[Cloning and functional characterization of the pinoresinol-lariciresinol reductase gene IiPLR2 in Isatis indigotica].

The pinoresinol-lariciresinol reductase (PLR), a crucial enzyme in the biosynthesis of lignans in plants, catalyzes a two-step reaction to produce lariciresinol and secoisolariciresinol. Lignans such as lariciresinol are the effective components of traditional Chinese medicine Radix Isatidis in exerting antiviral activity. In order to study the function of the key enzyme PLR in the biosynthesis of lariciresinol in Isatis indigotica, the original plant of Radix Isatidis, IiPLR2 was cloned from I. indigotica, with a full length of 954 bp, encoding 317 amino acids. Multiple sequence alignment showed that IiPLR2 contained a conserved nicotinamide adenine dinucleotide phosphate (NADPH)-binding motif. The phylogenetic tree showcased that IiPLR2 shared the same clade with AtPrR1 from Arabidopsis thaliana. The prokaryotic expression vector pET32a-IiPLR2 was constructed and then transformed into Escherichia coli BL21(DE3) competent cells for protein expression. The purified enzyme IiPLR2 could catalyze the conversion of pinoresinol to lariciresinol and the conversion of lariciresinol to secoisolariciresinol. The cloning, sequencing, and catalytic functional analysis of IiPLR2 in this study enrich the understanding of this kind of functional proteins in I. indigotica and supplement the biosynthesis pathways of lignans. Moreover, this study provides a functional module for further research on metabolic regulation and synthetic biology and lays a foundation for comprehensively revealing the relationship between the spatial structures and catalytic functions of such proteins.

High production of enantiopure (R,R)-2,3-butanediol from crude glycerol by Klebsiella pneumoniae with an engineered oxidative pathway and a two-stage agitation strategy.

BACKGROUND: (R,R)-2,3-butanediol (BDO) is employed in a variety of applications and is gaining prominence due to its unique physicochemical features. The use of glycerol as a carbon source for 2,3-BDO production in Klebsiella pneumoniae has been limited, since 1,3-propanediol (PDO) is generated during glycerol fermentation. RESULTS: In this study, the inactivation of the budC gene in K. pneumoniae increased the production rate of (R,R)-2,3-BDO from 21.92 ± 2.10 to 92.05 ± 1.20%. The major isomer form of K. pneumoniae (meso-2,3-BDO) was shifted to (R,R)-2,3-BDO. The purity of (R,R)-2,3-BDO was examined by agitation speed, and 98.54% of (R,R)-2,3-BDO was obtained at 500 rpm. However, as the cultivation period got longer, the purity of (R,R)-2,3-BDO declined. For this problem, a two-step agitation speed control strategy (adjusted from 500 to 400 rpm after 24 h) and over-expression of the dhaD gene involved in (R,R)-2,3-BDO biosynthesis were used. Nevertheless, the purity of (R,R)-2,3-BDO still gradually decreased over time. Finally, when pure glycerol was replaced with crude glycerol, the titer of 89.47 g/L of (R,R)-2,3-BDO (1.69 g/L of meso-2,3-BDO), productivity of 1.24 g/L/h, and yield of 0.35 g/g consumed crude glycerol was achieved while maintaining a purity of 98% or higher. CONCLUSIONS: This study is meaningful in that it demonstrated the highest production and productivity among studies in that produced (R,R)-2,3-BDO with a high purity in Klebsiella sp. strains. In addition, to the best of our knowledge, this is the first study to produce (R,R)-2,3-BDO using glycerol as the sole carbon source.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

The critical role of residues Phe120 and Val161 of (2 R,3 R)­2,3­butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site-directed mutagenesis.

NAD+-dependent (2 R,3 R)­2,3­butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium-chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)­2,3­butanediol (RR-BD) and meso-2,3­butanediol (meso-BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent KM values of the Phe120Ala mutant for RR-BD and meso-BD increased 36- and 369-fold, respectively; the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 586- and 3528-fold, respectively; and the apparent KM values of the Val161Ala mutant for RR-BD and meso-BD increased 4- and 37-fold, respectively, the catalytic efficiencies of this mutant with RR-BD and meso-BD decreased approximately 3- and 28-fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR-BD; 7.3-fold with meso-BD) due to an increase in KM (sixfold for RR-BD; 24-fold for meso-BD) and a slight increase (2.8-fold with RR-BD; 3.3-fold with meso-BD) in kcat. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.

Anti-inflammatory effect of lignans from flaxseed after fermentation by lactiplantibacillus plantarum SCB0151 in vitro.

Lignan, a beneficial constituent of Flaxseed (Linum usitatissimum L.) showed great interest in researchers because of its multiple functional properties. Nonetheless, a challenge arises due to the glycosidic structure of lignans, which the gut epithelium cannot readily absorb. Therefore, we screened 18 strains of Lactiplantibacillus plantarum, Lacticaseibacillus casei, Lactobacillus acidophilus, Lacticaseibacillus rhamnosus, Pediococcus pentosaceus, Pediococcus acidilactici, and Enterococcus durans to remove glycosides from flaxseed lignan extract enzymatically. Among our findings, Lactiplantibacillus plantarum SCB0151 showed the highest activity of ß-glucosidase (8.91 ± 0.04 U/mL) and higher transformed efficiency of Secoisolariciresinol (SECO) (8.21 ± 0.13%). The conversion rate of Secoisolariciresinol diglucoside (SDG) and the generation rate of SECO was 58.30 ± 3.78% and 32.13 ± 2.78%, respectively, under the optimized conditions. According to the LC-HRMSMS analysis, SECO (68.55 ± 6.57 µM), Ferulic acid (FA) (32.12 ± 2.50 µM), and Coumaric acid (CA) (79.60 ± 6.21 µM) were identified in the biotransformation products (TP) of flaxseed lignan extract. Results revealed that the TP exhibited a more pronounced anti-inflammatory effect than the flaxseed lignan extract. SECO, FA, and CA demonstrated a more inhibitory effect on NO than that of SDG. The expression of iNOS and COX-2 was significantly suppressed by TP treatment in LPS-induced Raw264.7 cells. The secretion of IL-6, IL-2, and IL-1ß decreased by 87.09 ± 0.99%, 45.40 ± 0.87%, and 53.18 ± 0.83%, respectively, at 60 µg/mL of TP treatment. Given these data, the bioavailability of flaxseed lignan extract and its anti-inflammatory effect were significantly enhanced by Lactiplantibacillus plantarum SCB0151, which provided a novel approach to commercializing flaxseed lignan extract for functional food.

Inactivation of hydrogenase-3 leads to enhancement of 1,3-propanediol and 2,3-butanediol production by Klebsiella pneumoniae.

Klebsiella pneumoniae can use glucose or glycerol as carbon sources to produce 1,3-propanediol or 2,3-butanediol, respectively. In the metabolism of Klebsiella pneumoniae, hydrogenase-3 is responsible for H2 production from formic acid, but it is not directly related to the synthesis pathways for 1,3-propanediol and 2,3-butanediol. In the first part of this research, hycEFG, which encodes subunits of the enzyme hydrogenase-3, was knocked out, so K. pneumoniae ΔhycEFG lost the ability to produce H2 during cultivation using glycerol as a carbon source. As a consequence, the concentration of 1,3-propanediol increased and the substrate (glycerol) conversion ratio reached 0.587 mol/mol. Then, K. pneumoniae ΔldhAΔhycEFG was constructed to erase lactic acid synthesis which led to the further increase of 1,3-propanediol concentration. A substrate (glycerol) conversion ratio of 0.628 mol/mol in batch conditions was achieved, which was higher compared to the wild type strain (0.545 mol/mol). Furthermore, since adhE encodes an alcohol dehydrogenase that catalyzes ethanol production from acetaldehyde, K. pneumoniae ΔldhAΔadhEΔhycEFG was constructed to prevent ethanol production. Contrary to expectations, this did not lead to a further increase, but to a decrease in 1,3-propanediol production. In the second part of this research, glucose was used as the carbon source to produce 2,3-butanediol. Knocking out hycEFG had distinct positive effect on 2,3-butanediol production. Especially in K. pneumoniae ΔldhAΔadhEΔhycEFG, a substrate (glucose) conversion ratio of 0.730 mol/mol was reached, which is higher compared to wild type strain (0.504 mol/mol). This work suggests that the inactivation of hydrogenase-3 may have a global effect on the metabolic regulation of K. pneumoniae, leading to the improvement of the production of two industrially important bulk chemicals, 1,3-propanediol and 2,3-butanediol.

Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.

A comparative study on flaxseed lignan biotransformation through resting cell catalysis and microbial fermentation by ß-glucosidase production Lactiplantibacillus plantarum.

BACKGROUND: Flax lignan has attracted much attention because of its potential bioactivities. However, the bioavailability of secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, depends on the bioconversion by the colon bacteria. Lactic acid bacteria (LAB) with ß-glucosidase activity has found wide application in preparing bioactive aglycone. RESULTS: LAB strains with good ß-glucosidase activity were isolated from fermented tofu. Their bioconversion of flax lignan extract was investigated by resting cell catalysis and microbial fermentation, and the metabolism of SDG by Lactiplantibacillus plantarum C5 following fermentation was characterized by widely targeted metabolomics. Five L. plantarum strains producing ß-glucosidase with broad substrate specificity were isolated and identified, and they all can transform SDG into secoisolariciresinol (SECO). L. plantarum C5 resting cell reached a maximum SDG conversion of 49.19 ± 3.75%, and SECO generation of 21.49 ± 1.32% (0.215 ± 0.013 mm) at an SDG substrate concentration of 1 mM and 0.477 ± 0.003 mm SECO was produced at 4 mm within 24 h. Although sixteen flax lignan metabolites were identified following the fermentation of SDG extract by L. plantarum C5, among them, four were produced following the fermentation: SECO, demethyl-SECO, demethyl-dehydroxy-SECO and isolariciresinol. Moreover, seven lignans increased significantly. CONCLUSION: Fermentation significantly increased the profile and level of flax lignan metabolites, and the resting cell catalysis benefits from higher bioconversion efficiency and more straightforward product separation. Resting cell catalysis and microbial fermentation of flax lignan extract by the isolated ß-glucosidase production L. plantarum could be potentially applied in preparing flax lignan ingredients and fermented flaxseed. © 2024 Society of Chemical Industry.

Comparison of Yield Characteristics, Chemical Composition, Lignans Content and Antioxidant Potential of Experimentally Grown Six Linseed (Linum usitatissimum L.) Cultivars.

Linseed represents a rich source of nutritional, functional and health-beneficial compounds. Nevertheless, the chemical composition and content of bioactive compounds may be quite variable and potentially affected by various factors, including genotype and the environment. In this study, the proximate chemical composition, lignans content and antioxidant potential of six experimentally grown linseed cultivars were assessed and compared. A diagonal cultivation trial in the University of South Bohemia Experimental Station in Ceské Budejovice, Czech Republic, was established in three subsequent growing seasons (2018, 2019 and 2020). The results showed that the cultivar and growing conditions influenced most studied parameters. The lack of precipitation in May and June 2019 negatively affected the seed yield and the level of secoisolariciresinol diglucoside but did not decrease the crude protein content, which was negatively related to the oil content. The newly developed method for lignans analysis allowed the identification and quantification of secoisolariciresinol diglucoside and matairesinol. Their content correlated positively with the total polyphenol content and antioxidant assays (DPPH and ABTS radical scavenging activity), indicating the significant contribution to the biofunctional properties of linseed. On the other hand, we did not detect minor linseed lignans, pinoresinol and lariciresinol. The results of this study showed the importance of cultivar and growing conditions factors on the linseed chemical composition and the lignans content, determining its nutritional and medicinal properties.

Systemic metabolic engineering of Enterobacter aerogenes for efficient 2,3-butanediol production.

2,3-Butanediol (2,3-BDO) is an important gateway molecule for many chemical derivatives. Currently, microbial production is gradually being recognized as a green and sustainable alternative to petrochemical synthesis, but the titer, yield, and productivity of microbial 2,3-BDO remain suboptimal. Here, we used systemic metabolic engineering strategies to debottleneck the 2,3-BDO production in Enterobacter aerogenes. Firstly, the pyruvate metabolic network was reconstructed by deleting genes for by-product synthesis to improve the flux toward 2,3-BDO synthesis, which resulted in a 90% increase of the product titer. Secondly, the 2,3-BDO productivity of the IAM1183-LPCT/D was increased by 55% due to the heterologous expression of DR1558 which boosted cell resistance to abiotic stress. Thirdly, carbon sources were optimized to further improve the yield of target products. The IAM1183-LPCT/D showed the highest titer of 2,3-BDO from sucrose, 20% higher than that from glucose, and the yield of 2,3-BDO reached 0.49 g/g. Finally, the titer of 2,3-BDO of IAM1183-LPCT/D in a 5-L fermenter reached 22.93 g/L, 85% higher than the wild-type strain, and the titer of by-products except ethanol was very low. KEY POINTS: Deletion of five key genes in E. aerogenes improved 2,3-BDO production The titer of 2,3-BDO was increased by 90% by regulating metabolic flux Response regulator DR1558 was expressed to increase 2,3-BDO productivity.

Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca.

L-Valine, a branched-chain amino acid with diversified applications, is biosynthesized with α-acetolactate as the key precursor. In this study, the metabolic flux in Klebsiella oxytoca PDL-K5, a Risk Group 1 organism producing 2,3-butanediol as the major fermentation product, was rearranged to L-valine production by introducing exogenous L-valine biosynthesis pathway and blocking endogenous 2,3-butanediol generation at the metabolic branch point α-acetolactate. After further enhancing L-valine efflux, strengthening pyruvate polymerization and selecting of key enzymes for L-valine synthesis, a plasmid-free K. oxytoca strain VKO-9 was obtained. Fed-batch fermentation with K. oxytoca VKO-9 in a 7.5 L fermenter generated 122 g/L L-valine with a yield of 0.587 g/g in 56 h. In addition, repeated fed-batch fermentation was conducted to prevent precipitation of L-valine due to oversaturation. The average concentration, yield, and productivity of produced L-valine in three cycles of repeated fed-batch fermentation were 81.3 g/L, 0.599 g/g, and 3.39 g/L/h, respectively.

Effects of 1,3-propanediol associated, or not, with butylene glycol and/or glycerol on skin hydration and skin barrier function.

OBJECTIVE: This study aimed to assess the effect of 1,3-propanediol at different concentrations (5%, 10%, or 15%), either applied alone or in combination with butylene glycol (BG) (5%) and/or glycerol (5%), on skin hydration and skin barrier function. The measurements were conducted using capacitance to determine skin hydration and trans epidermal water loss (TEWL) rates to evaluate skin barrier function. METHODS: A total of 30 healthy female subjects participated in the study. Capacitance and TEWL measurements were conducted at multiple time points, including before application and at 15 min, 2 and 8 h after the humectants were applied to the forearms of the subjects. All the subjects provided written informed consent. RESULTS: The 1,3-propanediol in all concentrations and in all combinations (with BG and/or glycerol) increased skin hydration and improved skin barrier function 15 min, 2 and 8 h after application. Glycerol increased the hydration performance of 1,3-propanediol. The application of 1,3-propanediol at a concentration of 15%, either alone or in combination with other humectants, reduced the TEWL to a greater extent than lower concentrations of 1,3-propanediol. Furthermore, the addition of glycerol to 1,3-propanediol 15% improved the skin barrier and reduced TEWL when compared with 1,3-propanediol alone and with the combination of 1,3-propanediol + BG. CONCLUSION: The humectants significantly improved skin hydration and reduced TEWL throughout the 8-h time course. The increase in 1,3-propanediol concentration, as well as its combination with glycerol, provided a greater benefit to the skin, improving both hydration and the skin barrier function.

OBJECTIF: Cette étude visait à évaluer l'effet sur l'hydratation de la peau et la fonction de barrière cutanée du 1,3-propanediol à différentes concentrations (5 %, 10 % ou 15 %), appliqué seul ou en association avec du butylène glycol (5 %) et/ou du glycérol (5 %). Les mesures ont été effectuées à l'aide de la capacitance pour déterminer l'hydratation de la peau et les taux de perte d'eau transépidermique (Trans Epidermal Water Loss, TEWL) pour évaluer la fonction de barrière cutanée. MÉTHODES: Au total, 30 sujets de sexe féminin en bonne santé ont participé à l'étude. Les mesures de la capacitance et de la TEWL ont été effectuées à plusieurs moments, y compris avant l'application, 15 minutes, 2 heures et 8 heures après l'application des produits humectant sur les avant-bras des sujets. Tous les sujets ont fourni un consentement éclairé écrit. RÉSULTATS: Le 1,3-propanediol, à toutes les concentrations et dans toutes les associations (avec le butylène glycol et/ou le glycérol), a augmenté l'hydratation de la peau et amélioré la fonction de barrière cutanée à 15 minutes, 2 heures et 8 heures après l'application. Le glycérol a augmenté les performances d'hydratation du 1,3-propanediol. L'application de 1,3-propanediol à une concentration de 15 %, seul ou en association avec d'autres produits humectant, a réduit la TEWL dans une plus grande mesure que des concentrations inférieures de 1,3-propanediol. En outre, l'ajout de glycérol au 1,3-propanediol 15 % a amélioré la barrière cutanée et réduit la TEWL par rapport au 1,3-propanediol seul et à l'association 1,3-propanediol + butylène glycol. CONCLUSION: Les produits humectant ont significativement amélioré l'hydratation de la peau et réduit la TEWL tout au long des 8 heures. L'augmentation de la concentration de 1,3-propanediol, ainsi que son association avec le glycérol, ont apporté un plus grand bénéfice à la peau, améliorant à la fois l'hydratation et la fonction de barrière cutanée.

Metabolic Engineering of Escherichia coli for De Novo Production of 1,2-Butanediol.

1,2-Butanediol (1,2-BDO) is an important platform chemical widely utilized in the synthesis of polyester polyols, plasticizers, cosmetics, and pharmaceuticals. However, no natural metabolic pathway for its biosynthesis has been identified, and biological production of 1,2-BDO from renewable bioresources has not been reported so far. In this study, we designed and experimentally verified a feasible non-natural synthesis pathway for the de novo production of 1,2-BDO from renewable carbohydrates for the first time. This pathway extends the l-threonine synthesis pathway by introducing two artificial metabolic modules to sequentially convert l-threonine into 2-hydroxybutyric acid and 1,2-BDO. Following key enzyme screening and enhancement of l-threonine synthesis module in the chassis microorganism, the best engineered Escherichia coli strain was able to produce 0.15 g/L 1,2-BDO using glucose as the sole carbon source. This work lays the foundation for the bioproduction of 1,2-BDO from renewable resources.

Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway.

Saccharomyces cerevisiae is a promising host for the bioproduction of higher alcohols, such as 2,3-butanediol (2,3-BDO). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO via glycolysis have been constructed. However, the specific 2,3-BDO production rates of engineered strains must be improved. To identify approaches to improving the 2,3-BDO production rate, we investigated the factors contributing to higher ethanol production rates in certain industrial strains of S. cerevisiae compared to laboratory strains. Sequence analysis of 11 industrial strains revealed the accumulation of many nonsynonymous substitutions in RIM15, a negative regulator of high fermentation capability. Comparative metabolome analysis suggested a positive correlation between the rate of ethanol production and the activity of the pyruvate-consuming pathway. Based on these findings, RIM15 was deleted, and the pyruvate-consuming pathway was activated in YHI030, a metabolically engineered S. cerevisiae strain that produces 2,3-BDO. The titer, specific production rate, and yield of 2,3-BDO in the test tube-scale culture using the YMS106 strain reached 66.4 ± 4.4 mM, 1.17 ± 0.017 mmol (g dry cell weight h)-1, and 0.70 ± 0.03 mol (mol glucose consumed)-1. These values were 2.14-, 2.92-, and 1.81-fold higher than those of the vector control, respectively. These results suggest that bioalcohol production via glycolysis can be enhanced in a metabolically engineered S. cerevisiae strain by deleting RIM15 and activating the pyruvate-consuming pathway.

Metabolic engineering of Escherichia coli for enhanced production of 1,3-butanediol from glucose.

1,3-Butanediol (1,3-BDO) finds versatile applications in the cosmetic, chemical, and food industries. This study focuses on the metabolic engineering of Escherichia coli K12 to achieve efficient production of 1,3-BDO from glucose via acetoacetyl-CoA, 3-hydroxybutyryl-CoA, and 3-hydroxybutyraldehyde. The accumulation of an intermediary metabolite (pyruvate) and a byproduct (3-hydroxybutyric acid) was reduced by disruption of the negative transcription factor (PdhR) for pyruvate dehydrogenase complex (PDHc) and employing an efficient alcohol dehydrogenase (YjgB), respectively. Additionally, to improve NADPH availability, carbon flux was redirected from the Embden-Meyerhof-Parnas (EMP) pathway to the Entner-Doudoroff (ED) pathway. One resulting strain achieved a record-high titer of 790 mM (∼71.1 g/L) with a yield of 0.65 mol/mol for optically pure (R)-1,3-BDO, with an enantiomeric excess (e.e.) value of 98.5 %. These findings are useful in the commercial production of 1,3-BDO and provide valuable insights into the development of an efficient cell factory for other acetyl-CoA derivatives.

A lesson from polybutylene succinate plastisphere to the discovery of novel plastic degrading enzyme genes in marine vibrios.

Polybutylene succinate (PBS) is an eco-friendly green plastic. However, PBS was shown as being non-biodegradable in marine environments, and up until now, only a limited number of PBS-degrading marine microbes have been discovered. We first set up in vitro PBS- and PBSA (polybutylene succinate adipate)-plastispheres to characterize novel PBS-degrading marine microbes. Microbial growth and oxygen consumption were observed in both PBS- and PBSA-plastispheres enriched with natural seawater collected from Usujiri, Hokkaido, Japan, and Vibrionaceae and Pseudoalteromonadaceae were significantly enriched on these films. Further gene identification indicated that vibrios belonging to the Gazogenes clade possess genes related to a PBS degrading enzyme (PBSase). The PBS degradation assay for six Gazogenes clade vibrios identified Vibrio ruber, Vibrio rhizosphaerae, and Vibrio spartinae as being capable of degrading PBS. We further identified the gene responsible for PBSase from the type strain of V. ruber, and the purified recombinant vibrio PBSase was found to have low-temperature adaptation and was active under high NaCl concentrations. We also provided docking models between the vibrio PBSase and PBS and PBSA units to show how vibrio PBSase interacts with each substrate compared to the Acidovorax PBSase. These results could contribute to a more sustainable society through further utilization of PBS in marine environments and plastic recycling.

Identification of the Microbial Transformation Products of Secoisolariciresinol Using an Untargeted Metabolomics Approach and Evaluation of the Osteogenic Activities of the Metabolites.

Secoisolariciresinol (SECO) is one of the major lignans occurring in various grains, seeds, fruits, and vegetables. The gut microbiota plays an important role in the biotransformation of dietary lignans into enterolignans, which might exhibit more potent bioactivities than the precursor lignans. This study aimed to identify, synthesize, and evaluate the microbial metabolites of SECO and to develop efficient lead compounds from the metabolites for the treatment of osteoporosis. SECO was fermented with human gut microbiota in anaerobic or micro-aerobic environments at different time points. Samples derived from microbial transformation were analyzed using an untargeted metabolomics approach for metabolite identification. Nine metabolites were identified and synthesized. Their effects on cell viability, osteoblastic differentiation, and gene expression were examined. The results showed that five of the microbial metabolites exerted potential osteogenic effects similar to those of SECO or better. The results suggested that the enterolignans might account for the osteoporotic effects of SECO in vivo. Thus, the presence of the gut microbiota could offer a good way to form diverse enterolignans with bone-protective effects. The current study improves our understanding of the microbial transformation products of SECO and provides new approaches for new candidate identification in the treatment of osteoporosis.

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol.

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation-reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from -17.0 kJ/mol to -29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation-reduction cascade at larger scales.

Metabolic engineering of Escherichia coli for biological production of 1, 3-Butanediol.

The production of 1,3-butanediol (1,3-BDO) from glucose was investigated using Escherichia coli as the host organism. A pathway was engineered by overexpressing genes phaA (acetyl-CoA acetyltransferase), phaB (acetoacetyl-CoA reductase), bld (CoA-acylating aldehyde dehydrogenase), and yqhD (alcohol dehydrogenase). The expression levels of these genes were optimized to improve 1,3-BDO production and pathways that compete with 1,3-BDO synthesis were disrupted. Culture conditions were also optimized, including the C: N ratio, aeration, induction time, temperature, and supplementation of amino acids, resulting in a strain that could produce 1,3-BDO at 257 mM in 36 h, with a yield of 0.51 mol/mol in a fed-batch bioreactor experiment. To the best of our knowledge, this is the highest titer of 1,3-BDO production ever reported using biological methods, and our findings provide a promising strategy for the development of microbial cell factories for the sustainable synthesis of other acetyl-CoA-derived chemicals.