Project description:Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.
Project description:3-Hydroxypropionic acid (3-HP) is a promising high value-added chemical. Acetyl-CoA carboxylase (Acc) is a vital rate-limiting step in 3-HP biosynthesis through the malonyl-CoA pathway. However, Acc toxicity in cells during growth blocks its ability to catalyze acetyl-CoA to malonyl-CoA. The balancing of Acc and malonyl-CoA reductase (MCR) expression is another an unexplored but key process in 3-HP production. To solve these problems, in the present study, we developed a method to mitigate Acc toxicity cell growth through Acc subunits (AccBC and DtsR1) expression adjustment. The results revealed that cell growth and 3-HP production can be accelerated through the adjustment of DtsR1 and AccBC expression. Subsequently, the balancing Acc and MCR expression was also employed for 3-HP production, the engineered strain achieved the highest titer of 6.8 g/L, with a high yield of 0.566 g/g glucose and productivity of 0.13 g/L/h, in shake-flask fermentation through the malonyl-CoA pathway. Likewise, the engineered strain also had the highest productivity (1.03 g/L/h) as well as a high yield (0.246 g/g glucose) and titer (up to 38.13 g/L) in fed-batch fermentation, constituting the most efficient strain for 3-HP production through the malonyl-CoA pathway using a cheap carbon source. This strategy might facilitate the production of other malonyl-CoA-derived chemical compounds in the future.
Project description:Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2'3'-cGAMP) formation was successfully developed. The 2'3'-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2'3'-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2'3'-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.
Project description:BackgroundItaconic acid, an unsaturated C5 dicarbonic acid, has significant market demand and prospects. It has numerous biological functions, such as anti-cancer, anti-inflammatory, and anti-oxidative in medicine, and is an essential renewable platform chemical in industry. However, the development of industrial itaconic acid production by Aspergillus terreus, the current standard production strain, is hampered by the unavoidable drawbacks of that species. Developing a highly efficient cell factory is essential for the sustainable and green production of itaconic acid.ResultsThis study employed combinatorial engineering strategies to construct Escherichia coli cells to produce itaconic acid efficiently. Two essential genes (cis-aconitate decarboxylase (CAD) encoding gene cadA and aconitase (ACO) encoding gene acn) employed various genetic constructs and plasmid combinations to create 12 recombination E. coli strains to be screened. Among them, E. coli BL-CAC exhibited the highest titer with citrate as substrate, and the induction and reaction conditions were further systematically optimized. Subsequently, employing enzyme evolution to optimize rate-limiting enzyme CAD and synthesizing protein scaffolds to co-localize ACO and CAD were used to improve itaconic acid biosynthesis efficiency. Under the optimized reaction conditions combined with the feeding control strategy, itaconic acid titer reached 398.07 mM (51.79 g/L) of engineered E. coli BL-CAR470E-DS/A-CS cells as a catalyst with the highest specific production of 9.42 g/g(DCW) among heterologous hosts at 48 h.ConclusionsThe excellent catalytic performance per unit biomass shows the potential for high-efficiency production of itaconic acid and effective reduction of catalytic cell consumption. This study indicates that it is necessary to continuously explore engineering strategies to develop high-performance cell factories to break through the existing bottleneck and achieve the economical commercial production of itaconic acid.
Project description:Melanization of members of the genus Trichosporon is poorly described. In this study, six strains, including two clinical isolates, from four different species (Trichosporon asahii, T. asteroides, T. inkin, and T. mucoides) were grown in culture media with or without L-dihydroxyphenylalanine (L-DOPA). Each strain produced a brownish pigment compatible with melanin when cultured in presence of L-DOPA, suggesting that these species are able to produce eumelanin. L-tyrosine was not able to elicit any type of pigment production on cultures. As eumelanin is produced by several fungi during parasitism, this pigment may contribute to Trichosporon virulence.
Project description:Pyrroloindolines are important structural units in nature and the pharmaceutical industry, however, most approaches to such structures involve transition-metal or photoredox catalysts. Herein, we describe the first tandem SET/radical cyclization/intermolecular coupling between 2-azaallyl anions and indole acetamides. This method enables the transition-metal-free synthesis of C3a-substituted pyrroloindolines under mild and convenient conditions. The synthetic utility of this transformation is demonstrated by the construction of an array of C3a-methylamine pyrroloindolines with good functional group tolerance and yields. Gram-scale sequential one-pot synthesis and hydrolysis reactions demonstrate the potential synthetic utility and scalability of this approach.
Project description:Although the enzyme catalytic nanoreactors reported so far have achieved excellent therapeutic efficacy, how to accurately exert enzyme activity in the tumor microenvironment to specifically kill tumor cells and avoid systemic oxidative damage would be an inevitable challenge for catalytic nanomedicine. At the present study, we fabricate an advanced biomimetic nanoreactor, SOD-Fe0@Lapa-ZRF for tumor multi-enzyme cascade delivery that combined specifically killing tumor cells and protect cells from oxidative stress. Methods: We first synthesized the FeNP-embedded SOD (SOD-Fe0) by reduction reaction using sodium borohydride. Next, SOD-Fe0 and Lapa cargo were encapsulated in ZIF-8 by self-assembly. In order to protect the cargo enzyme from digestion by protease and prolong blood circulating time, SOD-Fe0@Lapa-Z was further cloaked with RBC membrane and functionalized with folate targeting, resulting in the final advanced biomimetic nanoreactor SOD-Fe0@Lapa-ZRF. Results: Once internalized, ZIF-8 achieves pH-triggered disassembly in weakly acidic tumor microenvironment. The released SOD-Fe0 and Lapa were further endocytosed by tumor cells and the Lapa produces superoxide anion (O2 -•) through the catalysis of NQO1 that is overexpressed in tumor cells, while O2 -• is converted to H2O2 via SOD. At this time, the released ferrous ions from SOD-Fe0 and H2O2 are further transformed to highly toxic hydroxyl radicals (•OH) for specifically killing tumor cells, and there was no obvious toxicological response during long-term treatment. Importantly, SOD-Fe0@Lapa-ZRF enhanced the normal cell's anti-oxidation ability, and thus had little effect on the secretion of TNF-α, IL-6 and IL-1β pro-inflammatory cytokines, while effectively reversed the decreased activity of T-SOD and GSH-Px and remained stable MDA content after tumor treatment. In vitro and in vivo results indicate that the tumor microenvironment-responsive release multi-enzyme cascade have high tumor specificity and effective anti-tumor efficacy, and can protect cells from oxidative stress damage. Conclusion: The biomimetic nanoreactor will have a great potential in cancer nanomedicine and provide a novel strategy to regulate oxidative stress.
Project description:In this paper, an optimal semi-continuous process for vinegar production from edible alcohol through biotransformation by acetic acid bacteria (AAB) WUST-01 was developed. The optimized medium composition for the starting-up stage was glucose 5.1 g/L, yeast extract 26.2 g/L, and ethanol 11.9 mL/L, and the optimal ethanol for the following semi-continuous stage was 50 mL/L. In the semi-continuous biotransformation process, the optimal withdraw ratio was 50% of working volume with 12 h cycle time. With these conditions, the total acidity could reach to 77.3 g/L and the acidity productivity could reach to 3.0 g/(L h) in a 5 L reactor. Furthermore, it was investigated to strengthen vinegar synthesis through enhancing alcohol dehydrogenase and aldehyde dehydrogenase activity in AAB by ferrous ion and pueraria flower extract as the enzyme regulators. With these regulators, the vinegar synthesis efficiency can be improved 16.3 and 13.2% respectively.
Project description:Lactic acid is an intermediate-volume specialty chemical, used in the production of biodegradable polymers and other chemicals. Although lactic acid production process is well established, however, the cost of production is very high. Therefore, in this study; starchy biomass (cassava) was hydrolyzed with in-house enzyme cocktail prepared from Aspergillus foetidus MTCC508 and Bacillus subtilis RA10. Process optimization using Taguchi experimental design helped to optimize the most effective ratio of fungal and bacterial amylase for effective saccharification of cassava. A higher sugar yield of 379.63 mg/gds was obtained under optimized conditions, using 30 U/gds of bacterial enzyme and 90 U/gds of the fungal enzyme at pH 4 within 48 h of saccharification. Among 11 lactic acid bacteria isolated, Lactobacillus fermentum S1A and Lactobacillus farraginis SS3A produced the highest amount of lactic acid 0.81 g/g and 0.77 g/g, respectively, from the cassava hydrolysate. The study proved the potential renewable source of cassava biomass as a source for fermentable sugars that can be fermented to lactic acid with high yield. In future, this cost-effective and environmental-friendly bioprocess can be upscaled for industrial lactic acid production.
Project description:Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.