Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

Cytochrome P450 monooxygenase constitutes a significant group of oxidative enzymes that can introduce an oxygen atom in a high regio- and stereo-selectivity mode. We used the Bacillus megaterium cytochrome P450 BM3 (CYP450 BM3) and its variants namely mutant 13 (M13) and mutant 15 (M15) for the hydroxylation of diverse class of flavonoids. Among 20 flavonoids, maximum seven flavonoids were hydroxylated by the variants while none of these molecules were accepted by CYP450 BM3 in in vitro reaction. Moreover, M13 exhibited higher conversion of substrates than M15 and CYP450 BM3 enzymes. We found that M13 carried out regiospecific 3ʹ-hydroxylation reaction of naringenin with the highest conversion among all the tested flavonoids. The apparent K m and k cat values of M13 for naringenin were 446 µM and 1.955 s−1, respectively. In whole-cell biotransformation experiment with 100 µM of naringenin in M9 minimal medium with 2 % glucose in shake flask culture, M13 showed 2.14- and 13.96-folds higher conversion yield in comparison with M15 (16.11 %) and wild type (2.47 %). The yield of eriodictyol was 46.95 µM [~40.7 mg (13.5 mg/L)] in a 3-L volume lab scale fermentor at 48 h in the same medium exhibiting approximately 49.81 % conversion of the substrate. In addition, eriodictyol exhibited higher antibacterial and anticancer potential than naringenin, flavanone and hesperetin. We elucidated that eriodictyol being produced from naringenin using recombinant CYP450 BM3 and its variants from B. megaterium, which shows an approach for the production of important hydroxylated compounds of various polyphenols that may span pharmaceutical industries.

Ginsenosides are a group of glycosylated triterpenes isolated from Panax species. Ginsenosides are promising candidates for the prevention and treatment of cancer as well as food additives. However, owing to a lack of efficient approaches for ginsenoside production from plants and chemical synthesis, ginsenosides may not yet have reached their full potential as medicinal resources. In recent years, an alternative approach for ginsenoside production has been developed using the model yeast Saccharomyces cerevisiae and non-conventional yeasts such as Yarrowia lipolytica and Pichia pastoris. In this review, various metabolic engineering strategies, including heterologous gene expression, balancing, and increasing metabolic flux, and enzyme engineering, have been described as recent advanced engineering techniques for improving ginsenoside production. Furthermore, the usefulness of a systems approach and fermentation strategy has been presented. Finally, the present challenges and future research direction for industrial cell factories have been discussed.

Umbelliferone, also known as 7-hydroxycoumarin, is a phenolic metabolite found in many familiar plants. Its derivatives have been shown to have various pharmacological and chemo-preventive effects on human health. A uridine diphosphate glycosyltransferase YjiC from Bacillus licheniformis DSM 13, a cytochrome P450BM3 (CYP450 BM3) variant namely mutant 13 (M13) from Bacillus megaterium, and an O-methyltransferase from Streptomyces avermitilis (SaOMT2) were used for modifications of umbelliferone. Three umbelliferone derivatives (esculetin, skimmin, and herniarin) were generated through enzymatic and whole cell catalysis. To improve the efficiencies of biotransformation, different media, incubation time and concentration of substrate were optimized and the production was scaled up using a 3-L fermentor. The maximum yields of esculetin, skimmin, and herniarin were 337.10 μM (67.62%), 995.43 μM (99.54%), and 37.13 μM (37.13%), respectively. The water solubility of esculetin and skimmin were 1.28-folds and 3.98-folds as high as umbelliferone, respectively, whereas herniarin was 1.89-folds less soluble than umbelliferone. Moreover, the antibacterial and anticancer activities of herniarin showed higher than umbelliferone, esculetin and skimmin. This study proves that both native and engineered enzymes could be employed for the production of precious compounds via whole cell biocatalysis. We successfully produced three molecules herniarin, esculetin and skimmin in practical amounts and their antibacterial and anticancer properties were accessed. One of the newly synthesized molecules the present research suggests that the combinatorial biosynthesis of different biosynthetic enzymes could rapidly promote to a novel secondary metabolite.

Ginsenosides are major bioactive compounds present in the Panax species. Ginsenosides exhibit various pharmaceutical properties, including anticancer, anti-inflammatory, antimetastatic, hypertension, and neurodegenerative disorder activities. Although several commercial products have been presented on the market, most of the current chemical processes have an unfriendly environment and a high cost of downstream processing. Compared to plant extraction, microbial production exhibits high efficiency, high selectivity, and saves time for the manufacturing of industrial products. To reach the full potential of the pharmaceutical resource of ginsenoside, a suitable microorganism has been developed as a novel approach. In this review, cell biological mechanisms in anticancer activities and the present state of research on the production of ginsenosides are summarized. Microbial hosts, including native endophytes and engineered microbes, have been used as novel and promising approaches. Furthermore, the present challenges and perspectives of using microbial hosts to produce ginsenosides have been discussed.