A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris , we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S -adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris .

Oxygen limitation is regarded as a useful strategy to improve enzyme production by mycelial fungus like Aspergillus niger. However, the intracellular metabolic response of A. niger to oxygen limitation is still obscure. To address this, the metabolism of A. niger was studied using multi-omics integrated analysis based on the latest GEMs (genome-scale metabolic model), including metabolomics, fluxomics and transcriptomics. Upon sharp reduction of the oxygen supply, A. niger metabolism shifted to higher redox level status, as well as lower energy supply, down-regulation of genes for fatty acid synthesis and a rapid decrease of the specific growth rate. The gene expression of the glyoxylate bypass was activated, which was consistent with flux analysis using the A. niger GEMs iHL1210. The increasing flux of the glyoxylate bypass was assumed to reduce the NADH formation from TCA cycle and benefit maintenance of the cellular redox balance under hypoxic conditions. In addition, the relative fluxes of the EMP pathway were increased, which possibly relieved the energy demand for cell metabolism. The above multi-omics integrative analysis provided new insights on metabolic regulatory mechanisms of A. niger associated with enzyme production under oxygen-limited condition, which will benefit systematic design and optimization of the A. niger microbial cell factory.