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

Yang Gu,Yonglian Zhu,Peng Xu,Jingbo Ma

doi:10.1021/acssynbio.9b00468

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.

Highlights

Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation
Owing to the hard-wired, tightly complex feedback regulation[33] and lengthy reaction steps (>20 steps) of the de novo pathway, bioconversion by the Ehrlich pathway is considered as the preferred biological route to synthesize 2PE.[28]
In the Ehrlich pathway (Figure 1), L-phenylalanine is converted to 2-PE through four enzyme-dependent processes: (i) extracellular L-phenylalanine is internalized by amino permeases; (ii) L-phenylalanine is transaminated to phenylpyruvate by amino transferase with 2-oxoglutarate as the amine-receptor; (iii) phenylpyruvate is further decarboxylated to phenylacetaldehyde by phenylpyruvate decarboxylases; (iv) and phenylacetaldehyde is reduced to 2-PE by alcohol dehydrogenases or phenylacetaldehyde reductase with NADH as cofactor

Summary

Introduction

Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation. Substrate cofeeding could bypass the lengthy and highly regulated native metabolism and facilitates high carbon conversion rate. ACS Synthetic Biology pubs.acs.org/synthbio leveraged for product formation (namely, cosubstrate feeding).[26] One recent example is to use the acetate-driven acetylCoA metabolic shortcut to bypass the lengthy and highly regulated glycolytic pathway for efficient polyketides[27] and lipids production.[24] Such substrate doping strategy may present a metabolic advantage over a single substrate and lead to metabolic optimality beyond what can be achieved in single substrate fermentations. Metabolite doping or cosubstrate feeding can overcome intrinsic pathway limitations and achieve high carbon conversion efficiency and costefficiency. Taken together, refactoring critical enzymes and increasing metabolite trafficking as well as mitigating competitive pathways are effective strategies to improve the efficiency of overall pathway yield

Methods

Results

Conclusion