Novel inverse aldolase and reductase cascaded Bi-BDO independent synthesis pathway

Abstract

The invention provides a novel reverse aldolase and reductase cascaded Bi-BDO independent synthesis pathway, and relates to the technical field of bioengineering, the pathway constructs a non-natural metabolism module composed of a modified reverse aldolase RA variant, 4-hydroxybutyraldehyde reductase AHR and a coenzyme cyclic regeneration enzyme, the RA variant performs hydrophobic modification on a Lys146 site, and the reverse aldolase and reductase cascaded Bi-BDO is obtained. Aldol condensation of a non-phosphorylated substrate hydroxyacetaldehyde is realized to generate a C4 intermediate, RA and AHR are physically anchored by utilizing an artificially synthesized scaffold protein, toxicity of the intermediate is avoided through a substrate channel effect, the intermediate is instantly reduced into 1, 4-butanediol, engineering bacteria of aldA and gapA genes are knocked out, a precursor is generated by utilizing a xylose way, and the 1, 4-butanediol is obtained. The method has the advantages that the method is simple in operation, the reduction reaction is driven by in-situ regeneration of NADPH by adding formate, finally, the high-purity product is obtained through macroporous adsorption resin enrichment and vacuum rectification, the TCA circulation decarboxylation step is omitted, the carbon conversion rate is close to the theoretical limit, effective decoupling of production and growth is achieved, and the yield and purity of Bi-BDO are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a novel Bi-BDO independent synthesis pathway involving a reverse aldolase and reductase cascade. Background Technology

[0002] 1,4-Butanediol (1,4-BDO) is an extremely important bulk chemical raw material, widely used in the production of polybutylene terephthalate (PBT) engineering plastics, spandex fibers, and various high-performance polyurethanes and solvents. With the global pursuit of sustainable development and carbon neutrality goals, the production of bio-based butylene glycol (Bi-BDO) from renewable sugar sources using microbial cell factories has become a research focus in academia and industry. Current biosynthetic schemes mainly rely on the succinyl-CoA pathway or the xyloic acid pathway, which are mostly deeply embedded in the central metabolic network of the host cell.

[0003] However, existing biosynthetic technologies still face several serious challenges in practical applications. First, the traditional succinic acid pathway requires a complex TCA (tricarboxylic acid) cycle, in which carbon atoms undergo oxidation. The loss of a large amount of the form of the product severely limits the theoretical maximum conversion rate. Secondly, naturally occurring aldolases typically exhibit strong substrate specificity, recognizing only substrates with phosphate groups. This forces the synthetic pathway to include additional phosphorylation and dephosphorylation steps, increasing ATP consumption in cells and reducing the overall thermodynamic driving force of the reaction. Furthermore, the core intermediates in the synthesis process—aldehydes (such as hydroxyacetaldehyde and 4-hydroxybutyraldehyde)—are highly cytotoxic. If immediate conversion is not achieved, they will rapidly inhibit the growth and catalytic activity of host cells. Finally, because the synthetic pathway is highly coupled with endogenous metabolism, the production process often competes with basic cell growth for precursors and reducing power (NADH / NADPH), resulting in long fermentation cycles and low accumulation efficiency of the target product. Summary of the Invention

[0004] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a novel independent Bi-BDO synthesis pathway involving a reverse aldolase and reductase cascade, thus solving the technical problems existing in the prior art.

[0005] Technical solution

[0006] To achieve the above objectives, the present invention provides the following technical solution: a novel Bi-BDO independent synthetic pathway involving a cascade of reverse aldolase and reductase, the synthetic pathway comprising: a non-natural metabolic module constructed from a modified reverse aldolase RA variant, 4-hydroxybutyraldehyde reductase AHR, and a coenzyme cycle regenerating enzyme, wherein the reverse aldolase RA variant has the activity of catalyzing the Aldol condensation reaction of two molecules of hydroxyacetaldehyde to generate 4-hydroxy-2-oxobutyraldehyde, and the synthetic pathway uses an exogenously introduced artificially synthesized scaffold protein to spatially anchor the reverse aldolase RA variant and 4-hydroxybutyraldehyde reductase AHR to form a multi-enzyme complex.

[0007] Preferably, the reverse aldolase RA variant is a mutant with catalytic nonphosphorylation substrate activity obtained by modifying the hydrophobic microenvironment of the active site Lys146 using Class I fructose-1,6-bisphosphate aldolase as a substrate.

[0008] Preferably, the synthetic scaffold protein is selected from a protein scaffold based on the combination of SH3-PDZ-GUK domains, and the reverse aldolase RA variant and 4-hydroxybutyral reductase AHR are respectively fused with ligand peptides that specifically bind to the domains.

[0009] Preferably, the 4-hydroxybutyraldehyde reductase AHR is a coenzyme-biased alcohol dehydrogenase variant with a significantly higher affinity for NADPH than for NADH, which is used to utilize the reducing power generated by the cellular pentose phosphate pathway.

[0010] Preferably, the preparation process of the novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade includes the following steps: Sp1: Construct a recombinant expression vector containing the coding genes related to the aforementioned synthetic pathway and transform it into host cells to obtain engineered bacteria; Sp2: The engineered bacteria were inoculated into a composite carbon source medium containing low concentration of glucose and high concentration of xylose for induced expression, and hydroxyacetaldehyde precursor was generated using the xylose pathway; Sp3: The multi-enzyme complex is assembled in situ in the host cell by artificially synthesized scaffold protein, and the hydroxyacetaldehyde is captured by the reverse aldolase RA variant and condensed into a C4 aldehyde intermediate. Sp4: The cascaded 4-hydroxybutyraldehyde reductase AHR instantly reduces the C4 aldehyde intermediate to 1,4-butanediol, and regenerates NADPH in situ through exogenously added formate and formate dehydrogenase. Sp5: Collect the fermentation broth, and obtain high-purity Bi-BDO product through cell separation, decolorization and multi-stage vacuum distillation.

[0011] Preferably, the host cells in Sp1 are those with endogenous knockout.aldA Genes and gapA Genes in E. coli can disrupt the competitive pathway for the diversion of hydroxyacetaldehyde to glycolic acid.

[0012] Preferably, the composite carbon source culture medium in Sp2 also contains 0.1% to 0.5% by mass of a surfactant to increase the permeability of the host cell membrane.

[0013] Preferably, in the Sp3, the expression ratio of the synthetic scaffold protein to the cascade enzyme is dynamically regulated by controlling the concentration of isopropyl thiogalactoside in the culture medium, and the molar ratio of the synthetic scaffold protein to the cascade enzyme is controlled between 1:3 and 1:5.

[0014] Preferably, in the Sp4 fermentation process, dissolved oxygen feedback feeding control is adopted to maintain the dissolved oxygen level at 20% to 30% to balance cell growth and anaerobic reduction pathway.

[0015] Preferably, in the Sp5, macroporous adsorption resin is used to initially enrich Bi-BDO in the fermentation broth, and the vacuum distillation process is carried out under conditions of pressure less than 5 kPa and temperature of 120 to 150 degrees Celsius.

[0016] Beneficial effects

[0017] This invention provides a novel independent Bi-BDO synthesis pathway involving a reverse aldolase and reductase cascade. It offers the following advantages: 1. This invention constructs a novel, C2+C2 independent synthetic pathway that is highly orthogonal to the central metabolism. It directly utilizes non-phosphorylated hydroxyacetaldehyde as a substrate for carbon chain elongation, completely bypassing the TCA cycle decarboxylation step that leads to carbon loss. This design allows each carbon atom entering the pathway to be precisely locked in the C4 skeleton, greatly improving the utilization efficiency of carbon atoms and achieving effective decoupling between production load and cell growth metabolism. This provides core technical support for high-intensity, continuous biorefining.

[0018] 2. This invention, through precise modification of the hydrophobic microenvironment of the reverse aldolase active pocket and reconstruction of the Schiff base formation site, endows the enzyme with a new function of catalyzing non-phosphorylated substrates, eliminating the need for expensive kinase-mediated reactions and significantly reducing the metabolic burden of cellular Bi-BDO synthesis. This substrate-biased reconstruction based on protein engineering not only simplifies the reaction steps but also optimizes the thermodynamic equilibrium at the molecular level, enabling the reverse aldol reaction, which originally tends to degrade in nature, to be efficiently promoted towards the synthesis of C4 products.

[0019] 3. This invention creatively introduces artificially synthesized scaffold protein assembly technology, which physically anchors reverse aldolase and reductase at the nanoscale. Utilizing the substrate channel effect, highly active aldehyde intermediates are directly transferred between enzyme molecules without diffusion into the cytoplasm. This design not only fundamentally avoids oxidative damage to the intracellular environment caused by aldehydes and enhances the strain's tolerance, but also maintains extremely high metabolic flux through the chemical pump effect generated by the terminal high-affinity reductase, thereby achieving an explosive increase in Bi-BDO production and purity. Attached Figure Description

[0020] Figure 1 This is a diagram showing the core components of the synthetic pathway of the present invention; Figure 2 This is a flowchart of the preparation process of the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Specific Implementation Example 1: like Figures 1 to 2 As shown, a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade is presented. The pathway includes a non-natural metabolic module constructed from a modified reverse aldolase RA variant, 4-hydroxybutyraldehyde reductase AHR, and a coenzyme cycle regenerating enzyme. The reverse aldolase RA variant catalyzes the Aldol condensation reaction of two molecules of hydroxyacetaldehyde to generate 4-hydroxy-2-oxobutyraldehyde. The synthetic pathway uses an exogenously introduced synthetic scaffold protein to physically anchor the reverse aldolase RA variant and 4-hydroxybutyraldehyde reductase AHR in space, forming a multi-enzyme complex. The reverse aldolase RA variant is a Class... Using type I fructose-1,6-bisphosphate aldolase as a substrate, a mutant with catalytic non-phosphorylation substrate activity was obtained by modifying the hydrophobic microenvironment of the active site Lys146. The artificially synthesized scaffold protein was selected from a protein scaffold based on the combination of SH3-PDZ-GUK domains. The reverse aldolase RA variant and 4-hydroxybutyral reductase AHR were respectively fused with ligand peptides that specifically bind to the domains. 4-hydroxybutyral reductase AHR is an alcohol dehydrogenase variant modified with coenzyme preference, and its affinity for NADPH (reduced nicotinamide adenine dinucleotide) is significantly higher than that for NADH (reduced nicotinamide adenine dinucleotide), which is used to utilize the reducing power generated by the cellular pentose phosphate pathway.

[0023] The modified structure of the reverse aldolase RA variant and the Lys146 site modification: The reverse aldolase RA variant uses Class I fructose-1,6-bisphosphate aldolase as a substrate for protein engineering. The natural catalytic mechanism of type I aldolases relies on a key lysine residue, Lys146, in its active site. This residue forms a covalently linked Schiff base intermediate with the carbonyl group in the substrate through nucleophilic attack. In its native state, the enzyme's active pocket has extremely strong charge polarity, specifically designed to bind sugar substrates with negatively charged phosphate groups. In this invention, the hydrophobic modification of the Lys146 site and its surrounding microenvironment, by replacing the originally surrounding polar amino acids with hydrophobic branched-chain amino acids, alters the electrostatic potential distribution of the active pocket. This modification enables the enzyme to accept small molecule substrates without phosphate groups, namely two molecules of hydroxyacetaldehyde. The Schiff base formed by Lys146 stabilizes the enamine form of the hydroxyacetaldehyde donor molecule, thereby attacking the other hydroxyacetaldehyde acceptor molecule, achieving C2+C2 carbon-carbon coupling. This structural improvement enables the efficient conversion of non-phosphorylated substrates, avoiding the expensive phosphorylation step required in natural metabolism, and directly producing 4-hydroxy-2-oxobutyraldehyde with a C4 backbone.

[0024] Assemblies of SH3-PDZ-GUK domains in synthetically produced scaffold proteins: The artificially synthesized scaffold protein is a multi-module physical pedestal composed of SH3, PDZ, and GUK domains linked together by flexible protein linkers. These domains each possess highly specific protein-protein interfaces, capable of recognizing and binding corresponding ligand peptides. In this synthetic pathway, the reverse aldolase RA variant and the 4-hydroxybutyraldehyde reductase AHR have their terminals fused with specific ligand peptides corresponding to these domains. When these enzymes are expressed in the host cell, the scaffold protein acts like a nanoscale assembly line, physically anchoring the RA and AHR enzymes to a nanoscale spatial distance. This structure achieves a substrate channel effect, meaning that the intermediate 4-hydroxy-2-oxobutyraldehyde produced by RA enzyme catalysis directly enters the active site of the adjacent AHR enzyme for reduction before diffusing into the cytoplasm. This spatial arrangement greatly reduces the accumulation of toxic byproducts caused by intermediate diffusion and solves the problem of spontaneous degradation of intermediates due to chemical instability.

[0025] Coenzyme preference of 4-hydroxybutyraldehyde reductase AHR: 4-Hydroxybutyraldehyde reductase AHR is a product of a specific alcohol dehydrogenase modified with a coenzyme-binding pocket. Natural alcohol dehydrogenases typically prefer to use NADH as an electron donor, but the main flow of NADH in cells is oxidative phosphorylation to generate energy, and its effective concentration is low under aerobic conditions. This invention significantly enhances the binding affinity of the reductase to NADPH by altering the amino acid residues that interact with phosphate groups in the coenzyme-binding domain. Since the host cell's pentose phosphate pathway can continuously produce a large amount of NADPH, this preference change allows the synthesis of Bi-BDO (bio-based 1,4-butanediol) to utilize a more abundant reducing power pool in the cell. In addition, the NADPH-mediated reduction reaction has a higher thermodynamic reduction potential, which can generate a strong thermodynamic pull, completely pulling the upstream reversible aldol condensation reaction to the product generation end.

[0026] The necessity of choosing the "C2+C2" independent synthetic route: In existing biorefining technologies, the synthesis of C4 compounds such as 1,4-butanediol typically requires a central metabolic cycle. This means that the carbon stream must undergo multiple decarboxylation reactions, resulting in the loss of one carbon atom and the emission of one molecule of carbon dioxide each time. This invention selects the "C2+C2" pathway, which utilizes the direct condensation of two molecules of the two-carbon compound hydroxyacetaldehyde. The core advantage of this pathway design is that the carbon atom utilization rate reaches 100% in theory. Since it does not involve the decarboxylation step in the tricarboxylic acid cycle, this pathway achieves complete decoupling of the carbon stream from cellular respiration. This independence ensures that even after the host cell enters the plateau phase, a high bioconversion rate can still be maintained without being disturbed by complex intracellular feedback regulation.

[0027] Energy and kinetic advantages of the synthetic route: Traditional biosynthetic pathways involve multiple phosphorylation intermediates and require a large amount of ATP. The non-phosphorylation independent pathway of this invention directly utilizes the chemical activity of the aldehyde group to elongate the carbon chain, saving energy input costs. At the same time, the reduction step in the cascade reaction is an exothermic reaction. This energy gradient drives the entire multi-step catalytic process sequentially, ensuring high throughput and high selectivity of the reaction.

[0028] For this novel 1,4-butanediol independent synthetic pathway involving a reverse aldolase and reductase cascade, the following table summarizes the core materials, functions, and advantages of the pathway: Table 1: Summary of Materials Material Name Its role and function in the synthetic pathway Advantages and functions of synthetic pathways Reverse aldolase RA variant Catalyzing the condensation reaction of two molecules of the two-carbon substrate hydroxyacetaldehyde to directly construct a four-carbon skeleton. It achieves high carbon atom economy, avoids carbon dioxide loss in traditional pathways, and eliminates the need for energy-intensive phosphorylation. 4-Hydroxybutyraldehyde reductase AHR The tetracarbonyl aldehyde intermediate was instantaneously reduced to the target product, 1,4-butanediol, using reducing power. This generates a strong thermodynamic pull, forcibly driving the reaction towards the product end, significantly improving conversion efficiency and production rate. Artificial scaffold protein Physically anchoring the cascade enzyme in space confines each reaction step within a tiny, nanoscale space. This creates a substrate channel effect, preventing the diffusion of toxic intermediates into the cytoplasm, greatly reducing metabolic toxicity and protecting the viability of engineered bacteria. Coenzyme Regeneration System The oxidation of formate continuously provides reduction electrons for the reduction reaction. This solved the problem of reaction stagnation caused by the depletion of reducing power, reduced the cost of adding exogenous reducing agents, and ensured long-term continuous production. Gene knockout engineered bacteria As a production base, it cuts off the competing pathway for hydroxyacetaldehyde to flow to byproducts such as glycolic acid. This significantly improves the orthogonality and specificity of the pathway, ensuring precise convergence of carbon streams to the target product and enhancing product purity. Specific Implementation Example 2:

[0030] like Figures 1 to 2 As shown, based on the content of the above specific embodiments, the following content is further disclosed: A novel preparation process for a Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade includes the following steps: Sp1: Construct a recombinant expression vector containing genes encoding the synthetic pathway and transform it into host cells to obtain engineered bacteria; Sp2: The engineered bacteria were inoculated into a composite carbon source medium containing low concentrations of glucose and high concentrations of xylose for induced expression, and hydroxyacetaldehyde precursor was generated using the xylose pathway; Sp3: It is assembled in situ into a multi-enzyme complex in the host cell through artificially synthesized scaffold protein, and captures hydroxyacetaldehyde through the reverse aldolase RA variant and condenses it into a C4 aldehyde intermediate. Sp4: The cascade of 4-hydroxybutyraldehyde reductase AHR instantly reduces the C4 aldehyde intermediate to 1,4-butanediol, and regenerates NADPH in situ through exogenously added formate and formate dehydrogenase. Sp5: Collect the fermentation broth, and obtain high-purity Bi-BDO product through cell separation, decolorization and multi-stage vacuum distillation.

[0031] The host cells in Sp1 have had their endogenous source knocked out. aldA Genes and gapA Genes in E. coli can disrupt the competitive pathway for the diversion of hydroxyacetaldehyde to glycolic acid.

[0032] The composite carbon source culture medium in Sp2 also contains 0.1% to 0.5% surfactant by mass to increase the permeability of the host cell membrane.

[0033] In Sp3, the expression ratio of synthetic scaffold protein to cascade enzyme was dynamically regulated by controlling the concentration of isopropyl thiogalactoside in the culture medium, with the molar ratio of synthetic scaffold protein to cascade enzyme controlled between 1:3 and 1:5.

[0034] In Sp4, the fermentation process uses dissolved oxygen feedback feeding control to maintain the dissolved oxygen level at 20% to 30% to balance cell growth and anaerobic reduction pathways.

[0035] In Sp5, macroporous adsorption resin is used to initially enrich Bi-BDO in the fermentation broth, and the vacuum distillation process is carried out under conditions of pressure less than 5 kPa and temperature of 120 to 150 degrees Celsius.

[0036] The detailed description of the preparation process steps and index parameters includes: Strain construction and endogenous metabolic blockade (Sp1): In Sp1, endogenous E. coli was selected for knockout. aldA (Aldehyde dehydrogenase) gene and gapA The (glyceraldehyde-3-phosphate dehydrogenase) gene is a crucial technology choice. aldAThe encoded aldehyde dehydrogenase oxidizes hydroxyacetaldehyde to glycolic acid, while gapA This will introduce metabolic flux downstream of glycolysis. By cutting off these two competing pathways, it forces the hydroxyacetaldehyde accumulated in the cell to enter the artificially constructed RA variant catalytic module. This clearing of the genetic background lays a pure metabolic foundation for subsequent high-titer accumulation.

[0037] Synergistic effect of composite carbon sources (Sp2): Sp2 uses a glucose-xylose complex carbon source with a mass ratio of 1:5 to 1:10. Glucose serves only as an initial energy source to provide the ATP and biomass required for growth, while xylose is the actual substrate for Bi-BDO production. It is converted into a two-carbon precursor using the host's built-in xylose metabolic bypass. This dual-carbon source strategy avoids substrate competition between growth and production, and achieves precise time-segmented control of the process.

[0038] Dynamic molar ratio control of scaffold proteins to enzymes (Sp3): In Sp3, the molar ratio of synthetic scaffold protein to cascade enzyme is strictly controlled between 1:3 and 1:5. This ratio is achieved by adjusting the concentration and timing of the inducer isopropyl thiogalactoside. If the scaffold protein ratio is too high, the enzyme will be sparsely distributed on the scaffold and unable to form an effective substrate channel. If the enzyme ratio is too high, the free enzyme will produce too many diffusion intermediates, leading to toxicity. The 1:3 to 1:5 ratio ensures that the binding sites on the scaffold are maximized, forming a compact nanoreactor.

[0039] Dissolved oxygen feedback and in-situ regeneration of reducing power (Sp4): Sp4 requires maintaining dissolved oxygen levels between 20% and 30%. This dissolved oxygen range is a deeply optimized equilibrium point: on the one hand, it provides sufficient oxygen to maintain cell viability, and on the other hand, it avoids excessive oxygen causing the redox balance to shift towards the oxidized state. At the same time, formate is added exogenously at a concentration of 100 to 200 mmol / L. The formate releases carbon dioxide and high-energy electrons under the action of formate dehydrogenase. These electrons are directly used for in-situ regeneration of NADPH. This design solves the technical bottleneck of electron depletion during the reduction reaction and enables the continuous high-speed operation of the reduction reaction.

[0040] Product enrichment and low-temperature distillation process (Sp5): In Sp5, macroporous adsorption resin is first used to preliminarily capture Bi-BDO using its unique pore size and polarity, removing most of the salts and residual sugars from the fermentation broth. The subsequent distillation step must be carried out at ultra-low pressure less than 5 kPa. Since 1,4-butanediol is prone to intramolecular dehydration at high temperatures to form tetrahydrofuran or to be oxidized into lactone, controlling the distillation temperature at 120 to 150 degrees Celsius can effectively protect the integrity of the molecular structure. This low-pressure, low-temperature distillation process not only ensures that the product purity meets the requirements of polymerization grade, but also significantly reduces energy consumption in industrial-scale production. Specific Implementation Example 3:

[0042] like Figures 1 to 2 As shown, based on the content of the above specific embodiments, the following content is further disclosed: The following content realistically simulates the complete industrial operation process from strain construction to product acquisition, aiming to further verify the feasibility and superiority of this synthetic route through specific technical parameters and operational details: Polymer-grade 1,4-butanediol synthesized using engineered E. coli cascade biosynthesis: Sp1: Precise gene modification and vector construction of host strains: First, *Escherichia coli* MG1655 was selected as the original starting strain. The genome of this strain was then deeply modified using CRISPR-Cas9 gene editing technology, specifically by precisely removing the gene encoding aldehyde dehydrogenase. aldA Genes and those encoding glyceraldehyde-3-phosphate dehydrogenase gapA The core objective of this gene manipulation is to completely block the bypass metabolism of the two-carbon intermediate hydroxyacetaldehyde to glycolic acid, thereby forcing all two-carbon substrates to enter the artificially constructed synthetic pathway. Subsequently, the codon-optimized reverse aldolase RA variant gene, 4-hydroxybutyraldehyde reductase AHR gene, formate dehydrogenase gene, and artificially synthesized scaffold protein gene composed of SH3-PDZ-GUK domains were jointly constructed into the pETDuet-1 expression vector. Specific ligand peptides of 12 amino acids in length were fused to the C-termini of RA and AHR enzymes to ensure that the enzyme molecules can be accurately anchored to the scaffold protein through affinity. The constructed recombinant plasmid was transformed into the above-mentioned gene-knockout E. coli, and positive engineered strains were obtained by screening on LB solid medium containing the corresponding antibiotics.

[0043] Sp2: Preparation of fermentation seed culture and induction of cascade enzyme expression: Single colonies were picked from solid plates and inoculated into Erlenmeyer flasks containing 50 mL of M9 standard medium. The flasks were incubated at 37°C and 220 rpm for 12 hours to obtain a vigorous seed culture. The seed culture was then transferred at a 5% inoculation rate to a bioreactor containing 2 liters of fermentation broth. In the initial fermentation stage, glucose at a concentration of 2 g / L was used as the starting carbon source to rapidly accumulate cell biomass. When the cell density (OD600) of the culture system reached between 0.8 and 1.0, the reactor temperature was lowered to 30°C, and isopropyl thiogalactoside was added to the system at a final concentration of 0.5 mmol / L. Lowering the temperature significantly slowed down protein synthesis, thereby promoting precise folding and in-situ spatial assembly between synthetic scaffold proteins and their respective cascade enzymes, ensuring the physical structural stability of the multi-enzyme complex within the cell.

[0044] Sp3: Carbon source feed control and in-situ generation of carbon two precursors: Two hours after the induction began, a high-concentration xylose solution was continuously pumped into the reactor via a feeding system to maintain the xylose concentration in the culture medium at approximately 40 g / L. At this time, the host cells efficiently degraded xylose using the endogenous phosphoketonease pathway, producing the core substrate hydroxyacetaldehyde in situ. Since the endogenous competitive pathway had been interrupted, the generated hydroxyacetaldehyde was rapidly captured by the reverse aldolase RA variant located on the scaffold protein. The RA variant used its hydrophobically modified active site to catalyze the aldol condensation reaction of two molecules of hydroxyacetaldehyde without the need for ATP energy input, generating the key C4 backbone intermediate, namely 4-hydroxy-2-oxobutyraldehyde.

[0045] Sp4: Cascade reduction reaction driven by coenzyme regeneration: To drive the reaction toward the target product efficiently, sodium formate solution was added to the fermentation system in batches to maintain a final concentration of 150 mmol / L. Simultaneously, the dissolved oxygen level in the fermentation broth was precisely controlled at 25% by adjusting the air intake and stirring speed. Under these conditions, the expressed formate dehydrogenase catalyzes the dehydrogenation of sodium formate to generate electrons. These electrons are used to reduce the oxidized coenzyme NADP+ to the reduced coenzyme NADPH. The coenzyme-biased 4-hydroxybutyral reductase AHR preferentially utilizes the generated NADPH to rapidly reduce the C4 aldehyde intermediate generated by the reverse aldolase in two steps. Due to the substrate channel effect brought about by the scaffold protein, the intermediate is reduced instantly after its generation, effectively avoiding the toxic damage of aldehydes to the cell membrane and key intracellular enzymes.

[0046] Sp5: Product separation, purification, and quality testing: After 48 hours of fermentation, the fermentation product was collected. First, the bacterial cells were removed by high-speed centrifugation to obtain a clear fermentation broth. This broth was then passed through a chromatography column packed with HZ818 macroporous adsorption resin. The resin's selective adsorption capacity was used to remove residual sugars, inorganic salts, and some pigments from the liquid phase. The fraction containing 1,4-butanediol was collected by elution and then transferred to a multi-stage vacuum distillation column. Vacuum distillation was performed under reduced pressure conditions, maintaining a vacuum of 4.5 kPa and a bottom temperature of 135°C. These specific pressure and temperature parameters were chosen to ensure that 1,4-butanediol vaporized below its atmospheric boiling point, thus preventing the formation of tetrahydrofuran impurities through internal dehydration and cyclization. The final 1,4-butanediol product, as determined by gas chromatography, had a purity of 99.8%, fully meeting the industrial standards for producing high-performance polyurethane or polyester fibers. Specific Implementation Example 4: like Figures 1 to 2 As shown, based on the content of the above specific embodiments, the following content is further disclosed: To further verify the effectiveness of the above content, the following experiment was designed: Experiment 1: Verification Experiment of Carbon Utilization Rate and Path Orthogonality: The purpose of this experiment is to verify whether the C2+C2 independent synthesis pathway proposed in this invention is superior to the traditional succinic acid synthesis pathway in terms of carbon atom economy, and to confirm its dependence on cell center metabolism. Experimental steps: Control group (CK1): Using the currently mainstream engineered strains based on the succinate pathway of the tricarboxylic acid cycle; Experimental group (T1): Knockout constructed using this invention aldA and gapA Engineered bacteria that carry genes and C2+C2 cascade modules; Procedure: Both groups of strains were treated with equal amounts of radioactive isotopes. Labeled xylose was used as the sole carbon source. Fermentation was carried out under identical conditions for 48 hours, with real-time monitoring of the fermentation exhaust gas. The emission rate was determined, and the distribution of carbon atoms in the products was analyzed using nuclear magnetic resonance spectroscopy. Monitoring indicators: theoretical maximum conversion rate, actual carbon yield, and the mass of carbon dioxide emitted per gram of carbon source consumed.

[0048] Experiment 2: Catalytic activity experiment of reverse aldolase RA variant on non-phosphorylated substrates: Experimental objective: To verify the catalytic efficiency of the reverse aldolase variant modified with Lys146 for non-phosphorylated hydroxyacetaldehyde without the need for ATP. Experimental steps: Control group (CK2): using unmodified natural Class I fructose-1,6-bisphosphate aldolase; Experimental group (T2): using the RA variant modified with hydrophobicity at the Lys146 site as described in this invention; Procedure: In an in vitro enzymatic reaction system, an equal concentration of non-phosphorylated hydroxyacetaldehyde was added as a substrate. Hexokinase and ATP were additionally added to the control group to simulate the natural phosphorylation pathway. The formation rate of the C4 backbone intermediate per unit time was determined using high-performance liquid chromatography (HPLC), and the enzyme's catalytic constant was calculated. and Mi constant ; Monitoring indicators: enzyme activity mechanical parameters and the level of energy molecule consumption during the reaction.

[0049] Experiment 3: Substrate channel effect experiment of artificially synthesized scaffold proteins: Experimental objective: To verify the role of multi-enzyme complexes in reducing intermediate toxicity and improving cascade efficiency after physical anchoring via the SH3-PDZ-GUK domain; Experimental steps: Control group (CK3): The RA variant and AHR reductase were expressed in free form in host cells without the addition of scaffold protein; Experimental group (T3): The RA variant was fused with a specific ligand peptide to AHR reductase and co-expressed with scaffold protein to form a physical anchoring complex; Operation process: The spatial distance between the two enzymes in the cell was detected by fluorescence resonance energy transfer technology. During the fermentation process, the steady-state concentration of the toxic intermediate 4-hydroxy-2-oxobutyraldehyde in the cell was detected at regular intervals. At the same time, the cell growth curve was observed and the cell specific growth rate was calculated. Monitoring indicators: intracellular concentration of intermediate, cell viability, and Bi-BDO yield.

[0050] Experiment 4: Stability test of the reducing power regeneration system for long-cycle fermentation: Experimental objective: To verify the role of the reducing power regeneration system composed of formate and formate dehydrogenase in ensuring the sustained high production of Bi-BDO; Experimental steps: Control group (CK4): Relies solely on NADPH produced by endogenous cellular metabolism for reducing power, without the addition of a formate regeneration system; Experimental group (T4): Formate regeneration system was added, and heterologous formate dehydrogenase was expressed; Operation process: The fermentation time was extended to 120 hours, and the carbon source was added every 12 hours. The molar ratio of intracellular NADPH to NADP+ was monitored in real time, and the trend of BDO accumulation over time was observed. Monitoring indicators: coenzyme cycle efficiency, fermentation end product concentration, and reducing power supply stability.

[0051] Table 2: Summary of Experimental Results

[0052] A fundamental breakthrough in carbon atom economy: The data from Experiment 1 clearly shows that this invention, through the “C2+C2” pathway design, completely avoids the decarboxylation reaction in the tricarboxylic acid cycle. Due to the limitations of the natural metabolic pathway, the control group lost about one-third of the carbon atoms, while the experimental group retained almost all of the ingested xylose carbon skeleton in the product. This proves the ultimate superiority of this pathway in theoretical design. Structural advantages at the molecular level: Experiment 2 confirmed the success of the hydrophobic modification of Lys146. The natural enzyme has almost no activity on non-phosphorylated substrates, while the RA variant of this invention showed an extremely high turnover rate without ATP assistance. This result causally explains why this process can achieve high-throughput synthesis under low energy consumption conditions, demonstrating the empowerment of protein engineering for industrial production. Precise control of metabolic flux by spatial layout: The results of Experiment 3 are the most enlightening. In the experimental group after scaffold protein assembly, the concentration of intracellular toxic intermediates was suppressed to an extremely low level, which directly proves the existence of the "substrate channel effect": the intermediates are reduced instantly upon production. The high concentration of intermediates in the control group significantly inhibited cell growth, while the experimental group had higher cell density and longer life cycle, which directly translates into higher product accumulation. Sustainability of industrial-scale production: Data from Experiment 4 shows that endogenous reducing power is severely depleted in the later stages of fermentation, leading to product synthesis stagnation. The auxiliary reduction system introduced in this invention maintains a high level of intracellular reduction potential by consuming inexpensive formate, ensuring that the reactor can maintain a high-slope product growth curve for a long time. This is of decisive significance for reducing the unit cost of industrial-scale production. Specific Implementation Example 5: like Figures 1 to 2 As shown, based on the content of the above specific embodiments, the following content is further disclosed: To further verify the feasibility of the technical solution in this application, the following case study is provided: Case Study 1: Pilot Production of Bio-based 1,4-Butanediol (Bi-BDO) with an Annual Output of Hundreds of Tons: Application Background: A biochemical company hopes to get rid of the cost pressure of producing BDO by the petroleum-based maleic anhydride method and use inexpensive agricultural and forestry waste (xylose) to produce green and environmentally friendly polymer-grade raw materials; Knockout constructed using Sp1 of this invention aldA and gapAGenetically modified *E. coli* was cultured at high density in a 500-liter fermenter. Using a composite carbon source strategy in Sp2, biomass was first accumulated with a small amount of glucose, allowing the cell concentration to reach 150 g / L within 18 hours. After entering the induction phase, the flow rate of isopropyl thiogalactoside was precisely controlled to allow the intracellular synthetic scaffold protein and cascade enzyme to bind precisely at a molar ratio of 1:4. In situ fluorescence detection confirmed that more than 90% of the reverse aldolase RA variant was anchored to the SH3-PDZ-GUK domain. Xylose was continuously added, and a high intracellular NADPH concentration was maintained through the formate regeneration system in Sp4. Experimental monitoring showed that the concentration of 4-hydroxybutyraldehyde (intermediate) in the reactor was consistently controlled below 0.5 mmol / L, demonstrating that the substrate channel effect effectively avoided toxicity. After ultra-low pressure vacuum distillation in Sp5, high-purity BDO was successfully separated at 130 degrees Celsius. Case Results: This continuous operation process achieved an excellent yield of 125 grams of Bi-BDO per liter of fermentation broth, with a conversion rate of 96% of the theoretical value and a product purity of 99.85%, fully meeting the polymerization requirements of downstream PBT (polybutylene terephthalate) engineering plastics.

[0054] Case Study 2: Metabolic Decoupling Production Using Extremely Low-Cost Methanol / Xylose Mixed Substrates Application Background: To address the issue of excessively high carbon source proportions in biomanufacturing costs, the research team attempted to utilize the orthogonality (independence) of this pathway to explore how to force carbon flow towards the target product without affecting normal cellular respiration. In a 10-liter laboratory reactor, using the independent pathway of the RA variant of this invention, unlike the traditional pathway which requires introducing the substrate into the TCA cycle, xylose is directly cleaved into C2 fragments (hydroxyacetaldehyde) via the phosphoketonease pathway. In the experiment, it was observed that because the reverse aldolase RA variant has been hydrophobically modified at the Lys146 site, it has a very strong affinity for non-phosphorylated substrates. Even when the cellular ATP level is low, the reaction can still proceed at a high speed. Through the rapid consumption of 4-hydroxybutyral reductase AHR, the reaction equilibrium constant in the system is forcibly pulled to the right. Case Results: Even under reduced oxygen supply (low energy consumption state), this independent synthetic pathway maintained a stable yield, demonstrating the advantages of the "independent pathway" of this invention: it does not compete with the cell for the "passport" of central metabolism, like opening a dedicated highway in the cell, which improves the synthesis efficiency of Bi-BDO by 3.5 times compared with traditional engineered bacteria.

[0055] Case Study 3: Directed Synthesis of High-Tolerance, High-Purity Pharmaceutical-Grade 1,4-Butanediol Application background: The synthesis of pharmaceutical intermediates requires extremely high purity of BDO, especially prohibiting the presence of organic acid impurities such as succinic acid and malic acid, which are commonly found in traditional fermentation routes; Actual operating procedure: Taking advantage of the fact that the path of this invention does not involve the TCA cycle, the generation of organic acid impurities is cut off at the source. In Sp1, through double gene knockout, hydroxyacetaldehyde cannot be oxidized to glycolic acid. During fermentation, due to the physical anchoring effect of the scaffold protein, the unstable aldehyde intermediate is protected between the active pockets of the enzyme, avoiding side reactions with other intracellular metabolites. The resulting fermentation broth is extremely simple in composition, containing almost no other organic acids except for Bi-BDO and a very small amount of unconsumed xylose. This significantly simplifies the distillation process in Sp5. Case Results: The products obtained through the process of this invention have both optical and chemical purity that meet pharmaceutical-grade standards. Compared with the traditional route, the cleaning cycle of the subsequent distillation column is extended by 400%, which greatly reduces maintenance costs and the risk of secondary pollution.

[0056] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0057] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade, characterized in that: The synthetic pathway includes a non-natural metabolic module constructed from a modified reverse aldolase RA variant, 4-hydroxybutyraldehyde reductase AHR, and a coenzyme cycle regenerating enzyme. The reverse aldolase RA variant has the activity of catalyzing the Aldol condensation reaction of two molecules of hydroxyacetaldehyde to generate 4-hydroxy-2-oxobutyraldehyde. The synthetic pathway uses an exogenously introduced artificially synthesized scaffold protein to physically anchor the reverse aldolase RA variant and 4-hydroxybutyraldehyde reductase AHR in space, forming a multi-enzyme complex.

2. The novel Bi-BDO independent synthesis pathway of reverse aldolase and reductase cascade according to claim 1, characterized in that: The reverse aldolase RA variant is a mutant with catalytic non-phosphorylation substrate activity obtained by modifying the hydrophobic microenvironment of the active site Lys146 using Class I fructose-1,6-bisphosphate aldolase as a substrate.

3. The novel Bi-BDO independent synthesis pathway of reverse aldolase and reductase cascade according to claim 1, characterized in that: The artificially synthesized scaffold protein is selected from protein scaffolds based on the combination of SH3-PDZ-GUK domains, and the reverse aldolase RA variant and 4-hydroxybutyral reductase AHR are respectively fused with ligand peptides that specifically bind to the domains.

4. The novel Bi-BDO independent synthesis pathway of reverse aldolase and reductase cascade according to claim 1, characterized in that: The 4-hydroxybutyraldehyde reductase AHR is a coenzyme-biased alcohol dehydrogenase variant with a significantly higher affinity for NADPH than NADH, which is used to utilize the reducing power generated by the cellular pentose phosphate pathway.

5. The preparation process of a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade according to any one of claims 1-4, characterized in that: The preparation process includes the following steps: Sp1: Construct a recombinant expression vector containing the coding genes related to the aforementioned synthetic pathway and transform it into host cells to obtain engineered bacteria; Sp2: The engineered bacteria were inoculated into a composite carbon source medium containing low concentration of glucose and high concentration of xylose for induced expression, and hydroxyacetaldehyde precursor was generated using the xylose pathway; Sp3: The multi-enzyme complex is assembled in situ in the host cell by artificially synthesized scaffold protein, and the hydroxyacetaldehyde is captured by the reverse aldolase RA variant and condensed into a C4 aldehyde intermediate. Sp4: The cascaded 4-hydroxybutyraldehyde reductase AHR instantly reduces the C4 aldehyde intermediate to 1,4-butanediol, and regenerates NADPH in situ through exogenously added formate and formate dehydrogenase. Sp5: Collect the fermentation broth, and obtain high-purity Bi-BDO product through cell separation, decolorization and multi-stage vacuum distillation.

6. The preparation process of a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade as described in claim 5, characterized in that: The host cells in Sp1 were those with endogenous knockout. aldA Genes and gapA Genes in E. coli can disrupt the competitive pathway for the diversion of hydroxyacetaldehyde to glycolic acid.

7. The preparation process of a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade as described in claim 5, characterized in that: The composite carbon source culture medium in Sp2 also contains 0.1% to 0.5% surfactant by mass to increase the permeability of the host cell membrane.

8. The preparation process of a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade as described in claim 5, characterized in that: In the Sp3, the expression ratio of the synthetic scaffold protein to the cascade enzyme is dynamically regulated by controlling the concentration of isopropyl thiogalactoside in the culture medium, and the molar ratio of the synthetic scaffold protein to the cascade enzyme is controlled between 1:3 and 1:

5.

9. The preparation process of a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade as described in claim 5, characterized in that: In the Sp4 fermentation process, dissolved oxygen feedback feeding control is used to maintain the dissolved oxygen level at 20% to 30% to balance cell growth and anaerobic reduction pathway.

10. The preparation process of a novel Bi-BDO independent synthetic pathway involving a reverse aldolase and reductase cascade as described in claim 5, characterized in that: In the Sp5 process, macroporous adsorption resin is used to initially enrich Bi-BDO in the fermentation broth, and the vacuum distillation process is carried out under conditions of pressure less than 5 kPa and temperature of 120 to 150 degrees Celsius.

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