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Scholarly Works (3 results)

  • Thesis
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UC Riverside Electronic Theses and Dissertations (2022)

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.

    Cover page: Establishment of Phytosterol-Producing Yeast Platforms for Plant Enzyme Characterization and Brassinosteroids Biosynthesis ElucidationCreative Commons 'BY-NC-ND' version 4.0 license
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2020)
    With the rapid development of synthetic biology and metabolic engineering technologies, yeast has been generally considered as promising hosts for the bioproduction of secondary metabolites. Sterols are essential components of cell membrane, and are the precursors for the biosynthesis of steroid hormones, signaling molecules, and defense molecules in the higher eukaryotes, which are of pharmaceutical and agricultural significance. In this mini-review, we summarize the recent engineering efforts of using yeast to synthesize various steroids, and discuss the structural diversity that the current steroid-producing yeast can achieve, the challenge and the potential of using yeast as the bioproduction platform of various steroids from higher eukaryotes.
      Cover page: Yeast as a promising heterologous host for steroid bioproduction
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2023)
      Campesterol is a major phytosterol that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites, such as the phytohormone brassinosteroids. Recently, we established a campesterol-producing yeast strain and extended the bioproduction to 22-hydroxycampesterol and 22-hydroxycampest-4-en-3-one, the precursors to brassinolide. However, there is a trade-off in growth due to the disrupted sterol metabolism. In this study, we enhanced the growth of the campesterol-producing yeast by partially restoring the activity of the sterol acyltransferase and engineering upstream FPP supply. Furthermore, genome sequencing analysis also revealed a pool of genes possibly associated with the altered sterol metabolism. Retro engineering implies an essential role of ASG1, especially the C-terminal asparagine-rich domain of ASG1, in the sterol metabolism of yeast especially under stress. The performance of the campesterol-producing yeast strain was enhanced with the titer of campesterol to 18.4 mg/L, and the stationary OD600 was improved by ∼33% compared to the unoptimized strain. In addition, we examined the activity of a plant cytochrome P450 in the engineered strain, which exhibits more than 9-fold higher activity than when expressed in the wild-type yeast strain. Therefore, the engineered campesterol-producing yeast strain also serves as a robust host for the functional expression of plant membrane proteins.
        Cover page: Optimization of Campesterol-Producing Yeast Strains as a Feasible Platform for the Functional Reconstitution of Plant Membrane-Bound Enzymes
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