Valorization of Cabbage Waste as a Feedstock for Microbial Polyhydroxyalkanoate Production: Optimizing Hydrolysis Conditions and Polyhydroxyalkanoate Production.

Establishing a platform for the bioconversion of waste resources into value-added compounds is critical for achieving a sustainable and eco-friendly economy. Herein, we produced polyhydroxyalkanoate via microbial fermentation using cabbage waste as a feedstock and metabolically engineered Escherichia coli. For this, the hydrolysis conditions of cabbage waste were optimized by focusing on parameters such as substrate and enzyme concentrations to enhance the saccharification efficiency. The phaABC operon, which encodes key enzymes responsible for polyhydroxyalkanoate biosynthesis in Ralstonia eutropha H16, was overexpressed in E. coli. Using cabbage hydrolysate as the feedstock, this engineered E. coli strain could produce poly(3-hydroxybutyrate) with a polymer content of 26.0 wt % of dry cell weight. Moreover, malic acid in cabbage hydrolysate significantly enhanced poly(3-hydroxybutyrate) production; the addition of 0.5 g/L malic acid markedly increased poly(3-hydroxybutyrate) content by 59.9%. This study demonstrates the potential of cabbage waste as a promising raw material for the microbial production of polyhydroxyalkanoate.

DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability.

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

Characterization of a novel glutamate decarboxylase (GAD) from Latilactobacillus curvatus K285 isolated from Gat -Kimchi.

A lactic acid bacteria (LAB) producing γ-aminobutyric acid (GABA) was isolated from Gat-Kimchi, a Korean vegetable food. The isolate, K285, was identified as Latilactobacillus (formly Lactobacillus) curvatus. The gadB encoding glutamate decarboxylase (GAD) was cloned and an ORF encoding a protein of 451 amino acids was located. K285 GAD was smaller than other LAB GADs, and its amino acid sequence showed less than 80% homology with other LAB GADs, indicating the uniqueness of K285 GAD. The gadC encoding glutamate/GABA antiporter was located 75 bp upstream of gadB, indicating gadCB operon structure. The gadB was overexpressed in Escherichia coli and recombinant GAD was purified. Optimum pH and temperature of recombinant K285 GAD were pH 5.0 and 50 °C, respectively, and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 5.35 ± 0.27 mM and 0.041 ± 0.0008 mM/min, respectively. Lb. curvatus K285 might be useful for the production of foods enriched with GABA.

Some Important Metabolites Produced by Lactic Acid Bacteria Originated from Kimchi.

Lactic acid bacteria (LAB) have been used for various food fermentations for thousands of years. Recently, LAB are receiving increased attention due to their great potential as probiotics for man and animals, and also as cell factories for producing enzymes, antibodies, vitamins, exopolysaccharides, and various feedstocks. LAB are safe organisms with GRAS (generally recognized as safe) status and possess relatively simple metabolic pathways easily subjected to modifications. However, relatively few studies have been carried out on LAB inhabiting plants compared to dairy LAB. Kimchi is a Korean traditional fermented vegetable, and its fermentation is carried out by LAB inhabiting plant raw materials of kimchi. Kimchi represents a model food with low pH and is fermented at low temperatures and in anaerobic environments. LAB have been adjusting to kimchi environments, and produce various metabolites such as bacteriocins, γ-aminobutyric acid, ornithine, exopolysaccharides, mannitol, etc. as products of metabolic efforts to adjust to the environments. The metabolites also contribute to the known health-promoting effects of kimchi. Due to the recent progress in multi-omics technologies, identification of genes and gene products responsible for the synthesis of functional metabolites becomes easier than before. With the aid of tools of metabolic engineering and synthetic biology, it can be envisioned that LAB strains producing valuable metabolites in large quantities will be constructed and used as starters for foods and probiotics for improving human health. Such LAB strains can also be useful as production hosts for value-added products for food, feed, and pharmaceutical industries. In this review, recent findings on the selected metabolites produced by kimchi LAB are discussed, and the potentials of metabolites will be mentioned.

Characterization of a salt-resistant fibrinolytic protease of Bacillus licheniformis HJ4 isolated from Hwangseokae jeotgal, a traditional Korean fermented seafood.

Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27 kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84 h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27 kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively.

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr.

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40°C, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.