Dataset Information

Discovery of Several Novel Targets that Enhance ?-Carotene Production in Saccharomyces cerevisiae.

ABSTRACT: ?-Carotene is the precursor of vitamin A, and also exhibits multiple pharmaceutical functions by itself. In comparison to chemical synthesis, the production of ?-carotene in microbes by metabolic engineering strategy is relatively inexpensive. Identifying genes enhancing ?-carotene production in microbes is important for engineering a strain of producing higher yields of ?-carotene. Most of previous efforts in identifying the gene targets have focused on the isoprenoid pathway where the ?-carotene biosynthesis belongs. However, due to the complex interactions between metabolic fluxes, seemingly irrelevant genes that are outside the isoprenoid pathway might also affect ?-carotene biosynthesis. To this end, here we provided an example that several novel gene targets, which are outside the isoprenoid pathway, have improving effects on ?-carotene synthesis in yeast cells, when they were over-expressed. Among these targets, the class E protein of the vacuolar protein-sorting pathway (Did2) led to the highest improvement in ?-carotene yields, which was 2.1-fold to that of the corresponding control. This improvement was further explained by the observation that the overexpression of the DID2 gene generally boosted the transcriptions of ?-carotene pathway genes. The mechanism by which the other targets improve the production of ?-carotene is discussed.

SUBMITTER: Li J

PROVIDER: S-EPMC5471310 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Discovery of Several Novel Targets that Enhance β-Carotene Production in <i>Saccharomyces cerevisiae</i>.

Li Jia J Shen Jia J Sun Zhiqiang Z Li Jing J Li Changfu C Li Xiaohua X Zhang Yansheng Y

Frontiers in microbiology 20170615

β-Carotene is the precursor of vitamin A, and also exhibits multiple pharmaceutical functions by itself. In comparison to chemical synthesis, the production of β-carotene in microbes by metabolic engineering strategy is relatively inexpensive. Identifying genes enhancing β-carotene production in microbes is important for engineering a strain of producing higher yields of β-carotene. Most of previous efforts in identifying the gene targets have focused on the isoprenoid pathway where the β-carote ...[more]

PMID: 28663749

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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.

Project description:Replacement of petrochemical-based materials with microbially produced biodegradable alternatives calls for industrially attractive fermentation processes. Lignocellulosic materials offer non-edible alternatives for cultivated sugars, but require often use of expensive sugar releasing enzymes, such as β-glucosidases. These cellulose treatment costs could be reduced if microbial production hosts could use short cellodextrins such as cellobiose directly as their substrates. In this study, we demonstrate production of poly(hydroxybutyrate) (PHB) in yeast Saccharomyces cerevisiae using cellobiose as a sole carbon source. Yeast strains expressing PHB pathway genes from Cupriavidus necator and cellodextrin transporter gene CDT-1 from Neurospora crassa were complemented either with β-glucosidase gene GH1-1 from N. crassa or with cellobiose phosphorylase gene cbp from Ruminococcus flavefaciens. These cellobiose utilization routes either with Gh1-1 or Cbp enzymes differ in energetics and dynamics. However, both routes enabled higher PHB production per consumed sugar and higher PHB accumulation % of cell dry weight (CDW) than use of glucose as a carbon source. As expected, the strains with Gh1-1 consumed cellobiose faster than the strains with Cbp, both in flask and bioreactor batch cultures. In shake flasks, higher final PHB accumulation % of CDW was reached with Cbp route (10.0 ± 0.3%) than with Gh1-1 route (8.1 ± 0.2%). However, a higher PHB accumulation was achieved in better aerated and pH-controlled bioreactors, in comparison to shake flasks, and the relative performance of strains switched. In bioreactors, notable PHB accumulation levels per CDW of 13.4 ± 0.9% and 18.5 ± 3.9% were achieved with Cbp and Gh1-1 routes, respectively. The average molecular weights of accumulated PHB were similar using both routes; approximately 500 kDa and 450 kDa for strains expressing either cbp or GH1-1 genes, respectively. The formation of PHB with high molecular weights, combined with efficient cellobiose conversion, demonstrates a highly potential solution for improving attractiveness of sustainable polymer production using microbial cells.

Dataset Information

Discovery of Several Novel Targets that Enhance ?-Carotene Production in Saccharomyces cerevisiae.

Publications

Discovery of Several Novel Targets that Enhance β-Carotene Production in <i>Saccharomyces cerevisiae</i>.

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