Micropyrones A and B, two new α-pyrones from the actinomycete Microbacterium sp. GJ312 isolated from Glycyrrhiza uralensis Fisch

Abstract Two new α-pyrones, micropyrones A (1) and B (2), along with four known γ-pyrones, nocapyrone D (3), nocapyrone A (4), marinactinone A (5), and nocapyrone H (6), were isolated from the culture extract of actinomycete Microbacterium sp. GJ312, which was isolated from Glycyrrhiza uralensis. The structures of these compounds were identified by analysis of spectral data. They are the first α- and γ-pyrones reported from the genus Microbacterium. The antibacterial activity of all compounds against Staphylococcus aureus and methicillin resistant S. aureus was evaluated. However, none of them showed significant activity. This study represents the first phytochemical example of a Glycyrrhiza-derived actinomycete. Graphical Abstract


Introduction
The genus Microbacterium belongs to the family Microbacteriaceae. Members of this genus can be found in a wide range of environments including plants, soil, insects, human clinical specimens, marine environments, etc. Previous phytochemical investigation of the genus Microbacterium has led to the isolation of glycoglycerolipids (Wicke et al. 2000), peptaibols (Liu et al. 2015), indole (Niu et al. 2017), and cyclic peptides (Niu et al. 2017).
Plants of the genus Glycyrrhiza are belonging to the most widely applied herbs of traditional medicine in both Eastern and Western countries (Wang et al. 2020). The phytochemical investigation on Glycyrrhiza plants was extensively pushed on, and over 400 compounds have been isolated from this genus (Wang et al. 2020;Bao et al. 2021). However, the phytochemical evaluation on Glycyrrhiza-derived endophytes was limited. Up to now, a cyclic peptide (Wang et al. 2012) and a stearolactone (Wei et al. 2007) were isolated from Glycyrrhiza-derived rhizobia; four naphthoquinones (Shah et al. 2017) and two thiodiketopiperazine derivatives (Arora et al. 2016) were found in Glycyrrhiza-derived fungi. There is no chemical report about Glycyrrhiza-derived actinomycetes. Herein, we report the isolation, structural elucidation, and antibacterial activity of two new a-pyrones, micropyrones A (1) and B (2), along with four known c-pyrones, nocapyrone D (3) (Schneemann et al. 2010), nocapyrone A (4) (Schneemann et al. 2010), marinactinone A (5) (Wang et al. 2011), and nocapyrone H (6) (Lin et al. 2013), from the culture extract of an actinomycete Microbacterium sp. GJ312, which was isolated from G. uralensis.

Results and discussion
Micropyrone A (1) was assigned a molecular formula C 15 H 24 O 4 by (þ)-ESI-HRMS (m/z [M þ Na] þ 291.1572, calculated for C 15 H 24 O 4 Na þ : 291.1567, see Figure S7). Based on the small specific rotation value of [a] D 20 ¼ þ2.3 (c 0.1, CH 2 Cl 2 ), micropyrone A (1) might be a racemic mixture. Due to the limited amount, the racemic mixture was difficult to separate into the two enantiomers. The 13 C NMR spectrum (Table S1 & Figure  S3) showed signals for five nonprotonated carbons at d C 168.3, 166.2, 158.8, 109.4, and 109.1, two methines, three methylenes, five methyls including four ones at d C 18.9, 17.0, 10.2 and 10.1, and a methoxyl at d C 60.2. Above listed 13 C NMR data were characteristic for a-pyrone and were closely similar to those of nocapyrone R (Kim et al. 2014). After detailed analysis, the key difference was that a methylene in nocapyrone R was replaced by an oxymethine (d H 3.38 and d C 76.4) in 1. The heteronuclear multiple bond correlation (HMBC) spectrum (Figures S1 & S5) of 1 displayed correlations from two terminal methyls (CH 3 -12 and CH 3 -13) to oxymethine (d C 76.4), which suggested that oxymethine was located at C-10. Consequently, micropyrone A (1) was elucidated as 3,5-dimethyl-4-methoxyl-6-(4-hydroxy-5-methyl)hexyl-a-pyrone as shown in Figure 1. Micropyrone B (2) had the same molecular formula as micropyrone A (1) on the basis of (þ)-ESI-HRMS data ( Figure S12). All 15 carbon signals were well observed in the 13 C NMR spectrum (Table S1 & Figure S9), including six nonprotonated carbons, four methylenes, four methyls, and a methoxyl. It was also an a-pyrone. The 13 C NMR data of micropyrone B (2) were closely similar to those of micropyrone A (1). However, two doublets for two methyls of 1 in the 1 H NMR spectrum were absent in 2. Correspondingly, a singlet at d H 1.23 integrating for two methyls was observed in 2. These methyl protons displayed HMBC correlations (Figures S1 & S10) with a nonprotonated carbon at d C 70.8 and a methylene at d C 43.4, which indicated that a hydroxyl was attached at C-11. Therefore, micropyrone B (2) was elucidated as 3,5-dimethyl-4methoxyl-6-(5-hydroxy-5-methyl)hexyl-a-pyrone.
Four known compounds were identified as nocapyrone D (3) (Schneemann et al. 2010), nocapyrone A (4) (Schneemann et al. 2010), marinactinone A (5) (Wang et al. 2011), and nocapyrone H (6) (Lin et al. 2013) by comparing their spectroscopic data with literature values. The chemical structures of compounds 1-6 are shown in Figure  1. All compounds are characterized as dimethylated and methoxylated pyrones containing an aliphatic chain. Compounds 1 and 2 are a-pyrones, while 3-6 are c-pyrones. This class of aand c-pyrones are widespread in Nocardiopsis species (Schneemann et al. 2010;Lin et al. 2013;Kim et al. 2014). Notably, their occurrence in Microbacterium genus was not reported to the best of our knowledge. This study represents the first phytochemical example of a Glycyrrhiza-derived actinomycete.
Staphylococcus aureus (SA) and methicillin resistant S. aureus (MRSA) were inhibited by the extract of strain GJ312 ( Figure S13). Compounds 1-6 were evaluated for antibacterial activity against SA and MRSA. However, none of these compounds showed significant activity against SA and MRSA (MIC > 100 lg/mL). Kanamycin (MIC ¼ 1.6 lg/ mL) and vancomycin (MIC ¼ 1.6 lg/mL) were chosen as the standard drugs against SA and MRSA, respectively.

Bacterial material and fermentation
The strain GJ312 was isolated from a stem sample of Glycyrrhiza uralensis collected from Tianshui County, Gansu Province, China. We analyzed the partial 16S rRNA sequence similarity by blasting GenBank database. The highest similar result was Microbacterium saccharophilum K-1, and the other similar strains were also within the genus Microbacterium, so we named our strain GJ312 as Microbacterium sp. GJ312. The strain was fermented in TSA medium by adding 16 g malt extract and 4 g yeast extract per liter on a rotary shaker with 150 rpm at 28 C for 8 days. The TSA medium was composed of 15 g tryptone, 5 g soybean peptone, 5 g NaCl, and 1 L tap water.

Antibacterial assay
The antimicrobial activity against MRSA and SA was determined according to previously reported methods of the microplate Alamar Blue assay (Fu et al. 2014).

Disclosure statement
No potential conflict of interest was reported by the authors.