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Engineering Saccharomyces cerevisiae for geranylgeraniol overproduction by combinatorial design.

ABSTRACT: Combinatorial design is an effective strategy to acquire the optimal solution in complex systems. In this study, the combined effects of pathway combination, promoters' strength fine-tuning, copy numbers and integration locus variations caused by ?-integration were explored in Saccharomyces cerevisiae using geranylgeraniol (GGOH) production as an example. Two GGOH biosynthetic pathway branches were constructed. In branch 1, GGOH was converted from isopentenyl pyrophosphate (IPP) and farnesyl diphosphate (FPP). In branch 2, GGOH was derived directly from IPP and dimethylallyl pyrophosphate (DMAPP). Regulated by 10 combinations of 11 diverse promoters, a fusion gene BTS1-ERG20, a heterologous geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius (GGPPSsa) and an endogenous N-terminal truncated gene 3-hydroxyl-3-methylglutaryl-CoA reductase isoenzyme 1 (tHMGR), were incorporated into yeast by ?-integration, leading to a series of GGOH producing strains with yields ranging from 18.45?mg/L to 161.82?mg/L. The yield was further increased to 437.52?mg/L by optimizing the fermentation medium. Consequently, the GGOH yield reached 1315.44?mg/L in a 5-L fermenter under carbon restriction strategy. Our study not only opens large opportunities for downstream diterpenes overproductions, but also demonstrates that pathway optimization based on combinatorial design is a promising strategy to engineer microbes for overproducing natural products with complex structure.

SUBMITTER: Song TQ

PROVIDER: S-EPMC5678108 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Engineering Saccharomyces cerevisiae for geranylgeraniol overproduction by combinatorial design.

Song Tian-Qing TQ Ding Ming-Zhu MZ Zhai Fang F Liu Duo D Liu Hong H Xiao Wen-Hai WH Yuan Ying-Jin YJ

Scientific reports 20171108 1

Combinatorial design is an effective strategy to acquire the optimal solution in complex systems. In this study, the combined effects of pathway combination, promoters' strength fine-tuning, copy numbers and integration locus variations caused by δ-integration were explored in Saccharomyces cerevisiae using geranylgeraniol (GGOH) production as an example. Two GGOH biosynthetic pathway branches were constructed. In branch 1, GGOH was converted from isopentenyl pyrophosphate (IPP) and farnesyl dip ...[more]

PMID: 29118396

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Project description:β-ionone is a commercially attractive industrial fragrance produced naturally from the cleavage of the pigment β-carotene in plants. While the production of this ionone is typically performed using chemical synthesis, environmentally friendly and consumer-oriented biotechnological production is gaining increasing attention. A convenient cell factory to address this demand is the yeast Saccharomyces cerevisiae. However, current β-ionone titers and yields are insufficient for commercial bioproduction. In this work, we optimized S. cerevisiae for the accumulation of high amounts of β-carotene and its subsequent conversion to β-ionone. For this task, we integrated systematically the heterologous carotenogenic genes (CrtE, CrtYB and CrtI) from Xanthophyllomyces dendrorhous using markerless genome editing CRISPR/Cas9 technology; and evaluated the transcriptional unit architecture (bidirectional or tandem), integration site, and impact of gene dosage, first on β-carotene accumulation, and later, on β-ionone production. A single-copy insertion of the carotenogenic genes in high expression loci of the wild-type yeast CEN.Pk2 strain yielded 4 mg/gDCW of total carotenoids, regardless of the transcriptional unit architecture employed. Subsequent fine-tuning of the carotenogenic gene expression enabled reaching 16 mg/gDCW of total carotenoids, which was further increased to 32 mg/gDCW by alleviating the known pathway bottleneck catalyzed by the hydroxymethylglutaryl-CoA reductase (HMGR1). The latter yield represents the highest total carotenoid concentration reported to date in S. cerevisiae for a constitutive expression system. For β-ionone synthesis, single and multiple copies of the carotene cleavage dioxygenase 1 (CCD1) gene from Petunia hybrida (PhCCD1) fused with a membrane destination peptide were expressed in the highest β-carotene-producing strains, reaching up to 33 mg/L of β-ionone in the culture medium after 72-h cultivation in shake flasks. Finally, interrogation of a contextualized genome-scale metabolic model of the producer strains pointed to PhCCD1 unspecific cleavage activity as a potentially limiting factor reducing β-ionone production. Overall, the results of this work constitute a step toward the industrial production of this ionone and, more broadly, they demonstrate that biotechnological production of apocarotenoids is technically feasible.

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Engineering Saccharomyces cerevisiae for geranylgeraniol overproduction by combinatorial design.

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Engineering Saccharomyces cerevisiae for geranylgeraniol overproduction by combinatorial design.

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