All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10–C25) terpenes that are formed from polyisoprenyl diphosphates2–4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.

With the advent of the genomics era, heterologous gene expression has been used extensively as a means of accessing natural products (NPs) from environmental DNA samples. However, the heterologous production of NPs often has very low efficiency or is unable to produce targeted NPs. Moreover, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in native producers, especially in the cases of genome mining, it is also difficult to rationally and systematically engineer synthetic pathways to improved NPs biosynthetic efficiency. In this study, various strategies ranging from heterologous production of a NP to subsequent application of omics-guided synthetic modules optimization for efficient biosynthesis of NPs with complex structure have been developed. Heterologous production of spinosyn in Streptomyces spp. has been demonstrated as an example of the application of these approaches. Combined with the targeted omics approach, several rate-limiting steps of spinosyn heterologous production in Streptomyces spp. have been revealed. Subsequent engineering work overcame three of selected rate-limiting steps, and the production of spinosad was increased step by step and finally reached 1460 μg/L, which is about 1000-fold higher than the original strain S. albus J1074 (C4I6-M). These results indicated that the omics platform developed in this work was a powerful tool for guiding the rational refactoring of heterologous biosynthetic pathway in Streptomyces host. Additionally, this work lays the foundation for further studies aimed at the more efficient production of spinosyn in a heterologous host. And the strategy developed in this study is expected to become readily adaptable to highly efficient heterologous production of other NPs with complex structure.

ObjectiveThis study was carried out to investigate the possible application of Broussonetia papyrifera (B. papyrifera) silage as a functional feeding stuff in dairy cattle.MethodsSeventy-two Holstein cows were divided into four groups randomly and allocated to 6 pens with 3 individuals in each group and fed the original total mixed ratio (TMR) in the dairy farm or the new TMR with 5%, 10%, and 15% B. papyrifera silage, separately. Feed intake were recorded, milk and blood samples were collected, and milk composition, blood metabolites and milk fatty acids composition were measure at the end of the experiment.ResultsDry matter intake of cows decreased when they fed on diet with B. papyrifera, but no differences were observed in body condition score, milk yield, milk protein and lactose, feed efficiency and serum metabolites between groups. Both 10% or 15% of B. papyrifera silage in the diet significantly increased the immunoglobulin A (IgA) and IgG in serum, 15% of B. papyrifera silage increased the content of serum catalase, superoxide dismutase, total antioxidant capacity, and decreased the content of 8-hydroxy-2′-deoxyguanosine. Furthermore, 10% or 15% of B. papyrifera silage resulted in a significant decrease in the milk somatic cell count, and increased the polyunsaturated fatty acids content in the milk.ConclusionThe diets with 10% to 15% of B. papyrifera silage might enhance the immune and antioxidant function of dairy cows and increase the polyunstaturated fatty acid concentration in the milk.

The biosynthetic gene cluster of the fungal meroterpenoid emeridone F (8) was discovered in the genome of Aspergillus sp. TJ23, and its late-stage biosynthesis was elucidated by characterizing two α-ketoglutarate (αKG)-dependent dioxygenases, SptF and SptN. SptF catalyzes oxidative rearrangement followed by epoxidation, whereas SptN serves as the C-9 hydroxylase. Our study provides insight into the biosynthetic mechanisms of other andiconin (1)-derived natural products, exemplifying the important roles of αKG-dependent enzymes in the structural complexifications.

Spinosad is a potent insecticide that exhibits an excellent environmental and mammalian profile. However, spinosad production in the original producer, Saccharopolyspora spinosa, is insufficient for the huge global demand. Great efforts have been exerted to improve the production of spinosad. Strategies for spinosad overproduction in actinomycetes are reviewed in this article, including metabolic engineering of the precursor and spinosyn biosynthetic pathway, introduction of regulatory genes, genome-scale metabolic model-guided engineering, mutagenesis, genome shuffling, fermentation process optimization, omics analysis, and the heterologous biosynthesis of spinosad in other actinomycetes. Furthermore, highly productive industrial strains should be used as heterologous hosts for enhancing spinosad biosynthesis in the future. To accelerate the engineering process, the CRISPR/Cas9 system should be established in Sa. spinosa for large-scale genome editing. Notably, the regulatory mechanism of spinosad biosynthesis remains unclear. Thus, the combining multi-omics analysis with high-throughput screening of chemical elicitors would be a promising approach in characterizing the regulatory and signal transduction mechanisms and improving spinosad production in Sa. Spinosa.

BackgroundThe steadily increasing demand for diesel fuels calls for renewable energy sources. This has attracted a growing amount of research to develop advanced, alternative biodiesel worldwide. Several major disadvantages of current biodiesels are the undesirable physical properties such as high viscosity and poor low-temperature operability. Therefore, there is an urgent need to develop novel and advanced biodiesels.ResultsInspired by the proven capability of wax ester synthase/acyl-coenzyme A, diacylglycerol acyltransferase (WS/DGAT) to generate fatty acid esters, de novo biosynthesis of fatty acid branched-chain esters (FABCEs) and branched fatty acid branched-chain esters (BFABCEs) was performed in engineered Escherichia coli through combination of the (branched) fatty acid biosynthetic pathway and the branched-chain amino acid biosynthetic pathway. Furthermore, by modifying the fatty acid pathway, we improved FABCE production to 273 mg/L and achieved a high proportion of FABCEs at 99.3 % of total fatty acid esters. In order to investigate the universality of this strategy, Pichia pastoris yeast was engineered and produced desirable levels of FABCEs for the first time with a good starting point of 169 mg/L.ConclusionsWe propose new pathways of fatty acid ester biosynthesis and establish proof of concept through metabolic engineering of E. coli and P. pastoris yeast. We were able to produce advanced biodiesels with high proportions FABCEs and BFABCEs. Furthermore, this new strategy promises to achieve advanced biodiesels with beneficial low-temperature properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0270-7) contains supplementary material, which is available to authorized users.

Non-heme iron enzymes are versatile catalysts in the biosynthesis of medicinal natural products and have attracted increasing attention as practical catalytic tools in chemical synthesis due to their ability to perform chemically challenging transformations. The Fe(II)/αketoglutarate-dependent oxygenase TqaL catalyzes unusual aziridine formation from L-Val via cleavage of the unactivated C β −H bond. However, the mechanistic details as well as the synthetic potential of TqaL-catalyzed ring closure remain unclear. Herein, we show that the TqaL-catalyzed aziridination of L-Val proceeds with an atypical, mixed stereochemical course involving both the retention and inversion of the C3(C β ) stereocenter. It is also demonstrated that TqaL accepts L-Ile and L-allo-Ile to generate the same diastereomeric pairs of aziridine products via an enzyme-controlled, stereoconvergent process. Our mutagenesis studies reveal that the reaction type (aziridination versus hydroxylation) and the stereochemical outcome are regulated by Ile343 and Phe345. Proper substitutions of Ile343 or Phe345 also make TqaL highly active toward the oxidation of α-amino acid substrates. This work provides mechanistic insights into the stereoselectivity and substrate specificity of the TqaL reactions.

Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens-mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene-producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene-forming gene. Notably, by co-overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 μg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well-established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus.