Screening of Beauveria bassiana with high biocontrol potential based on ARTP mutagenesis and high-throughput FACS.

Beauveria bassiana is a promising biocontrol agent due to its entomopathogenic activities and residue-free characteristics. However, its susceptibility to abiotic stresses and naturally low virulence limit the effective application of this fungus. To effectively obtain fungal strains with high biocontrol potential, fluorescence-activated cell sorting (FACS) was used to screen mutant libraries generated by atmospheric and room temperature plasma (ARTP). Among about 8000 mutants obtained by ARTP mutagenesis, six candidate mutants were selected according to the forward scatter (FSC) signal readings of FACS. B6, with a 37.4% higher FSC reading than wild-type (WT), showed a 32.6% increase in virulence. It also presented a 13.5% decrease in median germinating time (GT50) and a 12.1% increase in blastospore production. Comparative analysis between insect transcriptional responses to B6 and WT infection showed that the immune response coupled with protein digestion and absorption progress was highly activated in B6-infected Galleria mellonella larvae, while fatty acid synthesis was suppressed after 3 days of infection. Our results confirmed the feasibility of sorting B. bassiana with high biocontrol potential via the combination of ARTP and FACS and facilitated the understanding of insect-pathogen interactions, highlighting a new strategy for modifying entomopathogenic fungi to improve the efficiency of biological control.

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases.

Sesquiterpenes comprise a diverse group of natural products with a wide range of applications in cosmetics, food, medicine, agriculture, and biofuels. Heterologous biosynthesis is increasingly employed for sesquiterpene production, aiming to overcome the limitations associated with chemical synthesis and natural extraction. Sesquiterpene synthases (STSs) play a crucial role in the heterologous biosynthesis of sesquiterpene. Under the catalysis of STSs, over 300 skeletons are produced through various cyclization processes (C1-C10 closure, C1-C11 closure, C1-C6 closure, and C1-C7 closure), which are responsible for the diversity of sesquiterpenes. According to the cyclization types, we gave an overview of advances in understanding the mechanism of STSs cyclization from the aspects of protein crystal structures and site-directed mutagenesis. We also summarized the applications of engineering STSs in the heterologous biosynthesis of sesquiterpene. Finally, the bottlenecks and potential research directions related to the STSs cyclization mechanism and application of modified STSs were presented.

Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering.

Kauralexin A1 (KA1) is a key intermediate of the kauralexin A series metabolites of maize phytoalexins. However, their application is severely limited by their low abundance in maize. In this study, an efficient biosynthetic pathway was constructed to produce KA1 in Saccharomyces cerevisiae. Also, metabolic and enzyme engineering strategies were applied to construct the high-titer strains, such as chassis modification, screening synthases, the colocalization of enzymes, and multiple genomic integrations. First, the KA1 precursor ent-kaurene was synthesized using the efficient diterpene synthase GfCPS/KS from Fusarium fujikuroi, and optimized to reach 244.36 mg/L in shake flasks, which displayed a 200-fold increase compared to the initial strain. Then, the KA1 was produced under the catalysis of ZmCYP71Z18 from Zea mays and SmCPR1 from Salvia miltiorrhiza, and the titer was further improved by integrating the fusion protein into the genome. Finally, an ent-kaurene titer of 763.23 mg/L and a KA1 titer of 42.22 mg/L were achieved through a single-stage fed-batch fermentation in a 5 L bioreactor. This is the first report of the heterologous biosynthesis of maize diterpene phytoalexins in S. cerevisiae, which lays a foundation for further pathway reconstruction and biosynthesis of the kauralexin A series maize phytoalexins.

Enzyme and Metabolic Engineering Strategies for Biosynthesis of α-Farnesene in Saccharomyces cerevisiae.

α-Farnesene, a type of acyclic sesquiterpene, is an important raw material in agriculture, aircraft fuel, and the chemical industry. In this study, we constructed an efficient α-farnesene-producing yeast cell factory by combining enzyme and metabolic engineering strategies. First, we screened different plants for α-farnesene synthase (AFS) with the best activity and found that AFS from Camellia sinensis (CsAFS) exhibited the most efficient α-farnesene production in Saccharomyces cerevisiae 4741. Second, the metabolic flux of the mevalonate pathway was increased to improve the supply of the precursor farnesyl pyrophosphate. Third, inducing site-directed mutagenesis in CsAFS, the CsAFSW281C variant was obtained, which considerably increased α-farnesene production. Fourth, the N-terminal serine-lysine-isoleucine-lysine (SKIK) tag was introduced to construct the SKIK∼CsAFSW281C variant, which further increased α-farnesene production to 2.8 g/L in shake-flask cultures. Finally, the α-farnesene titer of 28.3 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Transcriptome Analysis and Identification of Sesquiterpene Synthases in Liverwort Jungermannia exsertifolia.

The liverwort Jungermannia exsertifolia is one of the oldest terrestrial plants and rich in structurally specific sesquiterpenes. There are several sesquiterpene synthases (STSs) with non-classical conserved motifs that have been discovered in recent studies on liverworts; these motifs are rich in aspartate and bind with cofactors. However, more detailed sequence information is needed to clarify the biochemical diversity of these atypical STSs. This study mined J. exsertifolia sesquiterpene synthases (JeSTSs) through transcriptome analysis using BGISEQ-500 sequencing technology. A total of 257,133 unigenes was obtained, and the average length was 933 bp. Among them, a total of 36 unigenes participated in the biosynthesis of sesquiterpenes. In addition, the in vitro enzymatic characterization and heterologous expression in Saccharomyces cerevisiae showed that JeSTS1 and JeSTS2 produced nerolidol as the major product, while JeSTS4 could produce bicyclogermacrene and viridiflorol, suggesting a specificity of J. exsertifolia sesquiterpene profiles. Furthermore, the identified JeSTSs had a phylogenetic relationship with a new branch of plant terpene synthases, the microbial terpene synthase-like (MTPSL) STSs. This work contributes to the understanding of the metabolic mechanism for MTPSL-STSs in J. exsertifolia and could provide an efficient alternative to microbial synthesis of these bioactive sesquiterpenes.