Saccharomyces cerevisiae is an important platform organism for synthesis of chemicals and fuels. However, the promoters used in most pathway engineering studies in S. cerevisiae have not been characterized and compared in parallel under multiple conditions that are routinely operated in laboratory and the number of known promoters is rather limited for the construction of large biochemical pathways. Here a total of 14 constitutive promoters from S. cerevisiae were cloned and characterized using a green fluorescent protein (GFP) as a reporter in a 2 µ vector pRS426, under varying glucose and oxygen concentrations. The strengths of these promoters varied no more than sixfold in the mean fluorescence intensity of GFP, with promoter TEF1p being the strongest and promoter PGI1p the weakest. As an example of application for these promoters in metabolic engineering, the genes involved in xylan degradation and zeaxanthin biosynthesis were subsequently cloned under the control of promoters with medium to high strength and assembled into a single pathway. The corresponding construct was transformed to a S. cerevisiae strain integrated with a D-xylose utilizing pathway. The resulting strain produced zeaxanthin with a titer of 0.74 ± 0.02 mg/L directly from birchwood xylan.

Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL’s) from esterases ~100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P <0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL’s. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzyme are catalytically promiscuous, but HNL1 is especially so.

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The solubility of 4-nitrophthalimide in different solvents are of great importance for the design of its purification process via crystallization. The work reported new solubility data for 4-nitrophthalimide in 12 pure solvents of methanol, ethanol, isopropanol, cyclohexanone, acetone, acetonitrile, ethyl acetate, 2-butanone, chloroform, 1,4-dioxane benzyl alcohol and N,N-dimethylformamide. They were determined by a high-performance liquid chromatography at T = (273.15 to 323.15) K under pressure of 0.1 MPa. The 4-nitrophthalimide solubility in the selected solvents increased with the temperature increase. At a given temperature, the solubility of 4-nitrophthalimide is largest in N,N-dimethylformamide and lowest in chloroform. The solubility data in the these solvents ranked as N,Ndimethylformamide > cyclohexanone > (1,4-dioxane, acetone, 2-butanone, benzyl alcohol) > ethyl acetate > acetonitrile > methanol > ethanol > isopropanol > chloroform. The experimental solubility data were correlated by modified Apelblat equation, λh equation, Wilson model, and NRTL model. The obtained values of root-mean-square deviation and relative average deviation are all less than 16.17 × 10 −4 and 1.58%, respectively. The modified Apelblat equation achieved the best correlating results in totally.

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