p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation

Abstract

Actinosynnema species produce diverse natural products with important biological activities, which represent an important resource of antibiotic discovery. Advances in genome sequencing and bioinformatics tools have accelerated the exploration of the biosynthetic gene clusters (BGCs) encoding natural products. Herein, the completed BGCs of dnacin B1 were first discovered in two Actinosynnema pretiosum subsp. auranticum strains DSM 44131T (hereafter abbreviated as strain DSM 44131T) and X47 by comparative genome mining strategy. The BGC for dnacin B1 contains 41 ORFs and spans a 66.9 kb DNA region in strain DSM 44131T. Its involvement in dnacin B1 biosynthesis was identified through the deletion of a 9.7 kb region. Based on the functional gene analysis, we proposed the biosynthetic pathway for dnacin B1. Moreover, p-amino-phenylalanine (PAPA) unit was found to be the dnacin B1 precursor for the quinone moiety formation, and this was confirmed by heterologous expression of dinV, dinE and dinF in Escherichia coli. Furthermore, nine potential PAPA aminotransferases (APAT) from the genome of strain DSM 44131T were explored and expressed. Biochemical evaluation of their amino group transformation ability was carried out with p-amino-phenylpyruvic acid (PAPP) or PAPA as the substrate for the final product formation. Two of those, APAT4 and APAT9, displayed intriguing aminotransferase ability for the formation of PAPA. The proposed dnacin B1 biosynthetic machinery and PAPA biosynthetic investigations not only enriched the knowledge of tetrahydroisoquinoline (THIQ) biosynthesis, but also provided PAPA building blocks to generate their structurally unique homologues.

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

“…It was considered to activate the carboxyl group of AHBA to the phosphoryl moiety by the connection of AMP, and then to transfer the AHBA-AMP substrate to CoA [15,19]. To gain genetic insight into the involvement of mitE in mitomycin C biosynthesis, we carried out a direct in-frame deletion of mitE on pJQK401 by PCR technology [20,21]. The affording ∆mitE recombinants, named DJ01A-C, were confirmed by PCR and sequencing analysis (Figure S1).…”

Two strategies to improve the supply of PKS extender units for ansamitocin P-3 biosynthesis by CRISPR–Cas9

“…Actinosynnema spp. with a truly high guanine-cytosine (GC) content (73.9%) is well known for producing ansamitocin, as well as a variety of secondary metabolites such as actinosynneptide and dnacin (Martin et al 2014 ; Wang et al 2019a ; Zhong et al 2019 ; Kashyap et al 2019 ; Hu et al 2020 ). Actinosynnema genome editing relies on the homologous recombination either via single or double-crossover events, hence its genetic modification is time- and labor-intensive (Yu et al 2002 ; Fan et al 2016b ; Ning et al 2017 ; Wang et al 2020b ).…”