Introducing carbon assimilation in yeasts using photosynthetic directed endosymbiosis.

Conversion of heterotrophic organisms into partially or completely autotrophic organisms is primarily accomplished by extensive metabolic engineering and laboratory evolution efforts that channel CO2 into central carbon metabolism. Here, we develop a directed endosymbiosis approach to introduce carbon assimilation in budding yeasts. Particularly, we engineer carbon assimilating and sugar-secreting photosynthetic cyanobacterial endosymbionts within the yeast cells, which results in the generation of yeast/cyanobacteria chimeras that propagate under photosynthetic conditions in the presence of CO2 and in the absence of feedstock carbon sources like glucose or glycerol. We demonstrate that the yeast/cyanobacteria chimera can be engineered to biosynthesize natural products under the photosynthetic conditions. Additionally, we expand our directed endosymbiosis approach to standard laboratory strains of yeasts, which transforms them into photosynthetic yeast/cyanobacteria chimeras. We anticipate that our studies will have significant implications for sustainable biotechnology, synthetic biology, and experimentally studying the evolutionary adaptation of an additional organelle in yeast.

Discovery and biosynthesis of macrophasetins from the plant pathogen fungus Macrophomina phaseolina.

3-Decalinoyltetramic acids (DTAs) are a class of natural products with chemical diversity and potent bioactivities. In fungal species there is a general biosynthetic route to synthesize this type of compounds, which usually features a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) and a lipocalin-like Diels-Alderase (LLDAse). Using a synthetic biology approach, combining the bioinformatics analysis prediction and heterologous expression, we mined a PKS-NRPS and LLDAse encoding gene cluster from the plant pathogenic fungus Macrophomina phaseolina and characterized the cluster to be responsible for the biosynthesis of novel DTAs, macrophasetins. In addition, we investigated the biosynthesis of these compounds and validated the accuracy of the phylogeny-guided bioinformatics analysis prediction. Our results provided a proof of concept example to this approach, which may facilitate the discovery of novel DTAs from the fungal kingdom.

Engineering artificial photosynthetic life-forms through endosymbiosis.

The evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined photosynthetic conditions. We anticipate that these genetically tractable endosymbiotic platforms can be used for evolutionary studies, particularly related to organelle evolution, and also for synthetic biology applications.

Heterologous expression of a natural product biosynthetic gene cluster from Cordyceps militaris.

Cordyceps is a genus of ascomycete fungi widely used in old Chinese medicine, and many investigations have focus on uncovering their biological activities. Until now, only a few compounds have been identified from Cordyceps, mainly due to their poor yield. So as to make full use of Cordyceps, we used the strategy of genome mining and heterologous expression to discover natural products (NPs) from Cordyceps militaris. Analysis of the genome sequence of Cordyceps militaris CM01 showed the presence of a cryptic gene cluster encoding a highly-reducing polyketide synthetase (HR-PKS), enoyl-reductase (ER) and cytochrome P450. Heterologous expression in Aspergillus nidulans enabled the identification of two new polyketides, cordypyrone A and B. Their structures were determined by 1D and 2D NMR techniques. They showed only modest activities against pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Mycobacteria tuberculosis and Bacillus cereus.

Isolation and characterization of a new cytotoxic polyketide-amino acid hybrid from Thermothelomyces thermophilus ATCC 42464.

Fungi are a rich source of novel anticancer compounds. Bioassay-guided isolation has led to the isolation of four polyketide-amino acid hybrid compounds with trans-fused decalin system from the fungus Thermothelomyces thermophilus ATCC 42464 (=Myceliophthora thermophila ATCC 42464): myceliothermophins A, B, E and F (1-4). The structure of the new compound (myceliothermophin F, compound 4) was clearly determined by a combination of nuclear magnetic resonance (NMR) analysis and high-resolution electrospray ionisation mass spectroscopy (HRESIMS). The new compound exhibited promising cytotoxicity against some cell lines derived from colorectal carcinoma, hepatic carcinoma and gastric carcinoma, indicating that compounds with trans-fused decalin system would be promising in the course of developing novel anticancer drugs.

Genome mining and biosynthesis of the Acyl-CoA:cholesterol acyltransferase inhibitor beauveriolide I and III in Cordyceps militaris.

Ascomycete fungi Cordyceps are widely used in traditional Chinese medicine, and numerous investigations have been carried out to uncover their biological activities. However, primary researches on the physiological effects of Cordyceps were committed using crude extracts. At present, there are only a few compounds which were comprehensively characterized from Cordyceps, partial owing to the low production. In order to scientifically take advantage of Cordyceps, we used the strategy of genome mining to discover bioactive compounds from Cordyceps militaris. We found the putative biosynthetic gene cluster of the acyl-CoA:cholesterol acyltransferase inhibitor beauveriolides in the genome of C. militaris, and produced the compounds by heterologous expression in Aspergillus nidulans. Production of beauveriolide I and III also was detected in both ferment mycelia and fruiting bodies of C. militaris. The possible biosynthetic pathway was proposed. Our studies unveil the active compounds of C. militaris against atherosclerosis and Alzheimer's disease and provide the enzyme resources for the biosynthesis of new cyclodepsipeptide molecules.