehydrogenase E. Both enzymes belong to the group of alcohol dehydrogenases. The deletion of genes pedH and pedE inhibits the production of glycerate, a by-product of shikimate-dependent catabolism, preventing any diversion of the flow away from the shikimate pathway. In a further aspect of the invention, the microorganism further comprises functional deletion of the gene pobA. The gene pobA encodes for p-hydroxybenzoate hydroxylase, which is responsible for the degradation of 4HB to protocatechuate (PCA). Protocatechuate can be further degraded to fuel in the citric acid cycle. Therefore, functional deletion of pobA reduces the carbon flow towards the citric acid cycle, and therefore the carbon molecules remain in the shikimate pathway. Thus, deletion of pobA will further optimize the production of an aromatic compound of interest. In a further aspect of the invention, the microorganism further comprises an altered anthranilate synthase (trpE) to reduce activity of trpE. For example, reduced activity of trpE is achieved by a point mutation P290S, which significantly reduces the activity of the enzyme. The gene trpE encodes for anthranilate synthase, which catalyzes the conversion of chorismate into anthranilate and pyruvate. Reduced activity of trpE decreases this conversion, and forces the cell to use other conversions to produce pyruvate. Thus, deletion of trpE can further optimize the production of an aromatic compound of interest. The terms "functional deletion" and "functional deleted" mean that a sufficient amount of the gene region is removed or otherwise damaged, e.g., by mutation or modification, so that the gene region is no longer capable of producing functional products of gene expression. Mutations or modifications that can result in functional deletions include, but are not limited to, nonsense mutations, such as introduction of premature stop codons and removal of canonical and non-canonical start codons, mutations that alter mRNA splicing or other transcriptional processing, or combinations thereof. If desired, the entire gene region can be removed. Methods to functionally inactivate an enzyme include the genomic alteration of the gene encoding the enzyme, including deletion of part or the complete gene. Deletion of part or the complete gene encoding a gene may be accomplished by a site-specific recombinase. Other genetic tools that exist for the deletion of genes in a cell or microorganism are, for example, CRISPR-Cas9, homologous recombination, and TALENs. In the present invention, the microorganism has a shikimate pathway-dependent catabolism, and is dependent of this pathway for growth, as the regular glycolysis pathways are blocked by deletions of crucial enzymes in this pathway. Therefore, the microorganism needs enzymes that support growth via the shikimate pathway. In an aspect of the invention, the microorganism overexpresses at least one gene encoding an enzyme capable of performing a reaction derived from the shikimate pathway that can support growth. Preferably, the gene that is overexpressed encodes for an enzyme that increases the release of pyruvate in the microorganism and co-produces an aromatic compound of interest. Pyruvate serves as an energy source for the microorganism. In some aspects of the invention, the overexpressed gene is ubiC. The gene ubiC encodes chorismate pyruvate-lyase, which catalyzes the conversion of chorismate into aromatic 4-hydroxybenzoate (4HB) and pyruvate (Figure 1). In some aspects of the invention, the overexpressed gene is hyg5. The gene hyg5 encodes for 3-hydroxybenzoate synthase, and catalyzes the conversion of chorismate into 3-hydroxybenzoate and pyruvate (Figure 2). In some aspects of the invention, the overexpressed gene comprises overexpression of a combination of menF and pchB. The gene menF encodes isochorismate synthase, which catalyzes the conversion of chorismate into isochorismate. The gene pchB encodes for isochorismate pyruvate lyase, which catalyzes the conversion of isochorismate into salicylate and pyruvate (Figure 2). In some aspects of the invention, the overexpressed gene is trpE. The gene trpE encodes for anthranilate synthase, which catalyzes the conversion of chorismate into anthranilate and pyruvate (Figure 2). Overexpression of trpE can enable the production of anthranilate / tryptophan-derived compounds. Overexpression of the enzymes encoded by the described genes, support the release of pyruvate together with the co-production of an aromatic compound. The production of pyruvate is necessary for the growth of the microorganism as energy source. Via these supported pathways the microorganism co-produces an aromatic compound of interest. In some aspects of the invention, the overexpressed gene is pobR. The gene pobR encodes for p-hydroxybenzoate hydroxylase transcriptional activator, which is atranscription factor. This transcription factor regulates the promoter used to transcribethe ubiC gene. The terms "overexpression" and "overexpressed" mean the artificial increased expression of a protein in the cell of an organism. The overexpressed protein can be an enzyme or a transcription factor or any other protein in the cell. There is an increased quantity of the protein in the cell. Overexpression may result from the presence of one or more exogeneous genes, additional copies of the gene, a different promotor with increased activity or from other conditions. For the purpose of this invention, a microorganism is modified to stably overexpress the protein of interest. Typically, overexpression refers to an increased level of expression compared to the endogenous expression level. In some embodiments, overexpression means an increased expression of at least 1.5-fold compared to the endogenous expression of the protein in the cell, preferably at least 2-fold compared to the endogenous expression of the protein in the cell. The present invention describes the deletion and overexpression of specific genes that occur in a broad spectrum of microorganisms. The gene name and sequences in the description and exemplary embodiments correspond to the microorganism Pseudomonas putida. In other microorganisms, the corresponding genes may have a different name or different sequence. A skilled person in the relevant art knows how to identify the corresponding genes in other microorganisms. For example, using existing gene databases, such as NCBI genes. Furthermore, a skilled person can align the sequences of the genes in Pseudomonas putida with the genetic information of other species to identify the corresponding genes. For example, a skilled person can identify a pyruvate kinase with the most homology of the pyruvate kinase (pyk) or pyruvate kinase A (pykA), as described herein. Alternatively, the skilled person can identify a gene as the corresponding gene, as it is located in the same bacterial operon. Bacterial operons are co-regulated gene clusters wherein genes are grouped under a common control mechanism. A gene in the same operon, especially in the same position in an operon, can be identified as the corresponding gene. To illustrate, as follows are a number of examples of corresponding genes in other organisms. The corresponding gene of pyk in Escherichia coli (E. coli) is pykF (pyruvate kinase 1). The corresponding gene of pykA in E. coli is pykA (pyruvate kinase 2). The corresponding gene of edd in E. coli is edd (phosphogluconate dehydratase). The corresponding gene of ppc in E. coli is ppc (phosphoenolpyruvate carboxylase). The corresponding gene of pobA in E. coli is pobA. The corresponding gene of trpE in E. coli is trpE. The corresponding genes of pyk in S. cerevisiae are CDC19 and PYK2. The corresponding gene of ppc in S. cerevisiae is ppc1. The corresponding genes of trpE in S. cerevisiae are Trp2 and Trp3. The corresponding gene of pyk in Corynebacterium species is pyk. The corresponding gene of ppc in Corynebacterium species is ppc. The corresponding gene of quiC in Corynebacterium species is qsuB. The corresponding gene of pobA in Corynebacterium species is pobA. The corresponding gene of trpE in Corynebacterium species is trpE. The present invention provides a microorganism and a method for adapting a microorganism. The adapted microorganism can be called a recombinant microorganism, as the microorganism has a subset of deletions and at least one overexpressed gene. In some cases, the microorganism can be called a synthetic strain. The microorganism of the invention can be used for the production of aromatic compounds or interest. These microorganisms are developed to produce high yields of aromatic compounds. A microorganism is an organism of microscopic size. Microorganisms occur in many variants and many species all over the world. They can have different habitats and live everywhere from the poles to the equator, deserts, geysers, rocks, and the deep sea. Microorganisms can be single-cell organisms or multicellular organisms. The majority of microorganism belongs to the bacteria family, Archaea family or to the Eukaryota family (multicellular organisms). Microorganisms are essential in human culture and health in many different ways. For example, microorganisms are used to produce fuel, enzymes and other bioactive compounds. In the present invention, microorganisms are used to produce valuable compounds, in particular aromatic compounds. Microorganisms have an innate ability to produce these aromatic compounds as a byproduct of their central metabolism. However, in a wild-type microorganism, the yields of these aromatic compounds are low. Therefore, production of an aromatic compound of interest is inefficient using a wild-type microorganism. Metabolic engineering is a tool to construct microbial cell factories that can efficiently and cost-effectively produce valuable products. Therefore, it is a crucial concept in industrial applications. As used herein, "microorganism" refers to an organism of microscopic size. It can be a single cellular or multicellular organism, and it can serve as a bioreactor that can be used to produce a compound of interest, preferably an aromatic compound of interest. The microorganism of the invention can be used in industrial biotechnology. Furthermore, the microorganism of the invention can be used in microbial cell factories, such as bioreactors and tissue culture facilities. In preferred embodiments, the microorganism is a single-cell microorganism, such as a bacterium or a fungus. Bacteria are suitable microorganisms for the present invention, as many tools exist to modify bacteria, in particular for modifying the genes and for modifying gene expression. Furthermore, many bacteria are suitable for growing in suspension, which allows scaling up the number of bacteria to grow at the same time, and thereby scaling up the production of the aromatic compound of interest. Fungi are suitable microorganisms for the present invention, as fungi are excellent organisms that act as cell factories for the production of compounds. Fungi are robust, and naturally produce many compounds. Furthermore, recombinant DNA techniques have become available to modify fungal properties. In preferred embodiments, the microorganism is a gram-negative bacterium. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. The Gram-negative bacteria have a hard, protective outer shell that protects them from their surroundings. Gram-negative bacteria are suitable for culturing in bioreactors at large scale, and are therefore suitable for the present invention. Gram-negative bacteria, include for example, Escherichia coli, as well as many pathogenic bacteria, such as Pseudomonas aeruginosa, Chlamydia trachomatis, and Yersinia pestis. In some embodiments, the microorganism is a bacterium, belonging to any of the following species: Agrobacterium species, Alcaligenes species, Alicyclobacillus species, Anabaena species, Anacystis species, Arthrobacter species, Azobacter species, Bacillus species, Brevibacterium species, Chromatium species, Clostridium species, Corynebacterium species, Cupriavidus species, Enterobacter species, Erwinia species, Escherichia species, Lactobacillus species, Lactococcus species, Mesorhizobium species, Methylobacterium species, Microbacterium species, Phormidium species, Pseudomonas species, Rhodobacter species, Rhodopseudomonas species, Rhodospirillum species, Rhodococcus species, Salmonella species, Serratia species, Shigella species, Staphylococcus species, Streptomyces species, Synnecoccus species, or Zymomonas species. For example, the microorganism may belong to a bacterial species, e.g., the Escherichia species, such as E. coli, Pseudomonas species, such as P. putida, Clostridium species, such as C. butyricum, Xanthomonas species, such as X. campestris, Corynebacterium species, such as C. glutamicum, or Deinococcus species, such as D. radiorans. In some embodiments, the microorganism is a fungus, for example, belonging to Aspergillus species, Candida species, Chrysosporium species, Cryptococcus species, Fusarium species, Kluyveromyces species, Neotyphodium species, Neurospora species, Penicillium (e.g., P. chrysogenum) species, Pichia species, Saccharomyces species, Trichoderma species, Xanthophyllomyces species, or Yarrowia species. In some embodiments, the microorganism is a yeast, for example, belonging to Saccharomyces species, e.g., S. cerevisiae, or Aspergillus species, e.g., A. niger. In preferred embodiments, the microorganism is a cell line, such as a mammalian cell line. Other organisms with a history of safe use in industrial biotechnology can also be explored. In some embodiments, the microorganism is Pseudomonas putida. P. putida is a Gram-negative soil bacterium and is an industrially relevant bacterium. P. putida has emerged as a host organism for microbial laboratory work with elaborated techniques for cultivation and genetic manipulation available. In some embodiments, the microorganism is Pseudomonas putida KT2440. Thismicroorganism is a potential candidate for industrial biocatalysis. This bacterium has gained industrial interest particularly due to its natural high solvent tolerance, fast growth, low nutrient demands, and ability to withstand industrial conditions. Moreover, its main carbon catabolism is a cyclic architecture composed of the Entner-Doudoroff, the Embden-Meyerhof-Parnas, and the Pentose Phosphate Pathway, which can lead to catabolic overproduction of NADPH. This feature not only allows an increased oxidative stress endurance, which is frequently observed in an industrial setting, but it can also aid in establishing redox-demanding biocatalytic processes. In a further aspect of the invention, the microorganism as described herein can use any carbon source that can enter a regular metabolism. The microorganism can use glycerol, glucose, xylose and / or cellobiose, and typically requires a single carbon source. The microorganism uses the carbon source to produce energy and for growth of the cell as well as the production of an aromatic compound of interest. It is an advantage of these microorganisms that they are not in need of any special carbon sources in order to grow or develop. In a further aspect of the invention, the microorganism as described herein can sustain growth in minimal media without any additional carbon supplements. This feature is a particular advantage of the microorganism of the invention having an altered metabolic pathway. In a further aspect, the invention provides a method for adapting a microorganism to produce an aromatic compound of interest, wherein the microorganism is adapted by functionally deleting the following genes: pyruvate kinase (pyk), pyruvate kinase A (pykA); phosphogluconate dehydratase (edd); and phosphoenolpyruvate carboxylase (ppc). Functionally deleting these genes can be performed by any method known by a skilled person in the relevant art, including for example, the methods as described herein. The genes can be deleted simultaneously or alternately. There may be intermediate microorganisms or strains that comprise a single deletion or a subset of the deletions, which can be used to further develop a microorganism of the invention. In a further aspect, the microorganism is adapted to overexpress at least one gene encoding an enzyme capable of performing a reaction derived from the shikimate pathway that can support growth. Typically, the enzyme encoded by such a gene is involved in a pyruvate-releasing reaction that is particularly growth-supporting, may co-produce an aromatic compound of interest, and is preferably associated with the utilization of an intermediate from the shikimate pathway. Overexpression may be performed by any method known to a skilled person in the relevant art, for example, the methods as described herein. Induced overexpression of a gene can be performed before, during or after functionally deleting genes. In some embodiments, the overexpression is performed in an early stage of the adaptation, as this helps the organism to survive and grow. The early stage may be before any deletion is realized, after the first deletion is realized, before all functional deletions are realized, or anywhere else in the adaption process. In a further aspect, the invention provides a method for production of an aromatic compound by a microorganism, comprising the following steps. Providing a microorganism according to the invention, or a microorganism as produced by the method according to the invention. Incubating the microorganism in a growth medium, comprising a carbon source. The microorganism as described herein can use any carbon source that can enter a regular metabolism. The microorganism can use glycerol, glucose, xylose and cellobiose. The microorganism uses the carbon source to produce energy and for growth of the cell as well as the production of an aromatic compound of interest. It is an advantage of this microorganism that it is not in need of any special carbon source in order to grow or develop. The microorganism produces an aromatic compound of interest. This compound can be harvested from the microorganism by any method known to a skilled person in the relevant art. The harvested aromatic compound of interest may be purified and / or may be used for further synthesis steps. The compound of interest may be an intermediate molecule that can be used for further synthesis or a compound that can be used as produced. In a further aspect, the invention provides the use of an aromatic compound produced by a method according to the invention for the production of bioplastics, such as bio-polyesters and bio-polystyrenes, pharmaceuticals, agrochemicals, cosmetics, chemicals, such as dyes, detergents, or biofuel molecules. Also provided herein is the use of the microorganism of the invention for the production of an aromatic compound, typically any aromatic compound (of interest) as disclosed herein, such as 3-hydroxybenzoate, 4HB, or salicylate, for the production of bioplastics, such as bio-polyesters and bio-polystyrenes, pharmaceuticals, agrochemicals, cosmetics, chemicals, such as dyes, detergents, or biofuel molecules. In an aspect of the invention, the microorganism is engineered to produce an aromatic compound of interest. The microorganism is steered to use the shikimate pathway for the growth of the cell, and thereby steered to produce an aromatic compound of interest. For example, the aromatic compound of interest can be 4-hydroxybenzoate (4HB), salicylate, 3-hydroxybenzoate, or anthranilate. Microorganisms producing 4HB or salicylate are described in the exemplary embodiments. Other aromatic compounds of interest may be folates and aromatic amino acids, such as tryptophan, phenylalanine, and tyrosine. The produced aromatic compounds may be used as produced or maybe used as a precursor molecule, for example, as a precursor molecule for methylparaben, methyl salicylate, indigo, phenol, tryptophan, catechol, vanillin, or arbutus. In some aspects of the invention, the microorganism is further evolved using adaptive laboratory evolution (ALE). ALE is a powerful experimental technique that facilitates the establishment and optimization of new metabolic pathways in microbial hosts. This technique relies on subjecting microbial hosts to selection pressure in order to allow the emergence of specific mutations to adapt to the desired conditions. Therefore, in some embodiments, the microorganism further comprises evolutionary raised mutations. Laboratory evolution of a microorganism is an approach for the generation of evolved microorganisms with desired characteristics. The evolution aims to improve the performance of a microorganism in order to improve the biotechnological procedures. Laboratory evolution can be applied to many types of microorganisms, for example, bacteria, fungi and microalgae. A typical procedure of laboratory evolution is the culturing of an adapted microorganism strain for two months. During these two months, the strain will adapt itself to the culture conditions. After those months the strain has become a mixed population of different genotypes. The mutation can occur in the genomic DNA of the microorganism or in plasmid DNA in the microorganism. The most suitable clone can be selected from the mixed population (heterogenic population) and used for further steps. In an exemplary embodiment, a reporter is used to select the clones of interest after the evolution. For example, a responsive biosensor that produces a signal if the aromatic compound of interest is produced. The biosensor produces a signal in the presence of the aromatic compound of interest, whereby a stronger signal corresponds with a higher yield of the aromatic compound of interest. For example, the reporter is a GFP reporter, whereby colonies can be selected based on fluorescence. Laboratory evolution can be done in any culture facility, such as a bioreactor, cell culture facility, bacterial culture facility, or fungus culture facility. Typically, the culture conditions are kept the same over two months with regular administration of food and nutrients for the microorganism. In order to increase the mutation rate of the microorganism it is possible to apply mutagenic conditions to the culture during evolution. This can be done by applying a chemical or physical agent capable of inducing changes in DNA. For example, applying ultraviolet radiation, radioactive substances, x-rays, or mutagenic chemicals. An increased mutation rate may result more diversity and / or faster evolution of the cultured microorganism. After ALE and selecting clones of interest the genomes and plasmids of the most efficiently evolved strains can be analyzed. Analysis reveals the mutations that occurred during evolution. These mutation may contribute to the production of the aromatic compound on interest. In some aspects of the invention, the microorganism comprises a mutated miaA gene. Preferably, in some aspects of the invention the gene miaA is functionally deleted. MiaA is a tRNA dimethylallyltransferase and has been known to affect the expression of various genes related to the central and secondary metabolism. Deletion of miaA leads to growth of a microorganism of the invention without the need for evolution. In some embodiments, the microorganism of the invention in P. putida comprises a functional deletion of miaA. In some aspects of the invention, the microorganism comprises a mutated mexT gene. Preferably, in some aspects of the invention the gene mexT is functionally deleted. The mexT gene encodes a transcriptional regulator that repressed the quinolone biosynthetic pathway and the first reaction of tryptophan biosynthesis. In preferred embodiments, the microorganism of the invention further comprises functional deletion of miaA and mexT. Deletion of both genes leads to increased growth rates of the microorganism and a shorter lag phase. In some embodiments, the microorganism is P. putida, comprising a functional deletion of mexT. In other embodiments, the microorganism is P. putida, comprising a functional deletion of miaA and functional deletion of mexT. In some aspects of the invention, the microorganism comprises a mutated garK gene. The gene garK encodes a glycerate kinase. Further provided herein is a method for screening cells or an organism having an increased production of an aromatic compound of interest. The method comprises using a selection chemical environment. In a first step, a selection chemical environment is determined. The cells or organism are grown in the selection chemical environment. This is followed by selecting at least one cell or organism that shows higher growth than one or more of the other cells or organisms in the selection environment. Higher (or increased) growth in the selection environment indicates that the cell or organism contains increased levels of the aromatic compound of interest. The invention is further explained in the following examples. These examples do not limit the scope of the invention but merely serve to clarify the invention. EXAMPLES Example 1 MATERIAL and METHODS Plasmids, primers, and strains. All strains and plasmids used in the present study are listed in Table 2. Primers used for plasmid construction and gene deletions are listed in Table 3. Culture conditions and medium. P. putida and E. coli cultures were incubated at30 °C and 37 °C, respectively. For cloning purposes, both strains were propagated in a Lysogeny Broth (LB) medium containing 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract. For the preparation of solid media, 1.5 % (w / v) agar was added. Antibiotics, when required, were used at the following concentrations: kanamycin (Km) 50 μg / ml, gentamycin (Gm) 10 μg / ml, chloramphenicol (Cm) 50 μg / ml and apramycin (Apra) 50 μg / ml. All growth and production experiments were performed using M9 minimal medium (per Liter; 3.88 g K2HPO4, 1.63 g NaH2PO4, 2.0 g (NH4)2SO4, pH 7.0). The M9 media were supplemented with a trace elements solution (10 mg / L ethylenediaminetetraacetic acid (EDTA), 0.1 g / L MgCl2.6H2O, 2 mg / L ZnSO4∙7H2O, 1 mg / L CaCl2∙2H2O, 5 mg / L FeSO4∙7H2O, 0.2 mg / L Na2MoO4∙2H2O, 0.2 mg / L CuSO4∙5H2O, 0.4 mg / L CoCl2∙6H2O, 1 mg / L MnCl2∙2H2O). In these experiments, strains were precultured in 10 ml LB with corresponding antibiotics. Then, the cultures were washed twice in M9 media without a carbon source. Finally, the cultures were diluted to an OD600 of 0.1 to start the experiment. Flask experiments were performed in 250 ml Erlenmeyer flasks filled with 25 ml of M9 minimal medium with 40 (SDC characterization) or 200 (ALE experiment) mM glycerol, and the cultures were incubated in a rotary shaker at 200 rpm at 30 °C. Cloning procedures. Plasmids were constructed using the standard protocols of the SevaBrick assembly (Damalas et al., 2020). All DNA fragments were amplified using Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs). To construct the plasmids pSensor and pSensorEvo, the 4HB-responsive regulator and promoter (pobR / PpobA) were synthesized by Integrated DNA Technologies (IDT), and the ubiCE31Q / M34Vwas amplified from the genome of E. coli with primers to introduce the E31Q and M34V mutations. All parts were integrated into the pSB1C3 repository and subsequently assembled into pSEVAb83 following the standard procedures of the SevaBrick assembly. All plasmids were transformed via heat shock in chemically competent E. coli DH5α λpyr cells and via electroporation or conjugation in P. putida (Thermo Fisher Scientific). Transformants were selected on LB agar plates with corresponding antibiotics and colonies were tested by colony PCR with Phire Hot Start II DNA polymerase (Thermo Fisher Scientific). After extraction, all constructs were verified by Sanger DNA sequencing (MACROGEN Inc.). Genome modifications. Genomic modifications in this study were performed using the protocol described by Wirth et al. (2020). Homology regions of ± 500 bp were amplified up and downstream of the target gene from the genome of P. putida KT2440. Both regions were cloned into the non-replicative pGNW vector and propagated in E. coli DH5α λpir. Correct plasmids were transformed into P. putida by electroporation and selected on LB + Km plates. Successful co-integrations were verified by PCR. Hereafter, co-integrated strains were transformed with the pQURE6-H, and transformants were plated on LB + Gm containing 2 mM 3-methylbenzoic acid (3-mBz). This compound induces the XylS – dependent Pm promoter, regulating the I-SceI homing nuclease that cuts the integrated pGNW vector. Successful gene deletions were verified by PCR and Sanger sequencing (MACROGEN, Inc.). The P290S modification in trpE was verified by MASC PCR as described by Wang & Church, (2011). Hereafter, the pQURE6-H was cured by removing the selective pressure, and its loss was verified by sensitivity to gentamycin. Analytical methods. Cell growth was determined by measuring the optical density at 600 nm (OD600) using an OD600 DiluPhotometer spectrophotometer (IMPLEN) or a Synergy plate reader (BioTek Instruments). Analysis of glycerol and pyruvate in supernatants was performed using high-performance liquid chromatography (HPLC) (Thermo Fisher Scientific) equipped with an Aminex HPX-87H column. The mobile phase was 5 mM of H2SO4 at a flow rate of 0.6 ml / min, the column temperatures were held at 60 °C and the compounds were detected using a Shodex RI-101 detector (Shodex). The amount of produced 4HB was determined using HPLC (Shimadzu) with a C18 column (4.6 mm × 250 mm) and a UV / vis detector set at 472 nm. The mobile phase consisted of Milli-Q water (A), 100 mM formic acid (B), and acetonitrile (C) with a flow rate of 1 ml / min at 30 °C. Chromatographic separation of analytes was attained using the following gradient program: t = 0 – 5 min: A-55 %, B-10 %, and C-35 %; from t = 5 – 10 min ramp to A-10 %, B-10 %, and C-80 % and held until 15 min. Then from t = 15 – 16 min, the gradient was returned to A-55 %, B-10 %, and C-35 %, and maintained isocratic for a total run time of 18 min. For quantification, calibration curves were prepared using pure standards (99 % purity) purchased from Sigma-Aldrich. Adaptive laboratory evolution. Strain ΔPYR was inoculated in two 250 ml shake flasks with M9 minimal medium supplemented with 200 mM glycerol as the sole carbon source and incubated in a rotary shaker at 200 rpm at 30 °C. The starting cell density was set at OD600 = 0.1, and cells were diluted back to the same OD600 after they reached an OD600 of > 1.0. Evolved strains were selected on M9 agar plates with 200 mM glycerol and characterized in 200 μL of M9 medium with 40 mM glycerol using a Synergy plate reader (BioTek Instruments). Cell density (OD600) and GFP fluorescence (excitation 485 nm, emission 512 nm, gain 50) were measured over time using continuous linear shaking (567 cpm, 3mm), and measurements were taken every fifteen minutes. A customized Python script was programmed to calculate the growth rates taking the natural log of the OD600 values. Whole-genome sequencing. The genomic DNA of the evolved mutants was isolated from LB overnight cultures using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich St. Louis, MO). The extracted DNA was evaluated by gel electrophoresis and quantified by a NanoDrop spectrophotometer (Thermo Fisher Scientific). Samples were sent for Illumina sequencing to Novogene Co. Ltd. (Beijing, China). Raw Illumina reads were trimmed for low quality and adapters with fastp (v0.20.0). Mutations were identified by comparing the reads to the annotated reference genome of Pseudomonas putida KT2440 (GCF_000007565.2) using breseq (v0.35.5) (Barrick et al., 2014). Genome-scale metabolic modeling. Computational analysis was performed using COBRApy (version 0.18.1) and Python (version 3.6, Python Software Foundation). iJN1462 was used; the latest developed genome-scale model (GEM) of P. putida to rank pyruvate-releasing reactions and to simulate native and SDC metabolism (Nogales et al., 2020). In all simulations, glycerol was used as the sole carbon source with a maximum uptake rate of 3.95 mmol / gCDW / h (Nikel et al., 2014). All metabolic reactions able to produce pyruvate in the iJN1462 GEM were evaluated for their capacity to support growth as the sole pyruvate source. The upper and lower bounds of all pyruvate-releasing reactions were constrained to zero except for two essential reactions: ANS2 (trpE, pabA) and ADCL (pabC). Iteratively, the flux through each reaction was unconstrained making it the main available source of pyruvate in the model. The growth rate, represented by the BIOMASS_KT2440_WT3 reaction, was maximized. Reactions were ranked according to the predicted maximum growth rates relative to the wild-type growth rate (100%). iJN1462 was modified to correctly simulate SDC and wild-type metabolism (Table 4) (Batianis et al., 2022). When simulating SDC, a pFBA-like constraint was applied such that the sum of all the predicted fluxes cannot exceed the sum of all predicted fluxes in native metabolism. Besides, as a base for SDC simulation, the flux was constrained to zero through reactions EDD, PYK, AGPOP, ME2, OAAFC, SERD_L, CYSTL, CYSDS, MCITL2, LDH_2, and LDH_D2 to reproduce the result of the biosensor-assisted ALE that made CHRPL the main pyruvate source. Model modifications that allowed the simulation of the different SDC strains are presented in Table 5. Maximum theoretical 4HB yields were calculated maximizing the 4HB exchange reaction (EX_4hbz_e). The optimal metabolism of the SDC strains and their maximum growth rates were simulated maximizing biomass production (BIOMASS_KT2440_WT3). Additional in silico simulations were performed using GEMs for three different microbes: iBWG_1329 for E. coli (Monk et al., 2013), iCW773 for C. glutamicum (Feierabend et al., 2021), and Yeast9 for S. cerevisiae (Zhang, C. et al., 2024). In each model, key reactions involved in native pyruvate production were deactivated to replicate the SDC metabolic configuration. Specifically, the following reactions were constrained to zero flux: pyruvate kinase (PYK), phosphoenolpyruvate carboxylase (PPC), and phosphogluconate dehydratase (where applicable). The inactivated reactions per organism were E. coli (PYK, EDA, PPC), C. glutamicum (PYK, PPC), and S. cerevisiae (r_0962, r_0884). To simulate SDC, a lower bound constraint was applied to the shikimate-derived pyruvate-releasing reaction, specifically anthranilate synthase (E. coli, C. glutamicum: ANS and S. cerevisiae: r_0203), for each organism. The lower bounds were set at 1.3 mmol / gCDW / h for E. coli, 1.35 mmol / gCDW / h for C. glutamicum, and 0.9 mmol / gCDW / h for S. cerevisiae. The glycerol uptake rate was fixed at 6 mmol / gCDW / h for all simulations. pFBA was performed to predict the maximum growth rates and to assess the metabolic reconfiguration under SDC conditions. Statistical analysis. All reported experiments are derived from independent biological replicates. Figures represent the mean values of corresponding biological triplicates and the standard deviation. The level of significance of the difference when comparing results was evaluated by an unpaired Student’s t-test. RESULTS Metabolic design and in-silico assessment of SDC. Pyruvate is a key node in central metabolism predominantly produced in P. putida via the Entner-Doudoroff (ED) and lower Embden–Meyerhof–Parnas (EMP) pathway. Yet, apart from the main carbon metabolism, pyruvate is produced by several other innate reactions, some derived from the shikimate pathway. Therefore, to increase the flux through the shikimate pathway, the aim was to establish it as the main source of cellular pyruvate creating SDC. iJN1462, the genome-scale model (GEM) of P. putida, was used to find all the pyruvate-releasing reactions present. The ability of each of these reactions to support in silico growth as the sole pyruvate source using glycerol as a carbon source was analyzed (Figure 3A). The model predicted the highest growth rate using a shikimate pathway reaction when chorismate pyruvate lyase (CHRPL) was the sole pyruvate source. This reaction cleaves chorismate, the final product of the shikimate pathway, to pyruvate and 4-hydroxybenzoate (4HB) (Figure 3B). When CHRPL is the sole pyruvate source, the in- silico growth rate reduces by 17.8 % compared to the wild-type (P. putida KT2440). Still, it allows 26.1% faster growth than the other pyruvate-releasing shikimate reactions and was therefore chosen as the most efficient candidate to establish SDC. To further evaluate the feasibility of SDC, the overall chemical equations were calculated from glycerol to pyruvate in terms of its ability to generate reduced cofactors and ATP compared to the native metabolism. For this purpose, native and SDC metabolism were simulated with flux balance analysis (FBA) in which pyruvate production was set as the main objective using glycerol as the carbon source. During native metabolism, glycerol is equimolarly converted to pyruvate, generating reducing equivalents in the process. As P. putida is an aerobic bacterium, a large surplus of ATP can be generated in the electron transport chain. In contrast, the SDC metabolism is energetically poor compared to the native metabolism. The shikimate pathway is an anabolic pathway and requires the incorporation of NADPH and ATP to reach the end product chorismate. Moreover, there is a net production of CO2 reducing the pyruvate yield. Nonetheless, with the production of reducing equivalents, a surplus of 0.23 ATP can be generated for growth and maintenance. Creating a growth-coupled scenario to establish SDC. The in silico analysis demonstrates the feasibility of SDC. However, its prediction solely relies on stoichiometry. Microbial metabolism is tightly regulated, and a metabolic reconfiguration of this extent is impossible to attain through rational engineering. Adaptive laboratory evolution (ALE) is a microbial engineering method commonly used to achieve desired phenotypes that cannot be obtained using the rational approach (Wu et al., 2022). Therefore, to install CHRPL as the major pyruvate source, a pyruvate auxotrophic strain (ΔPYR) was established to allow for growth-coupled evolution (Figure 4A). At first, edd was deleted, encoding a 6-phosphogluconate dehydratase. This reaction is part of the ED pathway together with the sequential pyruvate-releasing step, encoded by eda, and the deletion of edd will render the whole pathway inactive. Next, pyruvate was decoupled from phosphoenolpyruvate (PEP), by deleting the pyruvate kinases encoded by pyk and pykA. The last major pyruvate node was removed by deleting phosphoenolpyruvate carboxylase, encoded by ppc. This reaction converts PEP into oxaloacetate, which subsequently can be converted to pyruvate by the oxaloacetate decarboxylase (PP_1389). It is important to note that the deletion of pyk, pykA, and ppc not only disrupts the flow to pyruvate but also increases the intracellular PEP pool for the shikimate pathway. At last, glpR was deleted, which encodes the transcriptional repressor of the glycerol catabolic operon. This final strain was termed ΔPYR, and was evaluated whether it exhibited pyruvate auxotrophy by growing it in glycerol minimal media supplemented with increasing concentrations of pyruvate. As expected, ΔPYR was not able to grow in glycerol minimal medium without the addition of pyruvate, indicating that biomass formation is exclusively dependent on its external supplementation (Figure 4B). Albeit this strain can serve as a base strain for ALE, in-silico simulations indicated additional pyruvate-releasing reactions (AGPOP, CYSTL, SERD_L, ME2, OAADC, CYSDS, MCITL2, LDH_D, LDH_D2) that are more favorable and can allow faster growth rates than when using CHRPL (Figure 3A). Although the removal of these reactions would benefit evolution, they might affect cellular fitness. Moreover, the other chorismate-derived pyruvate-releasing reactions cannot be removed, as this would render the strain auxotrophic for both tryptophan and folate (Figure 3B). Therefore, by only selecting on growth, there is an increased possibility that undesired phenotypes might develop. To circumvent this problem, a second layer of screening by incorporating a previously established 4HB – responsive sensor was implemented (Jha et al., 2019). This sensor constitutes a double mutant pobR enzyme from Acinetobacter baylyi ADP1, which binds to 4HB upon detection and expresses sfGFP. This biosensor was further adapted by including an LAA degradation tag, to decrease leakiness and increase tunability (Figure 4C). The sensor was cloned in a pSEVAb83 backbone and efficacy in P. putida KT2440 in glycerol minimal media supplemented with increasing concentrations of 4HB was tested. As expected, the sensor displayed dose-dependent sfgfp expression to exogenous 4HB concentrations indicating a strong selection method for ALE (Figure 4D). Using this method, superior strains can be isolated that exhibit fast growth rates and a high flux towards 4HB formation. Establishing SDC through laboratory evolution. The establishment of SDC requires rigorous rearrangement of the complete metabolic network. A pyruvate auxotrophic strain was established to make the shikimate pathway the major source for pyruvate and a 4HB – responsive sensor to select superior isolates. To further drive evolution, a feedback-inhibition-resistant CHRPL was incorporated (Jha et al., 2019). To maintain high levels of CHRPL, the corresponding gene (ubiC) was placed under the control of the constitutive J23100 promoter downstream of the biosensor in the same transcriptional operon. Through this design, the 4HB produced by CHRPL creates a positive feedback loop and will increase its own transcription, likely aiding the evolution of SDC. P. putida ΔPYR was equipped with the modified biosensor and started evolution by cultivating the strain in glycerol minimal medium in two independent experiments. Growth in the population emerged in the first phase after roughly 20-24 days in both experiments (Figure 5A). After this initial passage, growth rates quickly increased with the next passage, indicating that crucial adaptations occurred in the initial phase. In total, the cultures in the two independent experiments were serially diluted for about 50 days in 13-18 passages and plated on minimal media with glycerol. Forty colonies were selected per ALE experiment based on fluorescence and selected for further characterization. The isolated mutants showed a diverse range of growth rates and fluorescence profiles (Figure 6). Within the isolated population, fast-growing mutants with low fluorescent profiles were observed. Most likely, these strains evolved through alternative salvage pathways to restore growth. This finding highlights the importance of the implemented 4HB-responsive biosensor during evolution to select superior strains. From the heterogeneous mixture of mutants, SDC.1 was isolated (Figure 5B). This strain demonstrated fast growth among the isolated mutants (0.099 per hour), albeit significantly slower than the wild-type (0.210 per hour) (Figure 6A). However, SDC.1 displayed high levels of fluorescence among the isolated mutants and a 10-fold increase in relative fluorescence compared to the wild-type. This indicates that the fast-growing mutant SDC.1 carries a high flux through the shikimate pathway towards 4HB biosynthesis (Figure 6B). Genomic characterization of SDC. To elucidate the genetic basis of SDC, the genomes and plasmids of the most efficiently evolved strains were sequenced. Isolates that displayed high relative fluorescence were solely focused on. This is because these strains should display a high carbon flux through the shikimate pathway irrespective of the growth rate. From each evolution experiment, ten mutants were chosen. At first, the biosensor plasmids were sequenced to check for alterations. However, no mutations occurred in the plasmids of all twenty isolates, indicating that growth occurred solely due to genomic alterations. All isolates from the two individual evolution experiments contained mutations in the miaA and mexT genes at various positions (Figure 7A). MiaA is a tRNA dimethylallyl transferase and has been known to affect the expression of various genes related to the central and secondary metabolism (Koshla et al., 2019). In P. putida specifically, it was reported that the removal of miaA, dramatically increased the expression of the trpE and trpGDC genes, both involved in tryptophan biosynthesis (Figure 8C) (Olekhnovich & Gussin, 2001). MiaA has been further studied in Pseudomonas chlororaphis, where its inactivation led to the upregulation of the trp genes and aroF. The latter encodes a 3-deoxy-7-phosphoheptulonate synthase, which catalyzes the first reaction of the shikimate pathway (Yu et al., 2017). The mexT gene encodes a transcriptional regulator that has mostly been studied in Pseudomonas aeruginosa in which it has been shown to repress the entire quinolone biosynthetic pathway and the first reaction of tryptophan biosynthesis (Figure 7C) (Tian et al., 2009). Another major mutation that occurred in 12 out of the 20 isolates was a perturbation in garK, encoding a glycerate kinase. However, as the garK perturbation did not occur in all isolates, this one was deemed as non-universal and focused solely on miaA and mexT. To assess the impact of the universal mutations of miaA and mexT, the SDC.1 mutations of these genes were reverse-engineered into ΔPYR-carrying and their growth was assessed in glycerol minimal medium while expressing pSensorEvo. The mutations from the SDC.1 were selected for reverse engineering because they exhibited superior performance compared to the other isolates, as indicated by high relative fluorescence levels and growth rate. Remarkably, the miaA mutation alone was sufficient to permit growth in glycerol minimal medium, bypassing the need for further evolutionary adaptation (Figure 7B). While the mexT mutation did not independently confer growth, its removal in combination with miaA led to a reduced lag phase, highlighting a synergistic effect (Figure 7B). As the inactivation of only miaA allowed growth in minimal medium with glycerol in ΔPYR, it appears key in regulating the shikimate pathway and could be a potential target for metabolic engineering strategies in other organisms. To further investigate the impact of the miaA mutation on shikimate pathway flux, an experiment was conducted to assess 4HB production in the presence and absence of the mutation. For this purpose, two strains were used: the KT2440ΔglpR ΔpobA strain (control) and the KT2440ΔglpR ΔpobA ΔmiaA mutant strain (the control strain with the miaA mutation identified in evolution). Both strains carried the pSEVAb23 vector with ubiCE31Q / M34V, under the control of the BBa_23100 promoter. Cultures were grown in minimal medium containing 100 mM glycerol, and 4HB production was quantified after 48 hours. As shown in Figure 7C, the strain with the miaA mutation exhibited a 3.8-fold increase in 4HB titer at 48 hours, reaching 1.42 mM, and achieved a 3.1-fold higher yield of 0.02 mol / mol on glycerol, compared to the control strain. In terms of overall growth and glycerol consumption, the KT2440ΔglpR ΔpobA ΔmiaA strain displayed a 1.3-fold lower final cell density compared and 1.18-fold increased glycerol consumption compared to the control strain (Figure 7D). Further deletion of mexT in the ΔglpR ΔpobA ΔmiaA background did not result in a significant increase in 4HB production, with the titer and yield observed for the KT2440ΔglpR ΔpobA ΔmiaA ΔmexT strain (1.45 mM and 0.021 mol / mol, respectively) being almost equal to that of the KT2440ΔglpR ΔpobA ΔmiaA strain. However, while the mexT inactivation did not appear to enhance 4HB production in this genetic background, it did contribute to an increase in growth rate under the SDC configuration. These results support the hypothesis that the miaA mutation significantly enhances carbon flux through the shikimate pathway, favoring the production of 4HB, and highlight miaA as a valuable target in metabolic engineering strategies aimed at optimizing shikimate pathway flux in microbial hosts, such as P. putida. Evaluation of metabolic fluxes in SDC. Strain SDC.1 was selected from the heterogeneous mix based on its growth rate and fluorescence profile. Next, the aim was to determine the flux increase through the shikimate pathway of this strain compared to the wild type. Chorismate, the final product of the shikimate pathway, is equimolarly cleaved into 4HB and pyruvate by chorismate pyruvate lyase. Therefore, the aim was to quantify 4HB production to evaluate whether SDC.1 uses the shikimate pathway as its main metabolic route for growth. In theory, the higher the yield of 4HB, the more flux is diverted into the shikimate pathway. FBA analysis predicts a maximum pathway yield of 0.39 mol 4HB per mol glycerol. In this scenario, there is no bacterial growth, and all carbon, including the released pyruvate by CHRPL, is directed toward 4HB synthesis. Therefore, this was set as the attainable maximum. To assess 4HB production, the pobA gene was deleted in both the KT2440ΔglpR (wild type) and SDC.1, creating SDC.2 (Figure 7A). This gene encodes a p-hydroxybenzoate hydroxylase and is responsible for the degradation of 4HB to protocatechuate (PCA), which can be further degraded to fuel the TCA cycle. The removal of the pobA gene had a negligible effect on wild type’s growth. However, growth of SDC.2 was severely stunted, requiring 14 days to reach the stationary phase compared to 4 days for SDC.1 (Figure 8A). Moreover, a lower final cell density was observed indicating that SDC.2 metabolism is highly dependent on the activity of the shikimate pathway and that the produced 4HB cannot be recycled back for cell proliferation. The obtained yield for SDC.2 was 0.06 mol 4HB per mol glycerol, a 13.8-fold increase compared to the KT2440ΔglpR ΔpobA, confirming a significantly increased carbon flux through the shikimate pathway (Figure 8B). However, this flux only comprises 15.4 % of the predicted maximum theoretical flux, implying that other routes are still taking a significant portion of the intracellular fluxes. The shikimate pathway was particularly focused on to further optimize SDC.2. It was hypothesized that quiC, encoding 3-dehydroshikimate (3HDS) dehydratase, was siphoning-off carbon from the shikimate pathway towards the TCA cycle (Figure 7A). When growth was simulated for SDC.2, the model predicted that 54 % of the flux entering the shikimate pathway is redirected through this reaction, converting the shikimate pathway intermediate 3DHS to PCA, efficiently circumventing the pobA deletion. As such, SDC.3 was created by deleting quiC from SDC.2 and its growth and 4HB production were assessed. It became apparent that this reaction indeed was a major metabolic bypass. The SDC.3 strain displayed even further stunted growth compared to SDC.2, reaching the stationary phase after 10 days with a concomitant significantly lower biomass formation (Figure 8A). The final 4HB yield increased 2.4-fold to 0.14 mol 4HB per mol glycerol (Figure 8C), indicating a further increase in the flux through the final reactions of the shikimate pathway. Although the optimized strain SDC.3 demonstrated increased fluxes through the shikimate pathway, it only reaches 36.7 % of the attainable predicted maximum yield. Therefore, the SDC metabolism was further simulated to identify bottlenecks. Simulations predicted that 19.7 % of the total consumed glycerol is excreted as glycerate. As mentioned earlier, 12 out of 20 isolates contained mutations in the garK gene. The isolated SDC.1 and its derivates contain a 16 bp deletion within the gene resulting in a frameshift. The garK gene encodes glycerate kinase, which is responsible for the phosphorylation of glycerate to glycerate-2-phosphate, which can then enter main metabolism. It was hypothesized that the frameshift renders the enzyme inactive and allows glycerate to accumulate. Glycerate production in SDC.3 was quantified and confirmed the hypothesis, as 18.3 % of the total amount of glycerol was excreted as glycerate, decreasing the total carbon flux towards the shikimate pathway (Figure 8C). According to model simulations, pyruvate production from glycerol using SDC only yields 6.1 % of ATP compared to the ED pathway (as described below in the Energy generation by SDC section). Aerobic bacteria like P. putida generate most electron carriers in the TCA cycle, which can then feed the electron transport chain and produce ATP through oxidative phosphorylation. However, in the SDC strains, the connection between glycolysis and TCA is disrupted and the TCA cycle can only be reached through the ATP-consuming shikimate pathway. Model predictions show that in native metabolism, glycerol is catabolized to the glycolytic intermediate dihydroxyacetone-P (DHAP) (Figure 1). This process costs one molecule ATP per molecule glycerol and yields one molecule reduced quinone. However, in SDC, the model predicts NAD-dependent oxidation of glycerol to glycerate by AldB-1, PP_2694, or FrmA. Therefore, the activation of the alternative glycerol utilization pathway most likely serves as an additional source of reducing equivalents which can be oxidized in the electron transport chain to generate a proton motive force for additional ATP. In addition, the GarK enzyme also requires the utilization of ATP. Therefore, it is likely that garK has mutated to conserve additional ATP. Although the model predicts NAD-dependent oxidation of glycerol to glycerate, the first step in glycerate production from glycerol in P. putida is initiated by the PQQ-dependent alcohol dehydrogenases encoded by pedE and pedH (Wehrmann et al., 2020). Like NADH, PQQH2 gets oxidized in the electron transport chain, generating ATP. The ability to generate ATP by converting glycerol into glycerate, therefore, reduces the required SDC-dependent flux towards the TCA cycle. To abolish glycerate production, both pedH and pedE where deleted from SDC.3, creating SDC.4. This deletion extended the lag phase slightly but did not have a significant impact on the growth rate (Figure 8A). Glycerate production was completely abolished and revealed to be a major metabolic bottleneck as its removal increased the yield from 0.14 to 0.28 mol 4HB per mol glycerol (Figure 8D). This accounts for 71.0 % of the maximum predicted pathway yield and indicates that a significant flux in SDC.4 is diverted through the shikimate pathway. For further optimization, branching pathways from the shikimate pathway were focused on. Within the isolated mutants, mutations were discovered in the genes miaA and mexT and have previously shown their impact on establishing SDC. Both their encoded proteins have been reported to directly influence tryptophan biosynthesis by regulating anthranilate synthase (ANS), encoded by trpE in P. putida. It was hypothesized that the SDC.1 strain and its further derivatives may display increased ANS activity due to the mutations in miaA and mexT. Like CHRPL, the TrpE enzyme cleaves chorismate releasing pyruvate in the process. However, this reaction requires additional L-glutamine or ammonia as an amine donor, which according to our in-silico assessment results in lower growth rates (Figure 3A). Yet, the removal of this reaction is unwanted as it would render the strain auxotrophic for tryptophan. Therefore, the aim was set to reduce its activity by introducing a P290S point mutation in the trpE gene of SDC.4. This specific mutation has been reported to lower the activity of the TrpE enzyme (Lenzen et al., 2019). This new strain, termed SDC.5, has a slower growth rate (0.008 ± 0.0001 per hour) compared to SDC.4 (0.011 ± 0.0004 per hour) yet reached similar final cell densities (Figure 8A). However, the 4HB yield increased from 0.28 to 0.35 mol 4HB per mol glycerol (Figure 8D). This accounts for 89.2 % of the maximum predicted pathway yield and indicates that SDC.5 diverts most of its glycerol metabolism through the shikimate pathway. Genomic integration and characterization of a stable SDC strain. So far, the flux was quantified through the shikimate pathway using 4HB as output. By reaching the predicted maximum, it can be concluded that all major bottlenecks are removed and that SDC was successfully established. Therefore, the aim was set to produce a stable strain that could serve as a chassis for shikimate pathway-derived products. For this purpose, the feedback resistant ubiCE31Q / M34Vgene was integrated in SDC.5 under the control of a bicistronic design at the innocuous PP_5322 site (Chaves et al., 2020). Furthermore, the pobA gene was restored to create the final SDC.6 strain in which 4HB can once more fuel biomass production (Figure 9A). The growth characteristics of SDC.6 was determined and compared to the KT2440ΔglpR, as both strains have different pathways to convert glycerol to pyruvate. Moreover, SDC.1 was used as a second control to examine the impact of all further modifications on growth. As expected, the KT2440ΔglpR using the canonical glycerol metabolism grew fast and reached the stationary phase within 24 hours. The genomically stable SDC.6 strain had a lower specific growth rate than SDC.1, 0.038 ± 0.001 per hour versus 0.052 ± 0.007 per hour, respectively (Figure 9B). It was hypothesized that the lower growth rate of SDC.6 is the result of i) the absence of QuiC, which allowed a quick bypass in SDC.1 from the shikimate pathway toward the TCA cycle, ii) the removal of glycerate production, which allowed higher ATP generation, and iii) fewer pyruvate production due to elevated trpE activity (Figure 9A). Although slower, SDC.6 reached a final OD600 of 3.17, which is 14.5 % higher than SDC.1. This can be attributed to the removal of glycerate formation in SDC.6, which can now be funneled towards biomass production (Figure 9A). However, this removal likely increased the lag phase in SDC.6. The PedE and PedH enzymes are part of the alternative glycerol metabolism in P. putida and their removal has been demonstrated to strongly prolong the lag phase (Wehrmann et al., 2020). Yet, SDC.6 is still superior to SDC.1, as it is devoid of all the bottlenecks described above and can therefore lead to higher yields for shikimate pathway-derived products. Next, the model was used to calculate the maximum specific growth rate in SDC.6. For this analysis, the maximum glycerol uptake rate was set at 3.95 mmol / gCDW / h, the same as in KT2440. However, the model predicts a lower glycerol uptake rate for this strain, with a maximum of 3.36 mmol / gCDW / h, probably indicating a limitation in cofactor regeneration. Moreover, the model predicts a maximum specific growth rate of 0.16 per hour, which is 4.2-fold higher than the actual growth rate we observed. Therefore, there is still room for improvement to establish SDC.6 as a true chassis for shikimate pathway-derived products. Energy generation by SDC. Overall equations were calculated from glycerol to pyruvate to study the feasibility of SDC in terms of its ability to generate reduced cofactors and ATP compared to native metabolism. Native and SDC metabolism were simulated with flux balance analysis (FBA) with pyruvate production (EX_pyr_e) as maximization objective. Then, reduced models were generated containing only predicted active reactions of central carbon metabolism (Table 1), as well as exchange reactions for nadh_c, nadph_c, atp_c, nad_c, nadp_c, pyr_c, h_c, h2o_c, q8_c, q8h2_c, o2_c, coa_c, co2_c, and pi_c. To find the most efficient overall reaction in terms of ATP generation, pyruvate exchange was set as the objective to maximize, and the bounds of its exchange reaction were constrained to the optimal value. Then, the ATP exchange reaction was set as a new objective to maximize. A glycerol uptake of 1 mmol / gDW / h was used and the fluxes through the different exchange reactions are defined as coefficients in the overall equations. According to iJN1462, oxidation of NADH via NADH dehydrogenase (reaction NADH16pp) allows the export of 3 H+to the periplasm. The reduction of ubiquinone-8 (q8) to ubiquinol (q8h2), and oxidation of q8h2 by cytochrome oxidase (CYTVo3_4pp) allows the export of 4 H+to the periplasm. Generation of ATP by the ATP synthase (ATPS4rpp) requires the import of 4 H+ from the cytoplasm resulting in a yield of 1 ATP per oxidation of 1 q8h2 and 1.75 ATP per oxidation of NADH. Overall equation native metabolism (KT2440): 1 Glycerol + 1 Q8 + 1 NAD + 1 ATP + Pi ^ 1 PYR + 1 Q8H2 + 1 NADH + H++ 2 ATP Simplified equation (conversion of reduced cofactors to ATP in the respiratory chain): 1 Glycerol ^ 1 PYR + 3.75 ATP Overall equation SDC metabolism: 1 Glycerol + 1.8 Q8 + 1.4 NAD + 1.1 NADPH + 1.1 O2 + 2.1 ATP + 1.3 H2O --> 0.55 PYR + 1.8 Q8H2 + 1.4 NADH + 1.4 CO2 + 2.1 ADP + 2.1 Pi + 2.9 H+Simplified equation (assuming conversion of NADH to NADPH and generation of ATP by oxidation of reduced cofactors in the respiratory chain): 1 Glycerol + 1.1 O2 --> 0.55 PYR + 1.4 CO2 + 0.23 ATP Note: the O2 in this equation is not used in the respiratory chain but in the first two reactions converting 4HB into TCA intermediates In silico validation of shikimate-dependent catabolism across diverse microbial hosts. To evaluate the broad applicability of the SDC, in silico simulations were conducted using genome-scale modeling for a diverse set of organisms: the Gram- negative bacterium E. coli, the Gram-positive bacterium C. glutamicum, and the yeast S. cerevisiae. In these models, key enzymes were deactivated, including pyruvate kinase, phosphoenolpyruvate carboxylase, and phosphogluconate dehydratase (where applicable), to disrupt native glycolysis. To simulate the metabolic characteristics of SDC, a constraint was imposed ensuring that at least 10% of the total glycerol uptake flux was directed towards a shikimate-derived pyruvate-releasing reaction, specifically anthranilate synthase (ANS). Using pFBA, these modified strains were assessed whether they could achieve a growth phenotype where over 80% of intracellular pyruvate production was rerouted through the shikimate-derived reaction. The results, summarized in Table 6, demonstrate that all tested strains were capable of adapting to this configuration, albeit with reduced growth rates compared to their respective wild- type strains. In E. coli, the growth rate decreased by 66.7%, from 0.33 to 0.11. Similarly, in C. glutamicum, the growth rate was reduced by 68.8%, from 0.32 to 0.10, while in S. cerevisiae, the reduction was 60.7%, from 0.28 to 0.11. Despite the reductions, these findings strongly support that SDC can be effectively implemented across a wide range of microbial hosts. The consistent rerouting of carbon flux towards shikimate-derived pyruvate production highlights the metabolic flexibility of these organisms and indicate that the SDC strategy can be broadly applied to optimize the synthesis of pyruvate- derived compounds, offering a scalable platform for enhancing carbon flux towards valuable aromatic metabolites in diverse microbial systems. Table 1. Reactions included in the reduced models for obtaining general equations for pyruvate production in native and SDC metabolism. GLYCtex, Glycerol transport via diffusion (extracellular to periplasm); GLYCtpp, Glycerol transport via channel (periplasm); GLYK, Glycerol kinase; G3PD2, Glycerol-3-phosphate dehydrogenase (NADP); TPI, Triose-phosphate isomerase; GAPD, Glyceraldehyde-3-phosphate dehydrogenase; GAPDi_nadp, Glyceraldehyde-3-phosphate dehydrogenase (NAD(P)+) (phosphorylating); PGK, Phosphoglycerate kinase; PGM, Phosphoglycerate mutase; ENO, Enolase; EX_glyc_e, Glycerol exchange; TKT1, Transketolase; FBA, Fructose-bisphosphate aldolase; FBP, Fructose-bisphosphatase; TKT2, Transketolase; TALA, Transaldolase; RPE, Ribulose 5-phosphate 3-epimerase; RPI, Ribose-5-phosphate isomerase; DDPA, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase; DHQS, 3-dehydroquinate synthase; DHQTI, 3-dehydroquinate dehydratase, irreversible; SHK3Dr, Shikimate dehydrogenase; SHKK, Shikimate kinase; PSCVT, 3-phosphoshikimate 1-carboxyvinyltransferase; CHORS, Chorismate synthase; CHRPL, Chorismate pyruvate lyase; 4HBHYOX, 4 hydroxybenzoate NADPH:oxygen oxidoreductase 3 hydroxylating; PCADYOX, Protocatechuate 34 dioxygenase; MUCCY_kt, 3 carboxy cis-muconate cycloisomerase; 4CMLCL_kt, 4- carboxymuconolactone decarboxylase; 3OADPCOAT, 3-oxoadipate CoA-transferase; 3OXCOAT, 3-oxoadipyl-CoA thiolase; SUCDi, Succinate dehydrogenase (irreversible); FUM, Fumarase; MDH, Malate dehydrogenase; PPCK, Phosphoenolpyruvate carboxykinase; CS, Citrate synthase; ACONTa, Aconitase (half-reaction A, Citrate hydro-lyase); ACONTb, Aconitase (half-reaction B, Isocitrate hydro-lyase); SUCOAS, Succinyl-CoA synthetase (ADP-forming). Metabolism Reactions in reduced modelsNative GLYCtex, GLYCtpp, GLYK, G3PD2, TPI, GAPD, GAPDi_nadp, PGK,PGM, ENO, EX_glyc_e SDC Same as above and TKT1, FBA, FBP, TKT2, TALA, RPE, RPI, DDPA,DHQS, DHQTI, SHK3Dr, SHKK, PSCVT, CHORS, CHRPL, 4HBHYOX, PCADYOX, MUCCY_kt, 4CMLCL_kt, 3OADPCOAT, 3OXCOAT, SUCDi, FUM, MDH, PPCK, CS, ACONTa, ACONTb, SUCOAS Table 2. Strains and plasmids used in this study. Strains Description Source Escherichia coli Dh5α Cloning host: F-λ-endA1 glnX44(AS) thiE1 (Grant et al., recA1 relA1 spoT1 gyrA96(NalR) rfbC1 deoR 1990) nupG Φ80(lacZΔM15) Δ(argF-lac)U169 hsdR17(rKmK) CC118λpir Cloning host: araD139 Δ(ara-leu)7697 (Herrero et ΔlacX74 galE galK phoA20 thi− 1 rpsE al., 1990) rpoB(RifR) argE(Am) recA1, λpir lysogen Pseudomonas putida KT2440 Wild-type strain; mt-2 derivative cured of the(Bagdasarian TOL plasmid pWW0 et al., 1981)KT2440 ΔglpR KT2440 with scarless deletion of the glpRThis study geneKT2440 ΔpobA KT2440 with scarless deletion of the pobAThis study geneKT2440 ΔglpR ΔpobA KT2440 ΔglpR with scarless deletion of theThis study pobA geneKT2440 ΔglpR ΔpobA KT2440 ΔglpR ΔpobA ΔglpR with deletion ofThis study ΔmiaA the SDC.1 mutation in miaAKT2440 ΔglpR ΔpobA KT2440 ΔglpR ΔpobA ΔglpR ΔmiaA withThis study ΔmiaA ΔmexT deletion of the SDC.1 mutation in mexTKT2440 ΔPYR KT2440 with scarless deletion of the glpR,This study edd, pyk, pykA and ppc genesSDC.1 KT2440 ΔPYR mutant isolated afterThis study evolution in minimal medium with glycerol SDC.2 SDC.1 with a scarless deletion of the pobA This study geneSDC.3 SDC.2 with a scarless deletion of the quiCThis study geneSDC.4 SDC.3 with a scarless deletion of the pedEThis study and pedH genesSDC.5 SDC.4 with a P290S amino acid substitutionThis study in the trpE geneSDC.6 SDC.5 with the scarless introduction ofThis study BCD2-ubiCE31Q / M34Vand BBBa_J23100 – pobAKT2440 ΔPYR ΔmiaA KT2440 ΔPYR with deletion of the SDC.1This study mutation in miaAKT2440 ΔPYR ΔmexT KT2440 ΔPYR with deletion of the SDC.1This study mutation in mexTKT2440 ΔPYR ΔmiaA KT2440 ΔPYR with deletions of the SDC.1This study ΔmexT mutations in mexT and miaA PlasmidspGNW Plasmid for genome editing in Gram-negative(Wirth et al., bacteria: ori (R6K) neo, KmR2020)pGNW – edd pGNW for scarless deletion of edd This studypGNW – glpR pGNW for scarless deletion of glpR This studypGNW – pyk pGNW for scarless deletion of pyk This studypGNW – pykA pGNW for scarless deletion of pykA This studypGNW – ppc pGNW for scarless deletion of ppc This studypGNW – pobA pGNW for scarless deletion of pobA This studypGNW – quiC pGNW for scarless deletion of quiC This studypGNW – pedE pGNW for scarless deletion of pedE This studypGNW – pedH pGNW for scarless deletion of pedH This studypGNW – trpEP290S pGNW for scarless substitution of P290S inThis study trpE pGNW – BCD2-ubiC pGNW for scarless introduction of p14 –This study BCD2 - ubiCE31Q / M34VpGNW – p100 - pobA pGNW for scarless introduction ofThis study BBa_J23100 – BBa_B0034 - pobA pGNW – mexT::mut pGNW for scarless deletion of the SDC.1This study mutation in mexT pGNW – miaA::mut pGNW for scarless deletion of the SDC.1This study mutation in miaA pQURE6-H Helper plasmid; oriV(RK2), xylS, PmI-SceI;(Volke et al., GmR2020) pSB1C3 Repository vector, pUC19-derived pMB1 ori, IGEM CmRregistry pSB1C3_pobR-PPobA pSB1C3 with PPobA promoter and PobR(ΔL141,This study L220V)from Acinetobacter baylyi ADP1 pSB1C3_ubiCE31Q / M34V pSB1C3 with ubiC gene from Escherichia coliThis study encoding chorismate lyase with the E31Q and M34V amino acid substitutions pSEVAb83 oriV(pBBR1); ApraR(Damalas et al., 2020) pSensor oriV(pBBR1); PobR (ΔL141, L220V)This study / PPobA→sfgfp.laa; ApraRpSensorEvo oriV(pBBR1); PobR (ΔL141, L220V)This study / PPobA→sfgfp.laa; BBa_J23100→ubiCE31Q / M34VApraRpSEVAb23_UbiC oriV(pBBR1); KanR;This study BBa_J23100→ubiCE31Q / M34VTable 3. Primers used in this study. Primers Sequence ‘5 > 3’ Purpose HA1_edd_FW aggtctctcccgtggcactgaccgcgata Cloning theHA1_edd_RV aggtctctactggactccaggctaattgthomology arms for HA2_edd _FW aggtctctcagtgcaccaggtggtcgagscarless deletion of HA2_edd _RV aggtctcttcgaggtgctccagggccedd HA1_pykA_FW aggtctctcccgttttccagggtctgcaggcg Cloning theHA1_pykA_RV aggtctctgcacagactcctatagtgaagchomology arms for HA2_pykA_FW aggtctctgtgctccactcgtttcacagcacaaggscarless deletion of HA2_pykA_RV aggtctcttcgacctggcagccatcatcgaccpykA HA1_pyk_FW aggtctctcccgaggctgccgcggtcaaagc Cloning theHA1_pyk_RV aggtctcttcatgattgggcagtctcaagghomology arms for HA2_pyk_FW aggtctctatgagattgccgggggcgcgscarless deletion of HA2_pyk_RV aggtctcttcgacaatatggccgaccagggcagpyk HA1_ppc_FW aggtctctcccgtgctcaagttgtgcaggtgctgHA1_ppc_RV aggtctcttggcgctctctccgttgc Cloning theHA2_ppc_FW aggtctctgccagggcaagcccgggtcatghomology arms forHA2_ppc_RV aggtctcttcgacagctggccggcatccatgscarless deletion of ppcHA1_glpR_FW aggtctctcccgggcgtgaaggtagaccacatcatccCloning the g homology arms forHA1_glpR_RV aggtctctgggcggtcctttggggctgcscarless deletion ofHA2_glpR_FW aggtctctgcccgggctggtgggtgcatgccgglpRHA2_glpR_RV aggtctcttcgacacgatgtggggcgccttcgcHA1_pobA_FW aggtctctcccgcaggttcgctacaagggc Cloning theHA1_pobA_RV aggtctcttgttgttgtctctgttcggacghomology arms forHA2_pobA_FW aggtctctaacagggctgcgagctacgscarless deletion ofHA2_pobA_RV aggtctcttcgatcctccttggggagcattttpobAHA1_quiC_FW aggtctctcccgcgaaggcgccgatcgggc Cloning theHA1_quiC_RV aggtctctggcgggagttccttgttgtthomology arms forHA2_quiC_FW aggtctctcgcctcgcctgcaccggcctgtcscarless deletion ofHA2_quiC_RV aggtctcttcgacacgctggtggatgcagcgcquiCHA1_pedE_FW aggtctctcccggccccggacagcctggcgaa Cloning theHA1_pedE_RV aggtctctcctgcagcggggagcggccthomology arms forHA2_pedE_FW aggtctctcaggggttgcagttcccagtggagscarless deletion ofHA2_pedE_RV aggtctcttcgaccactgcctggggcaccacgtpedEHA1_pedH_FW aggtctctcccggcgccctgaccacgcccaac Cloning theHA1_pedH_RV aggtctctgacggctacctttggttttthomology arms forHA2_pedH_FW aggtctctcgtccgcctgcctactgccgcttgscarless deletion ofHA2_pedH_RV aggtctcttcgacgtttcgcgtatggccttgccpedGHHA1_trpEP290S_FW aggtctctcccgttggctacgactgcgtgcgctatg Cloning theHA1_trpEP290S_RV aggtctctcgagctgccgaccacatgghomology arms forHA2_trpEP290S_FW aggtctctctcgagcgaagtgttggtacgtgtcgagscarlessHA2_trpEP290S_RV aggtctcttcgacccggcctgcacatgcagctcsubstitution of P290S in trpE trpEP290S_Check_WTcatgtggtcggcagctcgcc Verification of the_FW scarless trpEP290S_Check_KI_ catgtggtcggcagctcgag substitution of FW P290S in trpEtrpEP290S_Check_RV ccggtcgaagacacacggccHA1_mexT_FW aggtctctcccgcgggctttgcccgttagacc Cloning theHA1_mexT_RV aggtctctggcgaactcggcgtcgtccghomology arms forHA2_mexT_FW aggtctctcgccttgctgatgccaaccctgctgpart deletion ofHA2_mexT_RV aggtctcttcgaccacaagctatcgggatccccaaagmexTHA1_miaA_FW aggtctctcccgccacggtgccgtgcgcgccaac Cloning theHA1_miaA_RV aggtctctgatcaacgccttgtaataghomology arms forHA2_miaA_FW aggtctctgatcgacatgccggccgccgatgcpart deletion miaAHA2_miaA_RV aggtctcttcgaccgggtagcgatgataccgcgpGNW_pobA_FW aggtctctcccgcaggttcgctacaagggc Cloning the pobApGNW_pobA_RV aggtctctcccgcaggttcgctacaagggcgene for scarless P100_pobA_FW aggtctctagtgctagctactagagaaagaggagaainsertion in SDC.5 atactagatgaaaactcaggttgc P100_pobA_RV aggtctctcactgtacctaggactgagctagccgtcaatgttgttgtctctgttcggacg ubiC E31Q / M34V_FW1 attggtctcagaattcgcggccgcttctagatgtcacaConstruction and ccccgcgtta cloning of ubiC E31Q / M34V_RV1 aggtctctatcctgcagcaacagccubiCE31Q / M34Vin ubiC E31Q / M34V_FW2 aggtctctggattccgtgacaaaacgttttgpSB1C3 ubiC E31Q / M34V_RV2 taaggtctcgactgcagcggccgctactagtattagtacaacggtgacgcc HA1_PP5322_FW aggtctctcccgcgacctggaggaccttgccc Cloning ofHA1_PP5322_RV aggtctcttgggccatcaatccagcttcubiCE31Q / M34Vwith a BCD2_PP5322_FW aggtctctcccactagaggcccattgacaaggctctcgbicistronic design cg for scarless BCD2_PP5322_RV aggtctctagaaaacctccttagcatgattaagatgintegration in BCD2_ubiC_FW aggtctctttctaatgtcacaccccgcgttaacSDC.5 BCD2_ubiC_RV aggtctctcgtaattattggggacccctggHA2_PP5322_FW aggtctcttacgatcggtcaggggttgccggHA2_PP5322_RV aggtctcttcgaccaggccatgggtgacccggTable 4. Modifications of P. putida GEM iJN1462 for simulations of native andthe SDC metabolism. Lb, lower bound of a reaction; ub, upper bound of a reaction; g3p_c, Glyceraldehyde 3-phosphate; 3pg_c, 3-Phospho-D-glycerate; AKGDb, Oxoglutarate dehydrogenase (dihydrolipoamide S-succinyltransferase); THRA, Threonine aldolase; LSERDHr, L-serine dehydrogenase; ACACT1r, Acetyl-CoA C-acetyltransferase; MACCOAT, 2-Methylacetyl-CoA thiolase; MMSAD3, Methylmalonate-semialdehyde dehydrogenase (malonic semialdehyde); ACALD, Acetaldehyde dehydrogenase (acetylating); ALDD2x, Aldehyde dehydrogenase (acetaldehyde, NAD); ALDD2y, Aldehyde dehydrogenase (acetaldehyde, NADP); KAT1, 3-ketoacyl-CoA thiolase; GAPDi_nadp, Glyceraldehyde-3-phosphate dehydrogenase (NAD(P)+) (phosphorylating). Reaction Modification Reason AKGDb Make irreversible (lb = 0) Avoid a cycle in which AKG isproduced from SUCCoA fixing CO2. Irreversible according to BioCyc.com THRA Make irreversible (lb = 0) Avoid threonine production via itsdegradation pathway Irreversible according to BioCyc.comLSERDHr Make irreversible (ub = 0) Avoid the use of THF relatedreactions as source of NAD(P)H Irreversible according to BioCyc.com ACACT1r Make irreversible (lb = 0) Make PDH essentialMACCOAT, Make irreversible (ub = 0) MMSAD3, ACALD, ALDD2x, ALDD2y, KAT1 GAPDi_nadp Correct: g3p_c + nadp_c +E.C number did not correspond h2o_c -> 3pg_c + h_c + with the correct reaction nadph_c Table 5. Model modifications to simulate different SDC strains. EDD, 6-phosphogluconate dehydratase; PYK, pyruvate kinase; AGPOP, DGTP:pyruvate 2-O-phosphotransferase; ME2, malic enzyme; OAADC, Oxaloacetate decarboxylase; SERD_L, L-serine deaminase; CYSTL, Cystathionine b-lyase; CYSDS, Cysteine Desulfhydrase; MCITL2, Methylisocitrate lyase; LDH_D, D-lactate dehydrogenase; LDH_D2, D-lactate dehydrogenase; 4HBHYOX, 4 hydroxybenzoate NADPHoxygen oxidoreductase 3 hydroxylating; DHSKDH, Dehydroshikimate dehydratase; GLYALDDr, D-Glyceraldehyde dehydrogenase; ALCD19, Alcohol dehydrogenase (glycerol). Strain Reactions constrained to 0SDC.1 EDD, PYK, AGPOP, ME2, OAADC, SERD_L, CYSTL, CYSDS, MCITL2,LDH_D, LDH_D2 SDC.2 EDD, PYK, AGPOP, ME2, OAADC, SERD_L, CYSTL, CYSDS, MCITL2,LDH_ D, LDH_D2, 4HBHYOX SDC.3 EDD, PYK, AGPOP, ME2, OAADC, SERD_L, CYSTL, CYSDS, MCITL2,LDH_ D, LDH_D2, 4HBHYOX, DHSKDH SDC.4 EDD, PYK, AGPOP, ME2, OAADC, SERD_L, CYSTL, CYSDS, MCITL2,LDH_ D, LDH_D2, 4HBHYOX, DHSKDH, GLYALDDr, ALCD19 SDC.5 EDD, PYK, AGPOP, ME2, OAADC, SERD_L, CYSTL, CYSDS, MCITL2,LDH_ D, LDH_D2, 4HBHYOX, DHSKDH, GLYALDDr, ALCD19 SDC.6 EDD, PYK, AGPOP, ME2, OAADC, SERD_L, CYSTL, CYSDS, MCITL2,LDH_ D, LDH_D2, DHSKDH, GLYALDDr, ALCD19 Table 6. Predicted growth rates and shikimate-derived pyruvate production in SDC-modified microbial models. Growth rate (after modifications) refers to the predicted growth rate after in silico modifications, which include deactivation of PYK,EDA, PPC (E. coli); PYK, PPC (C. glutamicum); r_0962, r_0884 (S. cerevisiae).Organism Model Target Growth rate Growth rate % of reaction (before (after total modificatio modification pyruvat ns) s) e produc ed E. coli iBWG_13 Anthranilate0.33 0.11 82%29 synthase (ANS) C.iCW773 Anthranilate 0.32 0.1 88%glutamicum synthase (ANS) S. cereavisae Yeast9 Anthranilate 0.28 0.11 81.6%synthase (r_0203) Example 2 Expanding shikimate pathway-dependent catabolism (SDC) through other pyruvate-releasing pathways. In addition to the 4HB biosynthetic pathway, SDC has the potential to be sustained through various pyruvate-releasing reactions derived from chorismate. The shikimate pathway features several side branches that originate from chorismate and release pyruvate, including conversions of chorismate into anthranilate, 4-amino-4-deoxychorismate to 4-aminobenzoic acid, and isochorismate into salicylate or 2,3-dihydroxy-2,3-dihydrobenzoic acid - all of which serve as precursors for a diverse range of valuable compounds (Averesch & Krömer, 2018). To showcase the modular aspect of SDC while expanding its application scope further, it was integrated with the salicylate biosynthetic pathway (Fig. 10A). Salicylate holds significant prominence as an aromatic monomer and functions as a precursor in the production of aspirin and lamivudine - an anti-HIV drug. Moreover, adipic acid can be obtained from salicylate via both chemical and microbial methods, which serves as a crucial constituent for synthesizing nylon-6,6. Salicylate is synthesized from chorismate in a pathway comprising two reactions, catalyzed by the isochorismate synthase (entC gene product) and isochorismate pyruvate lyase (pchB gene product) (Fig. 10). The salicylate operon is placed under the control of the strong inducible expression system RhaRS / PrhaBAD in vector pSEVAb83 resulting in plasmid pSal. Strains SDC.4 and SDC.5 lacking the pUbiC were transformed with pSal yielding strains SDC.4_sal and SDC.5_sal, respectively. The capacity of the strains to grow and produce salicylate was evaluated in M9 minimal medium with 40 mM of glycerol. As shown in Fig.10B, it was proved that both strains were able to grow on glycerol demonstrating that SDC remained functional even when coupled with other pyruvate-releasing reactions rather than the originally established chorismate lyase. The salicylate production yield reached 0.18 and 0.31 mol / mol for SDC.4_sal and SDC.5_sal respectively, representing 46.7 % and 81 % of the maximum theoretical pathway yield (Fig. 10C). This yield represents the highest ever reported for salicylate. However, it is important to note that in strain SDC_sal a flux portion is allocated towards the essential biosynthesis of 4HB thereby diminishing the total salicylate yield. Overall, it was demonstrated that alternative pyruvate-releasing reactions derived from the shikimate pathway can sustain SDC and lead to record-breaking production yields. Example 3 A unique combination of genetic modifications in Pseudomonas putida, forming the basis of a new metabolic architecture, is provided in Table 7. Table 7. Unique combination of genetic modifications. Gene Modification Type Effect / RoleubiC Overexpression Catalyzes conversion of chorismate to 4HBpobA Deletion Removal prevents diversion of 4HB to theprotocatechuate branch, thereby increasing the metabolic flux towards 4HB and salicylate production quiC Deletion Removal prevents diversion of chorismatetowards quinate degradation pathway pedH Deletion Eliminates byproductspedE Deletion Eliminates byproductstrpE Point mutation Removal prevents diversion of chorismatetowards anthranilate and eventually tryptophan edd Deletion Eliminates the Entner-Doudoroff pathway,steering carbon flux to the shikimate pathway pykA Deletion Removes a key glycolytic enzyme, pyruvatekinase, directing more carbon flux towards the shikimate pathway pyk Deletion Removes a key glycolytic enzyme, pyruvatekinase, directing more carbon flux towards the shikimate pathway ppc Deletion Removes a key glycolytic enzyme, pyruvatekinase, directing more carbon flux towards the shikimate pathway More than 10 Multiple genomic mutations arised through genomic mutations laboratory evolution REFERENCES:^ Asin-Garcia, E., Batianis, C., Li, Y., Fawcett, J. 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Engineering in Life Sciences, 17(1), 47–57. https: / / doi.org / 10.1002 / elsc.201600072 SEQUENCES Amino acid sequence of engineered UbiC (chorismate lyase) MSHPALTQLRALRYCKEIPALDPQLLDWLLLQDSVTKRFEQQGKTVSVTMIREG FVEQNEIPEELPLLPKESRYWLREILLCADGEPWLAGRTVVPVSTLSGPELALQKLGK TPLGRYLFTSSTLTRDFIEIGRDAGLWGRRSRLRLSGKPLLLTELFLPASPLY Amino acid sequence of engineered pobR (transcription factor) MEQHHQYLAHPHSSEEIRTEDYIAGLAKGLALLEAFGIDRQRLNVTQVAERTGIS RTAARRYLKTLKFLGYLDTDEHYFWLTHRVLRFSSSYLSSAHLPKVAQSFLNLLCAQ TSLTFSIVVLDEHEVVPVARSYLPQQDNRVSPYGMHLGNRLPAHATSTGKVLLSVLD REVQIEWIEKYGLKRLTPYTITDEHTFLETLDAVRQSDYCLSTEEHELGVIAIAVPVLN AQGLTIAALNCMSQTNRVQPQYLIDQVLPLLRNTANELRNLV Amino acid sequence of engineered menF (isochorismate synthase) MQSLTTALENLLRHLSQEIPATPGIRVIDIPFPLKDAFDALSWLASQQTYPQFYW QQRNGDEEAVVLGAITRFTSLDQAQRFLRQHPEHADLRIWGLNAFDPSQGNLLLPRL EWRRCGGKATLRLTLFSESSLQHDAIQAKEFIATLVSIKPLPGLHLTTTREQHWPDKT GWTQLIELATKTIAEGELDKVVLARATDLHFASPVNAAAMMAASRRLNLNCYHFYM AFDGENAFLGSSPERLWRRRDKALRTEALAGTVANNPDDKQAQQLGEWLMADDKN QRENMLVVEDICQRLQADTQTLDVLPPQVLRLRKVQHLRRCIWTSLNKADDVICLHQ LQPTAAVAGLPRDLARQFIARHEPFTREWYAGSAGYLSLQQSEFCVSLRSAKISGNVV RLYAGAGIVRGSDPEQEWQEIDNKAAGLRTLLQME Amino acid sequence of engineered pchB (isochorismate pyruvate lyase) MKTPEDCTGLADIREAIDRIDLDIVQALGRRMDYVKAASRFKASEAAIPAPERVA AMLPERARWAEENGLDAPFVEGLFAQIIHWYIAEQIKYWRQTRGAA Amino acid sequence of engineered pykA (pyruvate kinase II) MSIRRTKIVA TLGPASNSPE VIEQLILAGL DVARLNFSHG TPDEHKARAR LIRDIAAKNG RHVALLGDLQ GPKIRIAKFA NKRIELKIGD KFTFSTAHPL TEGNQDIVGI DYPDLVKDCG VGDELLLDDG RVVMRVETAT ADALHCVVII GGPLSDHKGI NRKGGGLTAP ALTEKDKADI KLAAEMDLDY LAVSFPRDAS DMEYARKLRD EAGGSAWLVA KIERAEAVAD DETLDKLIAA SDAVMVARGD LGVEIGDAEL IAIQKKIIQH ARRNNKAVIV ATQMMESMIQ NPMPTRAEVS DVANAVLDNT DAVMLSAESA AGSYPIEAVQ AMARICLGAE KHPTSQKSSH RLHTTFQRCD ESIALAAMYT ANHFPGVKAI IALTESGYTP LIMSRLRSHV PIFALSPHRA TQARASMFRG VYPIAFDPAA LPADKVSQAA VDELLKRGLV EQGDWVILTK GDSYHTIGGT NGMKILHVGD PLVG Amino acid sequence of engineered pyk (pyruvate kinase) MSIRRTKIVATLGPASNSPEVIEQLILAGLDVARLNFSHGTPDEHKARARLIRDIA AKNGRHVALLGDLQGPKIRIAKFANKRIELKIGDKFTFSTAHPLTEGNQDIVGIDYPD LVKDCGVGDELLLDDGRVVMRVETATADALHCVVIIGGPLSDHKGINRKGGGLTAPA LTEKDKADIKLAAEMDLDYLAVSFPRDASDMEYARKLRDEAGGSAWLVAKIERAEAV ADDETLDKLIAASDAVMVARGDLGVEIGDAELIAIQKKIIQHARRNNKAVIVATQMME SMIQNPMPTRAEVSDVANAVLDNTDAVMLSAESAAGSYPIEAVQAMARICLGAEKHP TSQKSSHRLHTTFQRCDESIALAAMYTANHFPGVKAIIALTESGYTPLIMSRLRSHVPI FALSPHRATQARASMFRGVYPIAFDPAALPADKVSQAAVDELLKRGLVEQGDWVILT KGDSYHTIGGTNGMKILHVGDPLVG Amino acid sequence of engineered ppc (phosphoenolpyruvate carboxylase) MTDIDVRLREDVHVLGELLGETIRQQHGDAFLQKIEDIRHSAKADRRGPGEQLSS TLADLAEEDLLPVARAFNQFLNLANMAEQYQLIRRRDADQPEPFEAQVLPELLGRLK QAGHSNDALARQLAKLDIQLVLTAHPTEVARRTLIQKYDAIAGQLAAQDHRDLTPAE RQQVRERLRRLIAEAWHTEEIRRTRPTPVDEAKWGFAVIEHSLWHAIPSHLRKVDKA LLEATGLRLPLEAAPIRFASWMGGDRDGNPNVTAAVTREVLLLARWMAADLFLRDID ALAAELSMQQANDTLRKQVGDSAEPYRAVLKQLRDRLRATRAWAHSALTSNQPAGA DVLVDNRELIAPLELCYQSLHECGMGVIAEGPLLDCLRRAVTFGLFLGRLDVRQDAA RHRDALTEITDYLGLGRYADWDEEQRIAFLQAELKNRRPLLPAHFKPQADTAEVLAT CREVAAAPAASLGSYVISMAGAASDVLAVQLLLKEAGLTRPMRVVPLFETLADLDNA GPVMQRLLGLPGYRAGLRGPQEVMIGYSDSAKDAGTTAAAWAQYRAQENLVRICAE HQVELLLFHGRGGTVGRGGGPAHAAILSQPPGSVAGRFRTTEQGEMIRFKFGLPGIA EQNLNLYLAAVLEATLLPPPPPQPAWREVMDQLAADGVQAYRSVVRENPDFVEYFR QSTPEQELGRLPLGSRPAKRRAGGIESLRAIPWIFGWTQTRLMLPAWLGWETALTNA LARGQGELLAQMREQWPFFRTRIDMLEMVLAKADAQIAEAYDERLVQPHLRPLGAH LRDLLSQSCQVVLGLTGQPVLLAHSPETLEFISLRNTYLDPLHRLQAELLARSRSREA ALDSPLEQALLVTVAGIAAGLRNTG Amino acid sequence of engineered quiC (3-dehydroshikimate dehydratase) MQRSIATVSLSGTLPEKLEAIAAAGFDGVEIFENDLLYYAGSPRQVRQMCADLGI AITLFQPFRDFEGCRRDRLQKNLDRAERKFDLMQELGTDLVLVCSNVQADALGDEQL LVDDLRLLGEHAGKRGLRIGYEALAWGRHVNTYQQVWNLVRQADHPALGVILDSFH TLSLKGDPSAIRDIPGDKIFFVQMADAPILAMDVLEWSRHFRCFPGQGEMDMAGFLA PILATGYRGPLSLEIFNDGFRAAPTRQNAADGLRSLLYLEEQTRLRLEQENTPIEPGVL FSPPPASAYDGVEFLEFAVDEAVGARLGNWLKRLGFAEAGKHRSKEVQLLRQGDINI VLNAEPYSFGHNFFEAHGPSLCATALRVKDQQAALKRATAFRGQPFRGLVGPNECEV PAVRAPDGSLLYLVEQGTAGHTLYDTDFSLDNNATATGGLRRIDHMALALPAESLDS WVLFYKSLFDFAADDEVVLPDPYGLVKSRALRSQCGTLRLPLNISENRNTAIAHALSS YRGSGVHHIAFDCDDIFREVARAKLAGVPLLEIPLNYYDDLAARFDFDDEFLSELAYY NVLYDRDAQGGELFHVYTEPFEERFFFEIIQRKAGYAGYGAANVAVRLAAMAKARSG AARKPVL Amino acid sequence of engineered pobA (p-hydroxybenzoate hydroxylase) MKTQVAIIGAGPSGLLLGQLLHKAGIDNIIVERQTAEYVLGRIRAGVLEQGTVDLL REAGVAERMDREGLVHEGVELLVGGRRQRLDLKALTGGKTVMVYGQTEVTRDLMQ AREASGAPIIYSAANVQPHELKGEKPYLTFEKDGRVQRIDCDYIAGCDGFHGISRQSIP EGVLKQYERVYPFGWLGLLSDTPPVNHELIYAHHERGFALCSQRSQTRSRYYLQVPL QDRVEEWSDERFWDELKARLPAEVAADLVTGPALEKSIAPLRSLVVEPMQYGHLFLV GDAAHIVPPTGAKGLNLAASDVNYLYRILVKVYHEGRVDLLAQYSPLALRRVWKGER FSWFMTQLLHDFGSHKDAWDQKMQEADREYFLTSPAGLVNIAENYVGLPFEEVA Amino acid sequence of engineered edd (phosphogluconate dehydratase) MHPRILEVTQRLVERSRATRERYLQLIRGAASEGPMRASLQCANFAHGVAGCGS EDKQTLRLMNAANVAIVSAYNDMLSAHQPYLHFPEQIKQALREVGSVGQFAGGVPA MCDGVTQGEPGMELAIASREVIAMSTAVALSHNMFDAALMLGICDKIVPGLMMGAL RFGHLPTIFVPGGPMVSGISNKQKADVRQRYAEGKASREELLESEMNSYHSPGTCTF YGTANTNQLVMEVMGLHLPGASFVNPYTPLRDALTAEAAQQVTRMTKASGSFMPLG EIVDEKALVNSIVALHATGGSTNHTLHIPAIAQAAGIQLTWQDMADLSEVVPTLSHVY PNGKADINHFQAAGGMAFLIRELLDAGLLHEDVNTVAGHGLRRYTQEPFLDNGKLV WREGPQHSLDESILRPVSRPFSAEGGLRVMEGNLGRGVMKVSAVAPEHQVVEAPAR VFQDQQSLADAFKAGELERDFVAVVRFQGPRCNGMPELHKLTPFLGVLQDRGYKVA LVTDGRMSGASGKIPAAIHVCPEAYDGGPLARVRDGDIVRVDGVEGTLRIMVSAEELA SRELPPAPQGNDLGCGRELFGFMRMAFSPAEQGASAFTSALEHLK Amino acid sequence of engineered pedH (phosphogluconate dehydratase) MTRSPRRPLFAVSLVLSAMLLAGAAHAAVSNEEILQDPKNPQQIVTNGLGVQGQ RYSPLDLLNVNNVKELRPVWAFSFGGEKQRGQQAQPLIKDGVMYLTGSYSRVFAVD ARTGKKLWQYDARLPDDIRPCCDVINRGVALYGNLVFFGTLDAKLVALNKDTGKVV WSKKVADHKEGYSISAAPMIVNGKLITGVAGGEFGVVGKIQAYNPENGELLWMRPT VEGHMGYVYKDGKAIENGISGGEAGKTWPGDLWKTGGAAPWLGGYYDPETNLILFG TGNPAPWNSHLRPGDNLYSSSRLALNPDDGTIKWHFQSTPHDGWDFDGVNELISFN YKDGGKEVKAAATADRNGFFYVLDRTNGKFIRGFPFVDKITWATGLDKDGRPIYNDA SRPGAPGSEAKGSSVFVAPAFLGAKNWMPMAYNKDTGLFYVPSNEWGMDIWNEGIA YKKGAAFLGAGFTIKPLNEDYIGVLRAIDPVSGKEVWRHKNYAPLWGGVLTTKGNLV FTGTPEGFLQAFNAKTGDKVWEFQTGSGVLGSPVTWEMDGEQYVSVVSGWGGAVP LWGGEVAKRVKDFNQGGMLWTFKLPKQLQQTASVKP Amino acid sequence of engineered pedE (phosphogluconate dehydratase) MTIRSLPALSPLALSVRVLLMAGSLALGNVATAASTPAAPAGKNVTWEDIANDHL TTQDVLQYGMGTNAQRWSPLAQVNDKNVFKLTPAWSYSFGDEKQRGQESQAIVSDG VVYVTGSYSRVFALDAKTGKRLWTYNHRLPDNIRPCCDVVNRGAAIYGDKIYFGTLD ARVIALDKRTGKVVWNKKFGDHSAGYTMTGAPVLIKDKTSGKVLLIHGSSGDEFGVV GQLFARDPDTGEEVWMRPFVEGHMGRLNGKDSTPTGDVKAPSWPDDPTTETGKVE AWSHGGGAPWQSASFDPETNTIIVGAGNPGPWNTWARTSKDGNPHDFDSLYTSGQV GVDPSTGEVKWFYQHTPNDAWDFSGNNELVLFDYKDKNGNVVKATAHADRNGFFY VVDRNNGKLQNAFPFVDNITWASHIDLKTGRPVENPGQRPAKPLPGETKGKPVEVSP PFLGGKNWNPMAYSQDTGLFYIPGNQWKEEYWTEEVNYKKGSAYLGMGFRIKRMY DDHVGTLRAMDPTTGKLVWEHKEHLPLWAGVLATKGNLVFTGTGDGFFKAFDAKT GKELWKFQTGSGIVSPPITWEQDGEQYIGVTVGYGGAVPLWGGDMAELTKPVAQGG SFWVFKIPSWDNKTAQR Amino acid sequence of engineered hyg5 (3-hydroxybenzoate synthase) MNPSSLVLNGLTSYFENGRARVVPPVGRNILGVVNYASVCEYPTLDHGYPELEIN MVAPTAEPFAEVWVTDAESEHGERDGITYAHDGEYFFCAGRVPPTGRYTEATRAAYV TMFELLEEFGYSSVFRMWNFIGDINRDNAEGMEVYRDFCRGRAEAFEQCRLEFDQF PAATGIGSRGGGIAFYLLACRSGGHVHIENPRQVPAYHYPKRYGPRAPRFARATYLPS RAADGVGGQVFVSGTASVLGHETAHEGDLVKQCRLALENIELVISGGNLAAHGISAG HGLTALRNIKVYVRRSEDVPAVREICREAFSPDADIVYLTVDVCRSDLLVEIEGVVM

Claims

Claims 1. A microorganism with an altered metabolic pathway for the production of an aromatic compound of interest, the microorganism comprising: i) functional deletion of pyruvate kinase (pyk) gene; functional deletion of pyruvate kinase A (pykA) gene; functional deletion of phosphogluconate dehydratase (edd) gene; and functional deletion of phosphoenolpyruvate carboxylase (ppc) gene, and ii) overexpression of at least one gene encoding an enzyme performing a reaction derived from the shikimate pathway that can support growth.

2. The microorganism according to claim 1, further comprising one or more of: functional deletion of dehydroshikimate dehydratase (quiC) gene; functional deletion of PQQ-dependent alcohol dehydrogenase H (pedH) gene; functional deletion of PQQ-dependent alcohol dehydrogenase E (pedE) gene; and functional deletion of phenoxybenzoate dioxygenase subunit alpha (pobA) gene.

3. The microorganism of claim 1 or 2, further comprising functional deletion of tRNA dimethylallyltransferase (miaA) gene.

4. The microorganism according to any one of claims 1-3, wherein the overexpression of at least one gene is selected from: - chorismate pyruvate-lyase (ubiC); - isochorismate synthase (menF) and isochorismate pyruvate lyase (pchB); and - 3-hydroxybenzoate synthase (hyg5); or - a combination thereof.

5. The microorganism according to any one of claims 1-4, further comprising an altered anthranilate synthase (trpE) to reduce activity of trpE, for example by a mutated trpE.

6. The microorganism of any one of claims 1-5 being a bacterium.

7. The microorganism of any one of claims 1-6 being a bacterium selected from the group consisting of Agrobacterium, Alcaligenes, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Cupriavidus, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Serratia, Shigella, Staphylococcus, Streptomyces, Synnecoccus, and Zymomonas.

8. The microorganism of any one of claims 1-7 being E. coli, P. putida, C. butyricum, X. campestris, C. glutamicum, or D. radiorans.

9. The microorganism of any one of claims 1-8 having an Entner-Doudoroff pathway.

10. The microorganism of any one of claims 1-9 being a Gram-negative bacterium.

11. The microorganism of any one of claims 1-10 being P. putida.

12. The microorganism of any one of claims 1-5 being a fungus.

13. The microorganism of claim 12 being selected from the group consisting of Aspergillus, Candida, Chrysosporium, Cryptococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma, Xanthophyllomyces, and Yarrowia.

14. The microorganism of claim 12 or 13 being S. cerevisiae or A. niger.

15. The microorganism of any one of claims 1-14, wherein the aromatic compound of interest is selected from the group consisting of 3-hydroxybenzoate, 4-hydroxybenzoate (4HB), and salicylate.

16. A method for adapting a microorganism to produce an aromatic compound of interest, wherein the method comprises: i) functionally deleting pyruvate kinase (pyk) gene; functionally deleting pyruvate kinase A (pykA) gene; functionally deleting phosphogluconate dehydratase (edd) gene; and functionally deleting phosphoenolpyruvate carboxylase (ppc) gene, and ii) overexpressing at least one gene encoding an enzyme performing a reaction derived from the shikimate pathway that can support growth.

17. The method according to claim 16, wherein the method further comprises one or more of: functionally deleting dehydroshikimate dehydratase (quiC) gene; functionally deleting PQQ-dependent alcohol dehydrogenase H (pedH) gene; functionally deleting PQQ-dependent alcohol dehydrogenase E (pedE) gene; and functionally deleting phenoxybenzoate dioxygenase subunit alpha (pobA) gene.

18. The method of claim 16 or 17, further comprising functionally deleting tRNA dimethylallyltransferase (miaA) gene.

19. The method according to any one of claims 16-18, wherein the overexpressing of at least one gene is selected from: - chorismate pyruvate-lyase (ubiC); - isochorismate synthase (menF) and isochorismate pyruvate lyase (pchB); and - 3-hydroxybenzoate synthase (hyg5); or- a combination thereof.

20. The method according to any one of claims 16-19, further comprising altering anthranilate synthase (trpE) to reduce activity of trpE, for example by mutating trpE.

21. A method for production of an aromatic compound of interest by a microorganism, comprising: i) providing a microorganism according to any one of claims 1-15, or a microorganism as produced by the method of any one of claims 16-20, and ii) incubating the microorganism in a growth medium, comprising a carbon source, such as glycerol, glucose, xylose, and / or cellobiose, and thereby producing an aromatic compound of interest.

22. The method of claim 21, wherein the produced aromatic compound is 3-hydroxybenzoate, 4-hydroxybenzoate (4HB), or salicylate.

23. The method of any one of claims 16-22, wherein the microorganism is a bacterium.

24. The method of claim 23, wherein the microorganism is a bacterium selected from the group consisting of Agrobacterium, Alcaligenes, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Cupriavidus, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Serratia, Shigella, Staphylococcus, Streptomyces, Synnecoccus, and Zymomonas.

25. The method of any one of claims 16-24, wherein the microorganism is E. coli, P. putida, C. butyricum, X. campestris, C. glutamicum, or D. radiorans.

26. The method of any one of claims 16-25, wherein the microorganism has an Entner-Doudoroff pathway.

27. The method of any one of claims 16-26, wherein the microorganism is a Gram-negative bacterium.

28. The method of any one of claims 16-27, wherein the microorganism is a Pseudomonas putida.

29. The method of any one of claims 16-22, wherein the microorganism is a fungus.

30. The method of claim 29, wherein the microorganism is a fungus selected from the group consisting of Aspergillus, Candida, Chrysosporium, Cryptococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma, Xanthophyllomyces, and Yarrowia.

31. The method of claim 29 or 30, wherein the microorganism is S. cerevisiae or A. niger.

32. Use of an aromatic compound produced by the method according to any one of claims 21-31, for the production of bioplastics, such as bio-polyesters and bio-polystyrenes, pharmaceuticals, agrochemicals, cosmetics, chemicals, such as dyes, detergents, or biofuel molecules.

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