is a diterpene natural sweetener isolated in a trace amount from the leaves of Stevia rebaudiana. Selective glycosylation of rubusoside, a natural product abundantly presented in various plants, is a feasible approach for the biosynthesis of rebaudioside KA. In this study, bacterial glycosyltransferase OleD was identified to selectively transfer glucose from UDPG to 2′-hydroxyl group with a β-1,2 linkage at 19-COO-β-D-glucosyl moiety of rubusoside for the biosynthesis of rebaudioside KA. To eliminate the use of UDPG and improve the productivity, a UDPG regeneration system was constructed as an engineered Escherichia coli strain to couple with the glycosyltransferase. Finally, rubusoside at 22.5 g/L (35.0 mM) was completely converted to rebaudioside KA by the whole cells without exogenous addition of UDPG. This study provides an efficient and scalable method for highly selective biosynthesis of rebaudioside KA.

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Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrugresistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N-and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs.IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides.

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Guided by an MS/MS-based molecular networking, six undescribed cassane diterpenoids and three known ones were isolated and identified from the seeds of Caesalpinia sappan. Their structures were unequivocally elucidated by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. Cytotoxic evaluation showed that phanginin JA exhibited significant antiproliferative activities against human non-small cell lung cancer (A549) cells with IC 50 values of 16.79 � 0.83 μM. Further flow cytometry analysis revealed that phanginin JA could exert apoptotic effect of A549 cells by arresting cell cycle in G0/G1 phase.