Customized optimization of metabolic pathways by combinatorial transcriptional engineering

Abstract

A major challenge in metabolic engineering and synthetic biology is to balance the flux of an engineered heterologous metabolic pathway to achieve high yield and productivity in a target organism. Here, we report a simple, efficient and programmable approach named 'customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER)' for rapid tuning of gene expression in a heterologous pathway under distinct metabolic backgrounds. Specifically, a library of mutant pathways is created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism followed by high-throughput screening/selection. To demonstrate this approach, a single round of COMPACTER was used to generate both a xylose utilizing pathway with near-highest efficiency and a cellobiose utilizing pathway with highest efficiency that were ever reported in literature for both laboratory and industrial yeast strains. Interestingly, these engineered xylose and cellobiose utilizing pathways were all host-specific. Therefore, COMPACTER represents a powerful approach to tailor-make metabolic pathways for different strain backgrounds, which is difficult if not impossible to achieve by existing pathway engineering methods.

Figure 1.

General scheme of the COMPACTER method for combinatorial pathway design. See text for details.

Figure 2.

Creation of a highly efficient xylose utilizing pathway in both laboratory and industrial S. cerevisiae strains via COMPACTER. Red circle: xylose; blue triangle: ethanol. (a) Scheme of the engineered fungal xylose utilizing pathway. (b) Xylose fermentation of eight randomly picked clones from the COMPACTER library. (c) Comparison of xylose consumption and ethanol production in the industrial strain. Open symbol: the reference industrial strain CTY-XWT; solid symbol: the mutant industrial strain CTY-X7. (d) Comparison of xylose consumption and ethanol production in the laboratory strain. Open symbol: the reference laboratory strain INV-XWT; solid symbol: the mutant laboratory strain INV-X3. (e) Pathway switching in the industrial strain. Open symbol: the industrial strain harboring the pathway from the mutant laboratory strain CTY-X3; solid symbol: the mutant industrial strain CTY-X7. (f) Pathway switching in the laboratory strain. Open symbol: the laboratory strain harboring the pathway from the mutant industrial strain INV-X7; solid symbol: the mutant laboratory strain INV-X3. (g) Relative expression levels of XR, XDH and XKS in the mutant industrial strain CTY-X7 and the reference industrial strain CTY-XWT measured using qPCR. The expression levels were normalized by making the expression level of XR as 1. (h) Relative expression levels of XR, XDH and XKS in the mutant laboratory strain INV-X3 and the reference laboratory strain INV-XWT measured using qPCR. The expression levels were normalized by making the expression level of XR as 1.

Figure 3.

Creation of a highly efficient cellobiose utilizing pathway in both laboratory and industrial S. cerevisiae strains via COMPACTER. Red circle: cellobiose; black square: OD (A₆₀₀); blue triangle: ethanol. (a) Scheme of the engineered cellobiose utilizing pathway. (b) Library screening on an YPAC agar plate. (c) Comparison of cellobiose consumption and ethanol production in 250 ml flask fermentations in the industrial strain. Open symbol: the reference industrial strain CTY-CWT; solid symbol: the mutant industrial strain CTY-C59. (d) Comparison of cellobiose consumption and ethanol production in 250 ml flask fermentations in the laboratory strain. Open symbol: the reference laboratory strain INV-CWT; solid symbol: the mutant laboratory strain INV-C3. (e) Pathway switching in the industrial strain. Open symbol: the industrial strain harboring the pathway from the mutant laboratory strain CTY-C3; solid symbol: the mutant industrial strain CTY-C59. (f) Pathway switching in the laboratory strain. Open symbol: the laboratory strain harboring the pathway from the mutant industrial strain INV-C59; solid symbol: the mutant laboratory strain INV-C3. (g) Relative expression levels of CDT and BGL in the mutant industrial strain CTY-C59 and the reference industrial strain CTY-CWT measured using qPCR. The expression levels were normalized by making the expression level of CDT as 1. (h) Relative expression levels of CDT and BGL in the mutant laboratory strain INV-C3 and the reference laboratory strain INV-CWT measured using qPCR. The expression levels were normalized by making the expression level of CDT as 1.