Xuhua Mo

Phosphopantetheinyl transferases (PPTases) catalyze the phosphopantetheinylation of carrier proteins involved in primary and secondary metabolism. The overexpression of PPTase has been demonstrated to be an effective strategy for activating cryptic biosynthetic pathways related to secondary metabolites. Over 30 biosynthetic gene clusters encoding polyketide or nor-ribosomal peptide compounds have been identified in Saccharothrix syringae NRRL B-16468. However, with the exception of nocamycin, all other metabolites encoded by these clusters remain unidentified. In this study, we first identified the PPTase genes present in the genome of S. syringae NRRL B-16468. The subsequent overexpression of the Sfp-type PPTase gene QFZ22139 led to the generation of a novel compound saccharoridine. Saccharoridine was isolated and characterized as a cyclopentane[c]pyridine based on high-resolution mass spectrometry and comprehensive nuclear magnetic resonance spectroscopic analysis. Additionally, saccharoridine exhibited no antibacterial or antitumor activity. This study presents the first cyclopentane[c]pyridine compound isolated from a bacterial source.

Terpenes make up a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity has been largely unexplored. Here, we report the discovery of grimophan, a C₁₇ terpene featuring a rare deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of noncanonical terpenes. These enzymes thus constitute valuable tools for enhancing biotechnological production levels of noncanonical C₁₆ and C₁₇ terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the observed Asp-rich motif D₉₂DMPLG₉₇ to map the effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to noncanonical C₁₇ terpenes and provides a basis for their targeted discovery by genome mining and heterologous expression and for engineering C₁₇ terpene structural frameworks.

α-Glucosidase inhibitory activity-guided isolation led to the discovery of ten alkaloids from the medicinal mushroom Vanderbylia robiniophila SY636, including six new compounds ((+)-1a, (-)-1b, (+)-2a, (-)-2b, 3, and (+)-4a) and four newly reported natural products ((-)-4b and 5-7). Compounds (+)-1a, (-)-1b, (+)-2a, and (-)-2b possess a unique lactam-furan ring system, whereas 3 features a rare tetrahydrobenzo[c]oxepine-furan ring system. (+)-1a, (-)-1b, (+)-4a, and (-)-4b exhibited notable α-glucosidase inhibitory activities, with IC₅₀ values of 160-330 μM (acarbose as the positive control, IC₅₀ = 660 μM). Guided by docking results of bioactive compounds with α-glucosidase, derivatives D1-D28 were designed and synthesized, among which D15 and D19 exhibited stronger activities (IC₅₀ = 28 and 130 μM). Molecular docking and kinetic studies suggested that D15 and D19 interact with α-glucosidase at a single binding site, exhibiting an uncompetitive or mixed type of inhibition mechanism. D19 also exhibited notable antioxidant activity (IC₅₀ = 35 μM), comparable to l-ascorbic acid, suggesting its dual role in combating the development of diabetes. This study provides a foundation for the development of natural medicines from V. robiniophila and offers valuable guidance for future structural optimization toward the discovery of more potent and safer antidiabetic agents.

Terpenes are a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity is largely unexplored. Here, we report the discovery of grimophan, an unprecedented C17 terpene featuring a deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of novel methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of non-canonical terpenes. These enzymes thus constitute valuable tools for increasing biotechnological production levels of terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the conserved Asp-rich motif D92DMPLG97 to map effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to non-canonical C17 terpenes, provides a basis for their targeted discovery by genome mining and heterologous expression, and for engineering C17 terpene structural frameworks.

Methylorubrum extorquens AM1, a native formate-utilizing bacterium, has exhibited limited capacity to tolerate formate. In this study, we employed an adaptive laboratory evolution (ALE) strategy to develop an evolved strain FT3 derived from M. extorquens AM1, with enhanced formate tolerance. When cultivated with a mixture of carbon sources containing 90 mM formate and 30 mM methanol, the FT3 strain exhibited 5.3 times higher optical density (OD ₆₀₀ ) compared to the parental strain. FT3 strain was shown to efficiently utilize both methanol and formate in experiments using ¹³ C-labeled carbon sources. Furthermore, the mechanism underlying the enhanced formate tolerance in FT3 strain was investigated through a combination of DNA re-sequencing, transcriptome analysis, and ALE-inspired gene manipulation experiments. The FT3 strain was identified as a hypermutant, and its enhanced formate tolerance was attributed to increased formate transport, an improved methanol oxidation pathway, and enhanced formate oxidation and assimilation pathways. In addition, gene overexpression experiments indicated the involvement of genes META1_0287* , META1_3027 , META1_3028 , META1_3029 , META1_1261 , META1_1418 , and META1_2965 in formate tolerance. Notably, the addition of formate resulted in a significant improvement in the generation of NADH and NADPH in the FT3 strain. Moreover, using the FT3 strain as a chassis, an improved 3-hydroxypropionic acid (3-HP) production of 2.47 g/L through fed-batch fermentation was achieved. This study provides an important foundation for further engineering of the evolved M. extorquens strain as an efficient platform for the co-utilization of methanol and formate in the production of reduced chemicals. IMPORTANCE: In the present study, we successfully obtained an evolved strain FT3 derived from M. extorquens AM1 with high formate tolerance using the ALE strategy. The FT3 strain was identified as a hypermutant, with its enhanced formate tolerance attributed to increased formate transport, an improved methanol oxidation pathway, and enhanced formate oxidation and assimilation pathways. Through transcriptome analysis and ALE-inspired gene manipulation experiments, we identified several genes that contribute to the FT3 strain’s tolerance to formate. The enhanced levels of reducing equivalents and the increased tolerance to 3-HP make FT3 a suitable chassis for 3-HP production, achieving an improved yield of 2.47 g/L through fed-batch fermentation. This study provides an important foundation for further engineering of the evolved M. extorquens strain as an efficient platform for the co-utilization of methanol and formate in the production of reduced chemicals.

Bioactivity screening revealed that the antifungal activities of EtOAc extracts from coculture broths of Trametes versicolor SY630 with either Vanderbylia robiniophila SY341 or Ganoderma gibbosum SY1001 were significantly improved compared to that of monocultures. Activity-guided isolation led to the discovery of five aromatic compounds (1-5) from the coculture broth of T. versicolor SY630 and V. robiniophila SY341 and two sphingolipids (6 and 7) from the coculture broth of T. versicolor SY630 and G. gibbosum SY1001. Tramevandins A-C (1-3) and 17-ene-1-deoxyPS (6) are new compounds, while 1-deoxyPS (7) is a new natural product. Notably, compound 2 represents a novel scaffold, wherein the highly modified p-terphenyl bears a benzyl substituent. The absolute configurations of those new compounds were elucidated by X-ray diffraction, ECD calculations, and analysis of physicochemical constants. Compounds 1, 2, and 5-7 exhibited different degrees of antimicrobial activity, and the antifungal activities of compounds 6 and 7 against Candida albicans and Cryptococcus neoformans are comparable to those of fluconazole, nystatin, and sphingosine, respectively. Transcriptome analysis, propidium iodide staining, ergosterol quantification, and feeding assays showed that the isolated sphingolipids can extensively downregulate the late biosynthetic pathway of ergosterol in C. albicans, representing a promising mechanism to combat antibiotic-resistant fungi.

Methylobacterium species, the representative bacteria distributed in phyllosphere region of plants, often synthesize carotenoids to resist harmful UV radiations. Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C₃₀ backbone. However, its exact structure remains unknown. In the present study, the carotenoid produced by M. extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopenedioc acid (1), a glycosylated C₃₀ carotenoid. Furthermore, the genes related to the C₃₀ carotenoid synthesis were investigated. Squalene, the precursor of the C₃₀ carotenoid, is synthesized by the co-occurrence of META1p1815, META1p1816 and META1p1817. Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1. By using gene deletion and gene complementation experiments, the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4′-diapolycopene-4,4′-dioic acid to carotenoid 1. In conclusion, the structure and biosynthetic genes of carotenoid 1 produced by M. extorquens AM1 were firstly characterized in this work, which shed lights on engineering M. extorquens AM1 for producing carotenoid 1 in high yield.

In our previous work, postredienes A-C, three unusual linear sesterterpenes with high antifungal activities, were isolated from Pleurotus ostreatus SY10 when cocultured with Trametes robiniophila SY636. However, their titers were low, and exploration of newly biosynthesized trace analogues is required. Herein, genome mining analysis predicted that 17 gene clusters are involved in terpenoid biosynthesis in P. ostreatus. Thus, coculture conditions for strains SY10 and SY636 were optimized using a single-factor test and Box-Behnken design. As a result, the titers of postredienes A-C were increased by over 2.5-fold, reaching 1.28 to 8.40 mg/L. Moreover, five new terpenoids, named postredienes D-H (1-5), were successfully isolated. Compound 1 exhibited activities against the human pathogenic fungi Candida albicans and Cryptococcus neoformans comparable to those of amphotericin B. Compound 2 represents a novel sesterterpene with a five-membered ring at C-7. The absolute configurations of 1-5 were elucidated by making the methoxyphenylacetic acid esters and acetonide derivatives, combined with ECD and NMR calculation. Two potential gene clusters and relevant biosynthetic pathways for 1-5 were subsequently proposed based on real-time reverse transcription-quantitative PCR (RT-qPCR) analysis. The current study provides new insights into the research of terpenoid biosynthesis genes in P. ostreatus and other basidiomycetes.

Over 30 biosynthetic gene clusters for natural tetramate have been identified. This highlight reviews the biosynthetic strategies for formation of tetramic acid unit for the first time, discussing the individual molecular mechanism in detail.