Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Aloe emodin 3-O-glucoside inhibits cell growth and migration and induces apoptosis of non-small-cell lung cancer cells via suppressing MEK/ERK and Akt signalling pathways.

AIMS: Non-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer with a high mortality rate. Glycosylation of phenolic compounds may increase water-solubility and pharmacological activities and reduce the toxicity of aglycones. This study aimed to evaluate and compare the anticancer effect of aloe emodin 3-O-glucoside (AE3G) and its aglycone, aloe emodin (AE), against NSCLC. MAIN METHOD: A human adenocarcinoma cell line (A549) and other human non-small cell lung carcinoma cell lines (NCI-H460 cells and NCI-H1299 cells) and BALB/c nu/nu xenograft mice harbouring A549 cells were used as the NSCLC models. Inhibition of cell migration, disruption of mitochondrial membrane potential (MMP), DNA fragmentation, and expression levels of apoptotic proteins were measured by western blot, wound healing assay, JC-1 staining, or TUNEL staining. Histopathological changes in tumour tissues were observed by H&E and TUNEL staining. RESULTS: With no significant cytotoxicity against noncancerous cells (Vero cells), AE3G (5-50 µM) significantly and more effectively inhibited the growth, attachment, migration, Bcl-2 expression, and activation of MEK/ERK and Akt signalling proteins and induced cytochrome c release and Bax expression in A549 cells than AE. AE3G also significantly decreased the growth of other NSCLC cells, NCI-H460 cells and NCI-H1299 cells. AE3G suppressed the mRNA expression of matrix metalloproteinases, MMP2 and MMP9, and augmented the collapse of the mitochondrial MMP, cleavage of caspases (caspase 9, 8, and 3) and PARP, and DNA fragmentation. Intraperitoneal injection of AE3G (13 and 26 mg/kg/day) reduced the tumour volume and weight and induced apoptotic cell death in tumour tissues of xenograft NSCLC mice. SIGNIFICANCE: The present study demonstrated that AE3G significantly and more effectively diminished human NSCLC cell growth and migration by triggering mitochondria-dependent intrinsic apoptosis than AE, providing AE3G as a new potent candidate to prevent or treat human NSCLC.

UPLC-PDA coupled HR-TOF ESI/MS2 -based identification of derivatives produced by whole-cell biotransformation of epothilone A using Nocardia sp. CS692 and a cytochrome P450 overexpressing strain.

Epothilone A, a microtubule-stabilizing agent used as therapeutics for the treatment of cancers, was biotransformed into three metabolites using Nocardia sp. CS692 and recombinant Nocardia overexpressing a cytochrome P450 from Streptomyces venezuelae (PikC). Among three metabolites produced in the biotransformation reaction mixtures, ESI/MS2 analysis predicted two metabolites (M1 and M2) as novel hydroxylated derivatives (M1 is hydroxylated at the C-8 position and M2 is hydroxylated at C-10 position), each with an opened-epoxide ring in their structure. Interestingly, metabolite M3 lacks an epoxide ring and is known as deoxyepothilone A, which is also called epothilone C. Metabolite M1 was produced only in PikC overexpressing strain. The endogenous enzymes of Nocardia sp. catalyzed hydroxylation of epothilone A to produce metabolite M2 and removed epoxide ring to produce metabolite M3. All the metabolites were identified based on UV-vis analysis and rigorous ESI/MS2 fragmentation based on epothilone A standard. The newly produced metabolites are anticipated to display novel cytotoxic effects and could be subjects of further pharmacological studies.

Exploring the Nucleophilic N- and S-Glycosylation Capacity of Bacillus licheniformis YjiC Enzyme.

YjiC, a glycosyltransferase from Bacillus licheniformis, is a well-known versatile enzyme for glycosylation of diverse substrates. Although a number of O-glycosylated products have been produced using YjiC, no report has been updated for nucleophilic N-, S-, and C- glycosylation. Here, we report the additional functional capacity of YjiC for nucleophilic N- and S- glycosylation using a broad substrate spectrum including UDP-α-D-glucose, UDP-N-acetyl glucosamine, UDP-N-acetylgalactosamine, UDP-α-D-glucuronic acid, TDP-α-L-rhamnose, TDP-α-D-viosamine, and GDP-α-Lfucose as donor and various amine and thiol groups containing natural products as acceptor substrates. The results revealed YjiC as a promiscuous enzyme for conjugating diverse sugars at amine and thiol functional groups of small molecules applicable for generating glycofunctionalized chemical diversity libraries. The glycosylated products were analyzed using HPLC and LC/MS and compared with previous reports.

Biocatalytic Synthesis of Non-Natural Monoterpene O-Glycosides Exhibiting Superior Antibacterial and Antinematodal Properties.

A promiscuous Bacillus glycosyltransferase (YjiC) was explored for the enzymatic synthesis of monoterpene O-glycosides in vitro and in vivo. YjiC converted seven monoterpenes into 41 different sugar-conjugated novel glycoside derivatives. The whole-cell biotransformation of the same set of monoterpenes exhibited robust enzyme activity to synthesize O-glucosyl derivatives from Escherichia coli. These newly synthesized selected monoterpene-O-glucosyl derivatives exhibited enhanced antibacterial activities against human pathogenic bacteria and antinematodal activities against pine wood nematode Bursaphelenchus xylophilus.

Correction to: Cascade biocatalysis systems for bioactive naringenin glucosides and quercetin rhamnoside production from sucrose.

The name of the author "Yamaguchi Tokutaro" is incorrect for the first and last name has been interchanged. The correct presentation is "Tokutaro Yamaguchi".

Cascade biocatalysis systems for bioactive naringenin glucosides and quercetin rhamnoside production from sucrose.

Two sustainable and cost-effective cascade enzymatic systems were developed to regenerate uridine diphosphate (UDP)-α-D-glucose and UDP-ß-L-rhamnose from sucrose. The systems were coupled with the UDP generating glycosylation reactions of UDP sugar-dependent glycosyltransferase (UGT) enzymes mediated reactions. As a result, the UDP generated as a by-product of the GT-mediated reactions was recycled. In the first system, YjiC, a UGT from Bacillus licheniformis DSM 13, was used for transferring glucose from UDP-α-D-glucose to naringenin, in which AtSUS1 from Arabidopsis thaliana was used to synthesize UDP-α-D-glucose and fructose as a by-product from sucrose. In the second system, flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from A. thaliana was used to transfer rhamnose from UDP-ß-L-rhamnose to quercetin, in which AtSUS1 along with UDP-ß-L-rhamnose synthase (AtRHM1), also from A. thaliana, were used to produce UDP-ß-L-rhamnose from the same starter sucrose. The established UDP recycling system for the production of naringenin glucosides was engineered and optimized for several reaction parameters that included temperature, metal ions, NDPs, pH, substrate ratio, and enzymes ratio, to develop a highly feasible system for large-scale production of different derivatives of naringenin and other natural products glucosides, using inexpensive starting materials. The developed system showed the conversion of about 37 mM of naringenin into three different glucosides, namely naringenin, 7-O-ß-D-glucoside, naringenin, 4'-O-ß-D-glucoside, and naringenin, 4',7-O-ß-D-diglucoside. The UDP recycling (RCmax) was 20.10 for naringenin glucosides. Similarly, the conversion of quercetin to quercetin 7-O-α-L-rhamnoside reached a RCmax value of 10.0.

Biotechnological Advances in Resveratrol Production and its Chemical Diversity.

The very well-known bioactive natural product, resveratrol (3,5,4'-trihydroxystilbene), is a highly studied secondary metabolite produced by several plants, particularly grapes, passion fruit, white tea, and berries. It is in high demand not only because of its wide range of biological activities against various kinds of cardiovascular and nerve-related diseases, but also as important ingredients in pharmaceuticals and nutritional supplements. Due to its very low content in plants, multi-step isolation and purification processes, and environmental and chemical hazards issues, resveratrol extraction from plants is difficult, time consuming, impracticable, and unsustainable. Therefore, microbial hosts, such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum, are commonly used as an alternative production source by improvising resveratrol biosynthetic genes in them. The biosynthesis genes are rewired applying combinatorial biosynthetic systems, including metabolic engineering and synthetic biology, while optimizing the various production processes. The native biosynthesis of resveratrol is not present in microbes, which are easy to manipulate genetically, so the use of microbial hosts is increasing these days. This review will mainly focus on the recent biotechnological advances for the production of resveratrol, including the various strategies used to produce its chemically diverse derivatives.