Nguyen Huy Thuan

Recent advances in biochemistry and biotechnological synthesis of avermectins and their derivatives

Avermectins (AVMs), produced by Streptomyces avermitilis MA-4680 (or ATCC 31267, NRRL 8165, NCBIM 12804), are 16-member macrocylic lactones that play very important functions as bactericidal and antiparasitic agents against nematodes and anthropods, as well as Mycobacterium tuberculosis H37Rv. Since its discovery in 1975, use of AVM has been widely spreading around the globe. To date, the whole genome sequence of S. avermitilis K139 has been acquired, in which the AVM biosynthetic gene cluster was the most highly investigated to mine the genes responsible for functional as well as regulatory roles. Therefore, significant progress has been achieved for understanding and manipulating the biosynthesis, improved production, regulation mechanism, side effects, as well as the resistance of AVMs and their derivatives. These findings will facilitate further strain improvement and biosynthesis of novel derivatives bearing stable and improved biological activities, as well as overcoming the resistance mechanism to open up a bright period for these compounds. In this review, we have summarized and analyzed the update in advanced progress in biochemistry and biotechnological approaches used for the production of AVMs and their derivatives.

Metabolic engineering of E. coli for the production of isoflavonoid‐7‐O‐methoxides and their biological activities

Isoflavonoid representatives such as genistein and daidzein are highly potent anticancer, antibacterial, and antioxidant agents. It have been demonstrated that methylation of flavonoids enhanced the transporting ability, which lead to facilitated absorption and greatly increased bioavailability. In this paper, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12‐derived metK encoding S‐adenosine‐l‐methionine (SAM) synthase (accession number: K02129) for enhancement of SAM as a precursor and Streptomyces avermitilis originated SaOMT2 (O‐methyltransferase, accession number: NP_823558) for methylation of daidzein and genistein as preferred substrates. The formation of desired products via biotransformation including 4′‐O‐methyl‐genistein and 4′‐O‐methyl‐daidzein was confirmed individually by using chromatographical methods such as high‐performance liquid chromatography, liquid chromatography/time‐of‐flight/mass spectrometry (LC‐TOF‐MS), and nuclear magnetic resonance (NMR), and NMR (1H and 13C). Furthermore, substrates concentration, incubation time, and media parameters were optimized using flask culture. Finally, the most fit conditions were applied for fed‐batch fermentation with scale‐up to 3 L (working volume) to obtain the maximum yield of the products including 164.25 µM (46.81 mg/L) and 382.50 µM (102.88 mg/L) for 4′‐O‐methyl genistein and 4′‐O‐methyl daidzein, respectively. In particular, potent inhibitory activities of those isoflavonoid methoxides against the growth of cancer line (B16F10, AGS, and HepG2) and human umbilical vein endothelial cells were investigated and demonstrated. Taken together, this research work described the production of isoflavonoid‐4′‐O‐methoxides by E. coli engineering, improvement of production, characterization of produced compounds, and preliminary in vitro biological activities of the flavonoids being manufactured.

Screening of Strong 1-Aminocyclopropane-1-Carboxylate Deaminase Producing Bacteria for Improving the Salinity Tolerance of Cowpea

Aim: To isolate rhizobacteria containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and evaluate the ability of selected bacteria for improving the growth of cowpea seedlings under salt stress conditions. Methods: This study isolates salt-tolerant rhizobacteria which have strong ability to produce ACC deaminase and the phytohormone indol-3-acetic acid (IAA). Inoculation experiments with selected bacteria strains were used to verify the plant growth promoting activity of bacteria under salt stress conditions. Result: Two isolates belong to Enterobacter cloacae and one isolate belongs to Pseudomonas sp. have been identified. Those rhizobacteria were found to be highly salt-tolerant at salinity level up to 10% NaCl. The selected bacterial strains were also capable to produce and secrete large amounts of ACC deaminase and the phytohormone IAA into the growth medium. Cowpea plants inoculated with ST3 strain revealed a significant increase in shoot length and shoot fresh weight over uninoculated control at the salinity level of 1.5% NaCl. Conclusion: Three rhizobacterial strains belonging to the genera Enterobacter and Pseudomonas have been isolated. All three bacterial strains were identified as moderate halophiles and they can produce high levels of ACC deaminase and IAA. The strain Pseudomonas sp. ST3 showed the possible ability to promote the growth of cowpea under salt stress conditions.

Characterization of sterol glucosyltransferase from Salinispora tropica CNB-440: Potential enzyme for the biosynthesis of sitosteryl glucoside

A sterol glucosyltransferase-encoded gene was isolated from Salinispora tropica CNB-440, a marine, sediment-dwelling, Gram positive bacterium that produces the potent anticancer compound, salinosporamide A. The full-length gene consists of 1284 nucleotides and encodes 427 amino acids with a calculated mass of 45.65kDa. The gene was then cloned and heterologously expressed in Escherichia coli BL21(DE3). The amino acid sequence shares 39% similarity with the glycosyltransferase from Withania somnifera, which belongs to glycosyltransferase family 1. Enzyme reactions were carried out with the various free sterols (acceptor) and NDP-sugars (donor). The purified protein only showed activity for glucosylation of β-sitosterol with UDP-D-glucose and TDP-D-glucose donors, and optimal activity at pH 7.5 and 37°C. Among these two donors, UDP-D-glucose was preferred.

Saccharopolyspora Species: Laboratory Maintenance and Enhanced Production of Secondary Metabolites

Saccharopolyspora spp. are aerobic, Gram‐positive, non‐acid‐fast, and non‐motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for the laboratory maintenance of Saccharopolyspora species, including growth in liquid medium, growth on solid agar, long‐term storage, and generation of a higher producer strain by mutagenesis. Saccharopolyspora spinosa ATCC 49460 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Saccharopolyspora spp. Saccharopolyspora spinosa is a producer of spinosad, a prominent insecticide with selective activity against various insects.