The tapping of municipal wastewater for potable reuse significantly enhances drinking water supply in drought-stricken regions worldwide. Membrane-based potable reuse treatment trains commonly employ ultraviolet-based advanced oxidation processes (UV-AOPs) to degrade trace organic contaminants in water to produce high-quality recycled water. Hydrogen peroxide (H 2 O 2 ) is used as the default photo-oxidant. Meanwhile, chloramines, which are added to prevent biofouling, pass through the membranes and impact the treatment efficiency of UV-AOP. Water reuse facilities therefore face the dilemma of optimizing H 2 O 2 (an added photo-oxidant) and chloramines (a carry-over photooxidant) doses. Utilizing a uniquely designed pilot-scale reactor and real-time recycled water, we evaluated treatment efficiencies of UV-AOP on six important indicator contaminants, with monochloramine (NH 2 Cl) and H 2 O 2 as photo-oxidants. Hydroxyl radical (HO • ) and reactive chlorine species, such as the chlorine atom (Cl • ) and chlorine dimer (Cl 2•− ), were the major reactive species. Overall, radicals generated from photolysis of NH 2 Cl alone achieved removal of indicator compounds, which can be further improved by optimizing UV fluence, i.e., the UV dose. Furthermore, the addition of H 2 O 2 enhanced HO • formation and improved contaminant removal. However, the addition of H 2 O 2 , when the background NH 2 Cl level was above 2 mg L −1 (as Cl 2 ), provided limited improvement in treatment efficiency. These trade-offs between chloramine and H 2 O 2 as oxidants, and the recommended optimization of the associated effective UV fluence, are critical for energy-efficient and costeffective potable reuse to address the challenges of global water scarcity.

As essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. For example, in this study, we engineered campesterol production in yeast using plant enzymes; although we were able to enhance the titer of campesterol to ~40mg/L by upregulating the mevalonate pathway, no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, many heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through repeated culture. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to establish a set of phytosterol-producing yeast strains with decent growth and titer of campesterol (~ 7mg/L), βsitosterol (~2mg/L), 22-hydroxycampesterol (~1mg/L), and 22-hydroxycampest-4-en-3-one (~4mg/L). This work resolves the technical bottlenecks in phytosterol-derived pathway reconstitution in the backer's yeast and opens up opportunities for efficient bioproduction and pathway elucidation of this group of phytochemicals.

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 steroidproducing yeast can achieve, the challenge and the potential of using yeast as the bioproduction platform of various steroids from higher eukaryotes.

As essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. For example, in this study, we engineered campesterol production in yeast using plant enzymes; although we were able to enhance the titer of campesterol to ~40mg/L by upregulating the mevalonate pathway, no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, many heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through repeated culture. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to establish a set of phytosterol-producing yeast strains with decent growth and titer of campesterol (~ 7mg/L), βsitosterol (~2mg/L), 22-hydroxycampesterol (~1mg/L), and 22-hydroxycampest-4-en-3-one (~4mg/L). This work resolves the technical bottlenecks in phytosterol-derived pathway reconstitution in the backer's yeast and opens up opportunities for efficient bioproduction and pathway elucidation of this group of phytochemicals.

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