Dataset Information

Refactoring Ehrlich Pathway for High-Yield 2-Phenylethanol Production in Yarrowia lipolytica.

ABSTRACT: Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation. Substrates with different entry points into the metabolic network have different energetic and redox statuses. Generally, substrate cofeeding could bypass the lengthy and highly regulated native metabolism and facilitates high carbon conversion rate. Aiming to efficiently synthesize the high-value rose-smell 2-phenylethanol (2-PE) in Y. lipolytica, we analyzed the stoichiometric constraints of the Ehrlich pathway and identified that the selectivity of the Ehrlich pathway and the availability of 2-oxoglutarate are the rate-limiting factors. Stepwise refactoring of the Ehrlich pathway led us to identify the optimal catalytic modules consisting of l-phenylalanine permease, ketoacid aminotransferase, phenylpyruvate decarboxylase, phenylacetaldehyde reductase, and alcohol dehydrogenase. On the other hand, mitochondrial compartmentalization of 2-oxoglutarate inherently creates a bottleneck for efficient assimilation of l-phenylalanine, which limits 2-PE production. To improve 2-oxoglutarate (aKG) trafficking across the mitochondria membrane, we constructed a cytosolic aKG source pathway by coupling a bacterial aconitase with a native isocitrate dehydrogenase (ylIDP2). Additionally, we also engineered dicarboxylic acid transporters to further improve the 2-oxoglutarate availability. Furthermore, by blocking the precursor-competing pathways and mitigating fatty acid synthesis, the engineered strain produced 2669.54 mg/L of 2-PE in shake flasks, a 4.16-fold increase over the starting strain. The carbon conversion yield reaches 0.702 g/g from l-phenylalanine, 95.0% of the theoretical maximal. The reported work expands our ability to harness the Ehrlich pathway for production of high-value aromatics in oleaginous yeast species.

SUBMITTER: Gu Y

PROVIDER: S-EPMC7308069 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Refactoring Ehrlich Pathway for High-Yield 2-Phenylethanol Production in <i>Yarrowia lipolytica</i>.

Gu Yang Y Ma Jingbo J Zhu Yonglian Y Xu Peng P

ACS synthetic biology 20200312 3

Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation. Substrates with different entry points into the metabolic network have different energetic and redox statuses. Generally, substrate cofeeding could bypass the lengthy and highly regulated native metabolism and facilitates high carbon conversion rate. Aiming to efficiently synthesize the high-value rose-smell 2-phenylethanol (2-PE) in <i>Y. lipolyti ...[more]

PMID: 32134637

Similar Datasets

Project description:BackgroundLong-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA), are essential for human health and have been widely used in the food and pharmaceutical industries. However, the limited availability of natural sources, such as oily fish, has led to the pursuit of microbial production as a promising alternative. Yarrowia lipolytica can produce various PUFAs via genetic modification. A recent study upgraded Y. lipolytica for DHA production by expressing a four-gene cluster encoding a myxobacterial PKS-like PUFA synthase, reducing the demand for redox power. However, the genetic architecture of gene expression in Y. lipolytica is complex and involves various control elements, offering space for additional improvement of DHA production. This study was designed to optimize the expression of the PUFA cluster using a modular cloning approach.ResultsExpression of the monocistronic cluster with each gene under the control of the constitutive TEF promoter led to low-level DHA production. By using the minLEU2 promoter instead and incorporating additional upstream activating UAS1B4 sequences, 5' promoter introns, and intergenic spacers, DHA production was increased by 16-fold. The producers remained stable over 185 h of cultivation. Beneficially, the different genetic control elements acted synergistically: UAS1B elements generally increased expression, while the intron caused gene-specific effects. Mutants with UAS1B16 sequences within 2-8 kb distance, however, were found to be genetically unstable, which limited production performance over time, suggesting the avoidance of long repetitive sequence blocks in synthetic multigene clusters and careful monitoring of genetic stability in producing strains.ConclusionsOverall, the results demonstrate the effectiveness of synthetic heterologous gene clusters to drive DHA production in Y. lipolytica. The combinatorial exploration of different genetic control elements allowed the optimization of DHA production. These findings have important implications for developing Y. lipolytica strains for the industrial-scale production of valuable polyunsaturated fatty acids.

Dataset Information

Refactoring Ehrlich Pathway for High-Yield 2-Phenylethanol Production in Yarrowia lipolytica.

Publications

Refactoring Ehrlich Pathway for High-Yield 2-Phenylethanol Production in <i>Yarrowia lipolytica</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets