Biosynthetic gene cluster profiling from North Java Sea Virgibacillus salarius reveals hidden potential metabolites.

Virgibacillus salarius 19.PP.SC1.6 is a coral symbiont isolated from Indonesia's North Java Sea; it has the ability to produce secondary metabolites that provide survival advantages and biological functions, such as ectoine, which is synthesized by an ectoine gene cluster. Apart from being an osmoprotectant for bacteria, ectoine is also known as a chemical chaperone with numerous biological activities such as maintaining protein stability, which makes ectoine in high demand in the market industry and makes it beneficial to investigate V. salarius ectoine. However, there has been no research on genome-based secondary metabolite and ectoine gene cluster characterization from Indonesian marine V. salarius. In this study, we performed a genomic analysis and ectoine identification of V. salarius. A high-quality draft genome with total size of 4.45 Mb and 4426 coding sequence (CDS) was characterized and then mapped into the Cluster of Orthologous Groups (COG) category. The genus Virgibacillus has an "open" pangenome type with total of 18 genomic islands inside the V. salarius 19.PP.SC1.6 genome. There were seven clusters of secondary metabolite-producing genes found, with a total of 80 genes classified as NRPS, PKS (type III), terpenes, and ectoine biosynthetic related genes. The ectoine gene cluster forms one operon consists of ectABC gene with 2190 bp gene cluster length, and is successfully characterized. The presence of ectoine in V. salarius was confirmed using UPLC-MS/MS operated in Multiple Reaction Monitoring (MRM) mode, which indicates that V. salarius has an intact ectoine gene clusters and is capable of producing ectoine as compatible solutes.

Physicochemical Characteristics and Antibacterial Activities of Freeze-Thawed Polyvinyl Alcohol/Andrographolide Hydrogels.

Andrographolide (AG) is one of the compounds in Andrographis paniculata, which has a high antibacterial activity. This paper reports the freeze-thaw method's use to synthesize polyvinyl alcohol (PVA) hydrogels loaded with AG and its characterization. From the morphological examination, the porosity of the PVA/AG hydrogel was found to increase with the increasing AG concentration. The swelling degree test revealed that the hydrogels' maximum swelling degrees were generally greater than 100%. The composite hydrogel with the highest fraction of andrographolide (PAG-4) showed greater weight loss than the hydrogel without AG (PAG-0). The molecular interaction between PVA and AG resulted in the narrowing of the band attributed to the O-H and C=O stretching bonds and the emergence of an amorphous domain in the composite hydrogels. The loading of AG disrupted the formation of hydroxyl groups in PVA and interrupted the cross-linking between PVA chains, which lead to the decrease of the compression strength and the crystallinity increased with increasing AG. The antibacterial activity of the composite hydrogel increased with increasing AG. The PAG-4 hydrogel had the highest antibacterial activity of 37.9 ± 4.6b %. Therefore, the PVA/AG hydrogel has the potential to be used as an antibacterial device.

The influence of extraction methods on rutin yield of cassava leaves (Manihot esculenta Crantz).

Rutin, a well-known bioflavonoid, was found abundantly in cassava leaves. In the present study, extraction techniques including maceration, boiling, reflux, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE) were optimised to increase the yield of rutin. Extraction parameters such as solvents, solid-liquid ratio, temperature, and time were optimised to give better extraction yields for each method. HPLC analysis showed a high content of rutin which is up to 2.4% per dry weight of cassava leaves. The extraction yields under optimised condition were found to be 16.00 ± 0.21, 20.38 ± 0.66, 22.33 ± 2.3, 24.49 ± 0.41, and 23.37 ± 1.00 g rutin per kg dry weight for maceration, boiling, reflux, UAE and MAE methods, respectively. Specifically, UAE reduced the extraction time to 90 min, using only 40-60% of aqueous ethanol. Meanwhile, MAE completed the extraction under 5 min and no significant differences in output was observed between the use of water and aqueous ethanol. Accordingly, with the extraction efficiency of up to 99 and 94%, respectively, both processes provided better results. The subsequent green purification using chilling method produced a typical cassava bioflavonoid containing 82% of rutin and 17% of nicotiflorin. This study informs a new abundant source of rutin and provides the optimum condition of extraction methods for high yield of rutin from cassava leaves.

The diversity of ursodeoxycholic acid precursors from bile waste of commercially available fishes, poultry and livestock in Indonesia

Ursodeoxycholic acid (UDCA), a secondary bile acid (BA), has been used as a drug to treat various liver diseases. UDCA is synthesised from cholic or chenodeoxycholic acid (CA/CDCA), two primary BAs frequently used as the starting materials. Nowadays, swine, cattle, and poultry bile are the main sources of those BAs. However, other commercial animals could be promising sources as well. We identified two livestock, two poultries, and eight fishes that are commercially cultivated in Indonesia. Four free BAs including CA, CDCA, deoxycholic acid (DCA), and lithocholic acid (LA) were identified for their occurrences using thin-layer chromatography and high-performance liquid chromatography. CA was detected in cow, duck, red tilapia, gourami, the common carp, and grouper, whereas CDCA was only detected in two poultries and the common carp. The occurrence of DCA was common and abundant in most tested animals. In contrast, the presence of LA was found to be very low in all samples. The biliary bile of tilapia has been found to contain a high abundance of free CA (43% of the total bile). A simple extraction was able to purify CA from biliary bile of tilapia. This is a new promising and competitive source of CA.

Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production.

BACKGROUND: Artemisinin, a sesquiterpene lactone endoperoxide isolated from the medicinal plant Artemisia annua L., is a choice and effective drug for malaria treatment. Due to the low yield of artemisinin in plants, there is a need to enhance the production of artemisinin from A. annua and biotechnological technique may be one of the methods that can be used for the purpose. AIM: To study the transformation efficiency of Agrobacterium tumefaciens in A. annua that could be applied to enhance the production of artemisinin by means of transgenic plants. SETTING AND DESIGNS: The factors influencing Agrobacterium-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain and effect of organosilicone surfactants. Three strains of A. tumefaciens, that is, LBA4404, GV1301, and AGL1 harboring the binary vector pCAMBIA 1303 have been used for transformation. The evaluation was based on transient ß-glucuronidase (GUS). MATERIALS AND METHODS: Plant cell cultures were inniatiated from the seeds of A. annua using the germination Murashige and Skoog medium. A. tumefaciens harboring pCAMBIA were tranformed into the leaves of A.annua cultures from 2-week-old-seedling and 2-month-old-seedling for 15 min by vacuum infiltration. Transformation efficiency was determinated by measuring of blue area (GUS expression) on the whole leaves explant using ImageJ 1.43 software. Two organosilicon surfactants, that is, Silwet L-77 and Silwet S-408 were used to improve the transformation efficiency. RESULTS: The transformation frequency with AGL1 strain was higher than GV3101 and LBA4404 which were 70.91, 49.25, and 45.45%, respectively. Effect of organosilicone surfactants, that is, Silwet L-77 and Silwet S-408 were tested on A. tumefaciens AGL1 and GV3101 for their level of transient expression, and on A. rhizogenes R1000 for its hairy root induction frequency. For AGL1, Silwet S-408 produced higher level of expression than Silwet L-77, were 2.3- and 1.3-fold, respectively. For GV3101, Silwet L-77 was still higher than Silwet S-408, were 1.5- and 1.4-fold, respectively. However, GV3101 produced higher levels of expression than AGL1. The area of GUS expression spots of AGL1, LBA4404, and GV3101 strains was 53.43%, 41.06%, and 30.51%, respectively. CONCLUSION: A. tumefaciens AGl1 strain was the most effective to be transformed in to A. annua than GV3101 and LBA4404 strain. Surfactant Silwet S-408 produced the highest efficiency of transformation.