Unveiling a CAAX Protease-Like Protein Involved in Didemnin Drug Maturation and Secretion.

The assembly line biosynthesis of the powerful anticancer-antiviral didemnin cyclic peptides is proposed to follow a prodrug release mechanism in Tristella bacteria. This strategy commences with the formation of N-terminal prodrug scaffolds and culminates in their cleavage during the cellular export of the mature products. In this study, a comprehensive exploration of the genetic and biochemical aspects of the enzymes responsible for both the assembly and cleavage of the acylated peptide prodrug scaffolds is provided. This process involves the assembly of N-acyl-polyglutamine moieties orchestrated by the nonribosomal peptide synthetase DidA and the cleavage of these components at the post-assembly stage by DidK, a transmembrane CAAX hydrolase homolog. The findings not only shed light on the complex prodrug mechanism that underlies the synthesis and secretion of didemnin compounds but also offer novel insights into the expanded role of CAAX hydrolases in microbes. Furthermore, this knowledge can be leveraged for the strategic design of genome mining approaches aimed at discovering new bioactive natural products that employ similar prodrug biochemical strategies.

Improving the catalytic efficiency of Pseudomonas aeruginosa lipoxygenase by semi-rational design.

Lipoxygenase (LOX) catalyzes the peroxidation of unsaturated fatty acids to produce hydroperoxides, which had been widely used in food, medicine and chemical industries due to its decoloration of food and conversion of renewable oils. Thus, higher catalytic activity and stability is desired for low-cost and expanded industrial applications of LOX. To improve the catalytic activity of LOX, a mutant library of Pseudomonas aeruginosa lipoxygenase (PaLOX) was firstly built via semi-rational design. The kcat/Km of mutant increased by 9.2-fold and the half-life (t1/2) at 50 °C increased by 4.6 min. Molecular dynamics (MD) simulation indicated that mutation reduced steric hindrance to substrate binding and increased the flexibility of the lid domain that covered the bound unsaturated fatty acid substrate. In addition, van der Waals interactions between the substrate and amino acid residues of the binding pocket increased and alkyl and Pi-alkyl interactions decreased, which might improve the flexibility and substrate binding affinity. These findings promoted understanding of the structure-function relationship of LOX and increase its catalytic efficiency and stability for further industrial application.

Engineering sigma factors and chaperones for enhanced heterologous lipoxygenase production in Escherichia coli.

BACKGROUND: Lipoxygenase (EC. 1.13.11.12, LOX) can catalyze the addition of oxygen into polyunsaturated fatty acids to produce hydroperoxides, which are widely used in the food, chemical, and pharmaceutical industries. In recent years, the heterologous production of LOX by Escherichia coli has attracted extensive attention. However, overexpressed recombinant LOX in E. coli aggregates and forms insoluble inclusion bodies owing to protein misfolding. RESULTS: In this study, a split green fluorescent protein-based screening method was developed to screen sigma (σ) factors and molecular chaperones for soluble LOX expression. Three mutant libraries of Skp, GroES, and RpoH was analyzed using the high-throughput screening method developed herein, and a series of mutants with significantly higher yield of soluble heterologous LOX were obtained. The soluble expression level of LOX in the isolated mutants increased by 4.2- to 5.3-fold. Further, the highest LOX activity (up to 6240 ± 269 U·g-DCW-1) was observed in E. coli REopt, with the regulatory factor mutants, RpoH and GroES. Based on RNA-Seq analysis of the selected strains, E. coli Eopt, E. coli Sopt, E. coli Ropt, and wild type, amino acid substitutions in σ factors and molecular chaperones regulated the expression level of genes related to gene replication, recombination, and repair. Furthermore, the regulatory factor mutants were identified to be beneficial to the soluble expression of two other heterologous proteins, amylase and bone morphological protein 12. CONCLUSION: In this study, a high-throughput screening method was developed for improved soluble LOX expression. The obtained positive mutants of the regulatory factor were analyzed and employed for the expression of other heterologous proteins, thus providing a potential solution for the inclusion-body protein.

Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system.

BACKGROUND: Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved. RESULTS: Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL-1 in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively. CONCLUSIONS: In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production.

Combinatorial strategy towards the efficient expression of lipoxygenase in Escherichia coli at elevated temperatures.

Lipoxygenases (LOXs) are a family of non-heme iron oxidoreductases, which catalyze the addition of oxygen into polyunsaturated fatty acids. They have applications in the food and medical industries. In most studies, the soluble expression of LOXs in microbes requires low temperature (< 20 °C), which increases the cost and fermentation time. Achievement of soluble expression in elevated temperatures (> 30 °C) would shorten the production phase, leading to cost-efficient industrial applications. In this study, a combinatorial strategy was used to enhance the expression of soluble LOXs, comprising plasmid stability systems plus optimized carbon source used for auto-induction expression. Plasmid stability analysis suggested that both active partition systems and plasmid-dependent systems were essential for plasmid stability. Among them, the parBCA in it resulted in the enzyme activity increasing by a factor of 2 (498 ± 13 units per gram dry cell weight (U/g-DCW) after 6-h induction). Furthermore, the optimized carbon source, composed of glucose, lactose, and glycerol, could be used as an auto-induction expression medium and effectively improve the total and soluble expression of LOX, which resulted in the soluble expression of LOX increased by 7 times. Finally, the soluble expression of LOX was 11 times higher with a combinatorial strategy that included both optimized plasmid partition and auto-induction medium. Our work provides a broad, generalizable, and combinatorial strategy for the efficient production of heterologous proteins at elevated temperatures in the E. coli system. KEY POINTS : • Soluble expression of lipoxygenase at 30 °C or higher temperatures is industrially beneficial. • Strategies comprise plasmid partition and optimized auto-induction medium with glucose, lactose, and glycerol as carbon source. • Combinatorial strategy further improved LOX soluble expression at 30 °C and 37 °C.

Biotransformation of cholesterol and 16,17-alpha epoxypregnenolone by novel Cladosporium sp. strain IS547.

Nowadays, there are a few steroid drugs or intermediates that have been obtained via the transformation of microorganisms, and many strains of transformed steroids have not been found yet. Therefore, it is very significant to screen for the strains that have the abilities to transform steroids to produce valuable products. This study has focused on the screen and identification of strains, the structural identification of converted products, and the optimization of transformation conditions, as well as the establishment of transformation systems. A soil microbiota was screened for strain involved in the biotransformation of steroids. A new isolate IS547 is capable of converting a variety of steroids (such as cholesterol, ergosterol, hydrocortisone, progesterone, pregnenolone, and 16,17-alpha-epoxypregnenolone). Based on the 18S rDNA gene sequence comparison, the isolate IS547 has been demonstrated to be very closely related to Cladosporium sp. genus. Present paper is the first report regarding the microbial transformation by Cladosporium sp. to produce active intermediates, which include 7-hydroxy cholesterol, 20-droxyl-16α,17α-epoxypregna-4-dien-3-one, 7-ketocholesterol, and 7-droxyl-16α,17α-epoxypregna-4-dien-3,20-dione. Under the optimum conditions, the yields of product 3 and product 4 were 20.58 and 17.42%, respectively, higher than that prior to the optimization. The transformation rate increased significantly under the optimum fermentation conditions. This study describes an efficient, rapid, and inexpensive biotransformation system for the production of active pharmaceutical intermediates.

Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1.

The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7ß-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0.

[Screening and optimization of cholesterol conversion strain].

OBJECTIVE: Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized. The transformation products were identified. Fermentation conditions were optimized for conversion. METHODS: Cholesterol was used as sole carbon source to isolate strain SE-1. Morphology, physiological and biochemical characteristics of strain SE-1 were studied. 16S rRNA gene was sequenced and subjected to phylogenetic analysis. Fermentation supernatants were extracted with chloroform, the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20. Their structures were identified by 1H-NMR and 13C-NMR. Fermentation medium including carbon and nitrogen, methods of adding substrates and fermentation conditions for Strain SE-1 were optimized. RESULTS: Strain SE-1 was a Gram-negative bacterium, exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis. The sequence analysis of 16S rRNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B. cepacia (Genbank No. U96927). The similarity was 99%. The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products, 7beta-hydroxycholesterol and the minor product was 7-oxocholesterol. The optimum culture conditions were: molasses 5%, (NH4 )2SO4 0.3%, 4% of inoculation, pH 7.5 and 36 degrees C. Under the optimum culture condition, the conversion rate reached 34.4% when concentration of cholesterol-Tween 80 was 1 g/L. Cholesterol 7beta-hydroxylation conversion rate under optimal conditions was improved by 20.8%. CONCLUSION: Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale.