Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6

Abstract

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.

Discovery of the cryptic function of terpene cyclases as aromatic prenyltransferases

“…2 ) 3 , 15 , 16 , both of which are required for coordination to the 3 Mg 2+ ions necessary for catalysis. To confirm the predicted coding sequence of AaTPS, genomic DNA of this gene was put under the control of the alcA promoter and transformed into A. alternata TPF6 via the Agrobacterium tumefaciens -mediated transformation method to generate mutant Alternaria alternata GB171 17 . Transcription data were gathered and the mRNA sequence of AaTPS was confirmed.…”

Core Microbiome of Medicinal Plant Salvia miltiorrhiza Seed: A Rich Reservoir of Beneficial Microbes for Secondary Metabolism?

“…Therefore, the genera Pseudomonas and Sphingomonas in the seed microbiome are likely to be important reservoirs of rhizosphere or endosphere microbiome. Regarding the shared fungal core taxa, genus Alternaria can be a potential plant pathogen, but it also includes beneficial endophytes as biocontrol agents or other active compounds producing microbes [ 49 , 50 , 51 , 52 ]. Therefore, Alternaria can affect the germination of seeds and the assemblage of plant microbiome, and consequently, the growth and fitness of plants.…”

A Fungal Diterpene Synthase Is Responsible for Sterol Biosynthesis for Growth

“…A previous study demonstrated that the biosynthesis of sterols started with IPP and DMAPP, two C5 isoprene units synthesized by the MVA pathway ( Engels et al, 2008 ; Cha et al, 2012 ; Bian et al, 2017 ). Total genome sequencing and mRNA sequencing of NK17 were performed to identify the MVA pathway genes in NK17 via protein sequence BLASTP on NCBI.…”

“…In fungi, terpenoids are usually synthesized from two C 5 units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) ( Li et al, 2015 ). These two units were condensed via the mevalonate (MVA) pathway and undergo a cyclization step to form terpenoid precursors by various terpene synthases, e.g., monoterpene synthase ( Lange, 2015 ), sesquiterpene synthase ( Wang et al, 2016 ), diterpene synthase, triterpene synthase ( Dhingra and Cramer, 2017 ), and even taxadiene synthase ( Bian et al, 2017 ). Due to bioactivities, many of them have been used in diverse fields, such as medicinal chemistry ( Austin et al, 1988 ), food additives ( Ravichandran et al, 2018 ), and biofuels ( Wu et al, 2017 ).…”