Phytosterols that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites such as the phytohormone brassinosteroids. Brassinosteroids are essential and universal plant hormones, regulating plant growth and development, also involved in biotic and abiotic defense. With the promising potential of agriculture application, the biosynthesis of brassinolide in plants remains elusive although it has been intensively studied in the past decades. The promiscuity of brassinosteroid biosynthetic enzymes and the low abundance of brassinosteroids in nature is likely the reasons. In this project, to decipher the biosynthesis of brassinosteroids and pave the way for other steroid nature products (e.g. withanolides, saponins) , we firstly reconstituted the biosynthetic pathway of phytosterols in a heterologous host Saccharomyces cerevisiae and engineered the yeast to produce the free phytosterols and incorporate the sterols in the cell membrane. The resulted phytosterol-producing yeast strains provided a platform for enhanced reconstitution of plant membrane enzymes. Secondly, we optimized the growth of the strain by engineering sterol esterification mechanism, tuning upstream FPP pathway, and performing retro genetic engineering. Thirdly, based on the phytosterol-producing yeast platform, a plant scaffold protein MSBP1 was characterized to play an important role in BR biosynthesis, which expanded our understanding of this multifaced scaffold protein, and more importantly, it brought up a possibility that brassinosteroid biosynthesis takes a place in the metabolome. This finding highlighted the role of non-enzymatic proteins in nature product biosynthesis and broaden our vision when analyze the biosynthetic pathway in nature products.

Nature has endowed microbes and plants with enormous power to synthesize complex natural products from simple building blocks. Compounds derived from natural sources that show biological activities are defined as natural products. Over the past century, natural products have played an indispensible role in the pharmaceutical discovery, and have become an important element of modern therapy. Aromatic polyketides are an important group of natural products that exhibit extraordinary medicinal activities. Actinomycetes and filamentous fungi are the two major producers of aromatic polyketides, especially polycyclic aromatic polyketides; and they employ distinct enzymatic system to synthesize these complex structures from malonyl-CoA. Fungi utilize iterative multidomain megasynthase of type I nonreducing polyketide synthases (NRPKSs), while actinomycetes employ bacterial type II PKSs that comprise a group of dissociated enzymes. Compared to the well-studied type II PKS, NRPKS has been much less understood.

In this study, we first identified the unique product template (PT) domain of NRPKS that is not shared among the other PKS families as the first ring aldol cyclase. Based on the understanding of PT domain function, we performed phylogenetic analysis of PT domains, which classified the NRPKSs into five major groups. Subsequently, we proposed a sequence-function correlation of PT domain that allowed us to predict the activities of unknown PTs uncovered from genome sequencing. These target PT domains were assayed with the PKS4 (a previously identified NRPKS) minimal PKS domains in trans or in cis, and the proposed PT functions were confirmed through such product based assays. According to the established sequence-function relationship, we conducted genome mining of A. niger for the biosynthetic locus of TAN-1612, which contains an interesting tetracyclic naphthacenedione core structure. Subsequent investigations of the TAN-1612 biosynthetic pathway uncovered a novel alpha-hydroxylation-dependent Claisen cyclization cascade that involves a bifunctional dimanganese metallo-beta-lactamase-type thioesterase (MbL-TE). Also, the identification of the TAN-1612 biosynthetic pathway revealed a decaketide synthase AdaA, which enabled us to realize the in vitro enzymatic total synthesis of several important aromatic decaketide intermediates including rabelomycin and tetracenomycin D1.

Fungal pathogens induce a variety of diseases both in plants and post-harvest food crops, resulting in significant crop losses for the agricultural industry. Although the usage of chemical-based fungicides is the most common way of controlling these diseases, they damage the environment, have the potential to harm human and animal life, and may lead to resistant fungal strains. As such, there is an urgent need for diverse and effective agricultural fungicides that are environmentally- and eco-friendly. In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and Glycine max (GmPGIP3), and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1, BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. Investigation on the sequence-function relationships of PvPGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities. This suggests it may be a possible target for the future engineering of PGIP with enhanced activity. We found that tPvPGIP2_5-8 and tGmPGIP3_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full-length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full-length PvPGIP2 and GmPGIP3 via Y2H. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 secreting yeast or purified PGIP proteins clearly slows down the growth of A. niger and B. cinerea. On plant assays utilizing detached leaves from N. benthamiana, application of both full length and tPvPGIP2_5-8 secreting yeast or purified proteins reduced the growth and infection rate of B. cinerea. Additionally, PvPGIP2 remains thermostable up to 42°C and retains its inhibitory activity against B. cinerea on pectin assays and in planta assays, delaying growth of the pathogenic fungi by up to one week.