High-value Derivatives Research Articles

Metabolic engineering of Escherichia coli for de novo synthesis of 1,4-butanediol

1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the de novo synthesis of 1,4-butanediol was achieved after introduction of the path into Escherichia coli W3110 (K-12) chassis cells. To further improve the synthesis efficiency of this path, we deleted the genes encoding lactate dehydrogenase A (LdhA) and pyruvate formate lyase B (PflB) to block the metabolic bypass. Furthermore, the expression of citrate synthase (GltAR163L) was up-regulated to increase the α-ketoglutarate metabolic flux. In addition, we improved the synthesis of the key cofactor NADPH and up-regulated the expression of sucA, car, and yqhD by substituting with strong promoters to increase the efficiency of supplying precursors to 1,4-butanediol synthesis. Eventually, the recombinant strain produced up to 770 mg/L of 1,4-butanediol within 48 h in a shake flask, and 4.22 g/L of 1,4-butanediol within 60 h in a 5 L fermenter with a yield of 12.46 mg/g glucose. Compared with the previously reported method, the novel path designed in this study for the de novo synthesis of 1,4-butanediol does not need acetyl coenzyme A and avoids the byproduct acetate or the addition of ammonia. Therefore, the outcome is expected to provide a new idea for the metabolic engineering of microbial chassis for the production of 1,4-butanediol and its high-value derivatives.

Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue

Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (Kcat and Km) for epimerization activity were 219.9 ± 5.6 s−1 and 261.9 ± 18.1 mM, respectively, representing the highest Kcat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.

Mild Strategy for the Preparation of Alkyl Sulfonyl Fluorides from Alkyl Bromides and Alcohols Using Photoredox Catalysis and Flow Chemistry.

Facile access to sp3-rich scaffolds containing a sulfonyl fluoride group is still limited. Herein, we describe a mild and scalable strategy for the preparation of alkyl sulfonyl fluorides from readily available alkyl bromides and alcohols using photoredox catalysis. This approach is based on halogen atom transfer (XAT), followed by SO2 capture and fluorination. The method features mild conditions enabling fast access to high-value derivatives and has been scaled up to 5 g using a continuous stirred tank reactor cascade.

Decrypting the programming of β-methylation in virginiamycin M biosynthesis

During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic β-ketones into β-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for β-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second β-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin β-branching programming. Furthermore, variations in this control – whether natural or by design – open up avenues for diversifying polyketide structures towards high-value derivatives.

Metabolic engineering of Escherichia coli for efficient production of L-5-hydroxytryptophan from glucose

Background5-hydroxytryptophan (5-HTP), the direct biosynthetic precursor of the neurotransmitter 5-hydroxytryptamine, has been shown to have unique efficacy in the treatment of a variety of disorders, including depression, insomnia, and chronic headaches, and is one of the most commercially valuable amino acid derivatives. However, microbial fermentation for 5-HTP production continues to face many challenges, including low titer/yield and the presence of the intermediate L-tryptophan (L-Trp), owing to the complexity and low activity of heterologous expression in prokaryotes. Therefore, there is a need to construct an efficient microbial cell factory for 5-HTP production.ResultsWe describe the systematic modular engineering of wild-type Escherichia coli for the efficient fermentation of 5-HTP from glucose. First, a xylose-induced T7 RNA polymerase-PT7 promoter system was constructed to ensure the efficient expression of each key heterologous pathway in E. coli. Next, a new tryptophan hydroxylase mutant was used to construct an efficient tryptophan hydroxylation module, and the cofactor tetrahydrobiopterin synthesis and regeneration pathway was expressed in combination. The L-Trp synthesis module was constructed by modifying the key metabolic nodes of tryptophan biosynthesis, and the heterologous synthesis of 5-HTP was achieved. Finally, the NAD(P)H regeneration module was constructed by the moderate expression of the heterologous GDHesi pathway, which successfully reduced the surplus of the intermediate L-Trp. The final engineered strain HTP11 was able to produce 8.58 g/L 5-HTP in a 5-L bioreactor with a yield of 0.095 g/g glucose and a maximum real-time productivity of 0.48 g/L/h, the highest values reported by microbial fermentation.ConclusionIn this study, we demonstrate the successful design of a cell factory for high-level 5-HTP production, combined with simple processes that have potential for use in industrial applications in the future. Thus, this study provides a reference for the production of high-value amino acid derivatives using a systematic modular engineering strategy and a basis for an efficient engineered strain development of 5-HTP high-value derivatives.

Rendered-Protein Hydrolysates as a Low-Cost Nitrogen Source for the Fungal Biotransformation of 5-Hydroxymethylfurfural

5-hydroxymethylfurfural (HMF) is a platform chemical that can be converted into a wide range of high-value derivatives. Industrially, HMF-based derivatives are synthesized via chemical catalysis. However, biocatalytic transformation has emerged as an attractive alternative. Significant advances have been made in the last years using isolated enzymes and whole-cell biocatalysts in HMF biotransformation. Nonetheless, one of the major bottlenecks is the cost of the process, mainly due to the microorganism growth substrate. In this work, biotransformation studies to transform HMF into 2,5-di(hydroxymethyl)furan (DHMF) were carried out with the fungus Fusarium striatum using low-cost protein hydrolysates. The protein hydrolysates were obtained from fines, an unexploited material produced during the rendering process of meat industry waste residues. Given the high content in the protein of fines, of around 46%, protein hydrolysis was optimized using two commercially available proteases, Alcalase 2.4 L and Neutrase 0.8 L. The maximum degree of hydrolysis (DH) achieved with Alcalase 2.4 L was 21.4% under optimal conditions of 5% E/S ratio, pH 8, 55 °C, and 24 h. On the other hand, Neutrase 0.8 L exhibited lower efficiency, and therefore, lower protein recovery. After optimization of the Neutrase 0.8 L process using the response surface methodology (RSM), the maximum DH achieved was 7.2% with the variables set at 15% E/S ratio, initial pH 8, 40 °C, and 10.5 h. Using these hydrolysates as a nitrogen source allowed higher sporulation of the fungus and, therefore, the use of a lower volume of inoculum (three-fold), obtaining a DHMF yield > 90%, 50% higher than the yield obtained when using commercial peptones. The presented process allows the transformation of animal co- and by-products into low-cost nitrogen sources, which greatly impacts the industrial feasibility of HMF biotransformation.

Establishment of Kluyveromyces marxianus as a Microbial Cell Factory for Lignocellulosic Processes: Production of High Value Furan Derivatives.

The establishment of lignocellulosic biorefineries is dependent on microorganisms being able to cope with the stressful conditions resulting from the release of inhibitory compounds during biomass processing. The yeast Kluyveromyces marxianus has been explored as an alternative microbial factory due to its thermotolerance and ability to natively metabolize xylose. The lignocellulose-derived inhibitors furfural and 5-hydroxymethylfurfural (HMF) are considered promising building-block platforms that can be converted into a wide variety of high-value derivatives. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. The potential of this yeast to reduce furfural and HMF at high inhibitory loads was disclosed and characterized. Our results associated HMF reduction with NADPH while furfural-reducing activity was higher with NADH. In addition, furans’ inhibitory effect was higher when combined with xylose consumption. The furan derivatives produced by K. marxianus in different conditions were identified. Furthermore, one selected isolate was efficiently used as a whole-cell biocatalyst to convert furfural and HMF into their derivatives, furfuryl alcohol and 2,5-bis(hydroxymethyl)furan (BHMF), with high yields and productivities. These results validate K. marxianus as a promising microbial platform in lignocellulosic biorefineries.

Redox-Switchable Biocatalyst for Controllable Oxidation or Reduction of 5-Hydroxymethylfurfural into High-Value Derivatives.

Biocatalytic upgrading of biomass-derived 5-hydroxymethylfurfural (HMF) into high-value derivatives is of great significance in green chemistry. In this study, we disclosed the successful utilization of whole-cell Paraburkholderia azotifigens F18 for its switchable catalytic performance in the on-demand catalysis of HMF to different value-added derivatives, namely, selective reduction to 2,5-bis(hydroxymethyl)furan (BHMF) or oxidation to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). Based on the fine-tuning of biochemical properties, the biocatalyst can proceed an efficient hydrogenation reaction toward HMF with a good selectivity of 97.6% to yield the BHMF at 92.2%. Noteworthily, BHMF could be further oxidized to HMFCA and 2,5-furandicarboxylic acid (FDCA) by the whole cell. To realize the on-demand syntheses of HMFCA, the genes encoding HMF oxidoreductase/oxidase of whole-cell F18 were then deleted to prevent the further conversion of HMFCA to FDCA, which led to a 10-fold decrease of FDCA. Thus, an HMF conversion of 100% with an HMFCA yield of 98.3% was finally achieved by the engineered whole cell at a substrate concentration of 150 mM. Moreover, HMFCA synthesis was efficiently prepared with an excellent selectivity of 96.3% and a yield of 85.1% even at a high substrate concentration of up to 200 mM.

Computer-aided search for a cold-active cellobiose 2-epimerase

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis, with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (kcat), Michaelis constant (Km), and catalytic efficiency (kcat/Km) using lactose as a substrate were 117.0 ± 7.7 s-1, 429.9 ± 57.3 mM, and 0.27 mM-1s-1, respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications.

One-pot synthesis of bio-fuel additives from glycerol and benzyl alcohol: Mesoporous MCM-41 supported iron (III) chloride as a highly efficient tandem catalyst

The demand for renewable resources, surplus availability, low cost and lower environmental impact has led to the utilization of bio-glycerol into high-value derivatives and functional chemicals for sustainability of the bio-diesel industry. The production of bio-fuel additives is of great interest as there is a gradual shift from petro-based chemicals to green chemicals. This work involves the direct synthesis of oxygenated fuel additives, 1,3-dioxalane and 1,3-dioxane from glycerol and benzyl alcohol over FeCl3/MCM-41 catalyst. The reaction progresses successfully to produce 1,3-dioxalane primarily by in-situ oxidation of benzyl alcohol to benzaldehyde and subsequent condensation reaction of benzaldehyde with glycerol. Glycerol primarily acted as green solvent to facilitate the oxidation of benzyl alcohol to benzaldehyde and as a fuel additive precursor to produce 1,3-dioxalane in the preceding reaction. FeCl3/MCM-41 catalyst presented high performance achieving 92% glycerol conversion with 75% selectivity to 1,3-dioxalane in a reaction performed at 100 °C for 60 min.

Biocatalytic Transformation of 5-Hydroxymethylfurfural into High-Value Derivatives: Recent Advances and Future Aspects

Catalytic transformation of biomass-derived 5-hydroxymethylfurfural (HMF) into a series of high-value derivatives, such as 2,5-diformylfuran, 5-hydroxymethyl-2-furancarboxylic acid, 5-formyl-2-furancarboxylic acid, 2,5-furandicarboxylic acid, 2,5-dihydroxymethylfuran, 5-alkanoyloxymethylfurfural, 5,5-bis(hydroxymethyl)furoin and 5-hydroxymethylfurfurylamine, is a very important research field in the biorefinery process. For a long time, chemocatalytic pathways have been the main transformation methods of HMF, and so, they have been widely studied in recent years. However, considering some unavoidable issues of chemocatalytic pathways, biocatalytic pathways with many advantages, such as higher selectivity, gentler condition, lower investment and environmental friendliness, should be a promising alternative, and unfortunately, they have not yet attracted enough attention until now. To better understand the current research status, this review primarily retrospects and describes the discovery and development...

Lactobionic Acid: Significance and Application in Food and Pharmaceutical

Lactose has long been used as a precursor for the manufacture of high-value derivatives with emerging applications in the food and pharmaceutical industries. This review focuses on the main characteristics, manufacturing methods, physiological effects and applications of lactobionic acid. Lactobionic acid is a product obtained from lactose oxidation, with high potential applications as a bioactive compound. Recent advances in tissue engineering and application of nanotechnology in medical fields have also underlined the increased importance of this organic acid as an important biofunctional agent.

PROGRESS ON POTENTIAL TECHNOLOGIES FOR GLYCEROL VALORIZATION INTO OXYGENATED FUEL ADDITIVES: A REVIEW

Glycerol has tremendous potential to be transformed into highvalue derivatives /functional chemicals. Demand for renewable sources, surplus availability, low cost and lower environmental impact have led to numerous selective processes for glycerol conversion into products for sustainability of bio-diesel industry. Bio-fuel derived platform molecules are in great demand as there is shift from petro-based chemicals to green chemicals and one of the main interests in the recent years is in the production of bio-fuel additives. This paper reviews different techniques and relevant catalysts used for the production of bio-fuel additives from different pathways such as esterification, etherification, acetalization and acetylation.

Production of succinic acid from sugarcane molasses supplemented with a mixture of corn steep liquor powder and peanut meal as nitrogen sources by Actinobacillus succinogenes.

The potential of using corn steep liquor powder (CSLP), peanut meal (PM), soybean meal (SM), cotton meal (CM) and urea as the substitute of yeast extract (YE) as the nitrogen source was investigated for producing succinic acid (SA). Actinobacillus succinogenes GXAS137 was used as the fermenting bacterium and sugarcane molasses was used as the main substrate. None of these materials were able to produce SA as high as YE did. The CSLP could still be considered as a feasible and inexpensive alternate for YE as the yield of SA produced using CSLP was second only to the yield of SA obtained by YE. The use of CSLP-PM mixed formulation (CSLP to PM ratio = 2·6) as nitrogen source produced SA up to 59·2 g l(-1) with a productivity of 1·2 g l(-1) h(-1). A batch fermentation using a stirred bioreactor produced up to 60·7 g l(-1) of SA at the same formulation. Fed-batch fermentation that minimized the substrate inhibition produced 64·7 g l(-1) SA. These results suggest that sugarcane molasses supplemented with a mixture of CSLP and PM as the nitrogen source could be used to produce SA more economically using A. succinogenes. Significance and impact of the study: Succinic acid (SA) is commonly used as a platform chemical to produce a number of high value derivatives. Yeast extract (YE) is used as a nitrogen source to produce SA. The high cost of YE is currently the limiting factor for industrial production of SA. This study reports the use of a mixture of corn steep liquor powder (CSLP) and peanut meal (PM) as an inexpensive nitrogen source to substitute YE. The results showed that this CSLP-PM mixed formulation can be used as an effective and economic nitrogen source for the production of SA.

New one-pot syntheses of ketals and acetals from oleic acid

Ketals derived from unsaturated fatty acids, such as oleic acid (OA), are widely used as intermediates in the synthesis of various polymers, biologically active compounds and other high-value derivatives. We propose the study of new conditions for one-pot syntheses of ketals and acetals from oleic acid, using acetone or different alcohols as both reactant and solvent. OA was oxidized in a batch reactor in the presence of hydrogen peroxide (H2O2, 30%) as an oxidant and a peroxo-tungsten anion supported on resin as the catalyst. We investigated the influence of several parameters of this process, using 9,10-dihydroxyoctadecanoic acid as the target molecule. The best operating conditions resulted in an OA conversion above 80%, with a good yield (60%) obtained for the OA/H2O2/{PO4[WO(O2)2]4}3− system supported on a resin, at 70°C for 16h in acetone. The hydroxyl moiety was oxidized, generating the corresponding aldehyde derivative, when alcohols were used as the solvent.