Dataset Information

Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production.

ABSTRACT:

Background

Vanillin represents one of the most widely used flavoring agents in the world. However, microbial synthesis of vanillin is hindered by the host native metabolism that could rapidly degrade vanillin to the byproducts.

Results

Here, we report that the industrial workhorse Saccharomyces cerevisiae was engineered by systematic deletion of oxidoreductases to improve the vanillin accumulation. Subsequently, we harnessed the minimal aromatic aldehyde reduction (MARE) yeast platform for de novo synthesis of vanillin from glucose. We investigated multiple coenzyme-A free pathways to improve vanillin production in yeast. The vanillin productivity in yeast was enhanced by multidimensional engineering to optimize the supply of cofactors (NADPH and S-adenosylmethionine) together with metabolic reconfiguration of yeast central metabolism. The final yeast strain with overall 24 genetic modifications produced 365.55 ± 7.42 mg l^-1 vanillin in shake-flasks, which represents the best reported vanillin titer from glucose in yeast.

Conclusions

The success of vanillin overproduction in budding yeast showcases the great potential of synthetic biology for the creation of suitable biocatalysts to meet the requirement in industry. Our work lays a foundation for the future implementation of microbial production of aromatic aldehydes in budding yeast.

SUBMITTER: Mo Q

PROVIDER: S-EPMC10771647 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Publications

Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production.

Mo Qiwen Q Yuan Jifeng J

Biotechnology for biofuels and bioproducts 20240106 1

<h4>Background</h4>Vanillin represents one of the most widely used flavoring agents in the world. However, microbial synthesis of vanillin is hindered by the host native metabolism that could rapidly degrade vanillin to the byproducts.<h4>Results</h4>Here, we report that the industrial workhorse Saccharomyces cerevisiae was engineered by systematic deletion of oxidoreductases to improve the vanillin accumulation. Subsequently, we harnessed the minimal aromatic aldehyde reduction (MARE) yeast pla ...[more]

PMID: 38184607

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Project description:Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

Project description:Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds.ImportanceThe final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.

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Project description:One of the key issues affecting yields of biovanillin in cell factories such as Escherichia coli is product toxicity, the mechanisms of which are poorly understood. To identify targets for engineering improved strains, we have studied mechanisms of vanillin toxicity in E. coli using a two-pronged apparoach: (i) a global proteomic analysis supported by multiple physiological experiments and mutant analyses (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome sequencing. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin. Upregulated proteins included enzymes capable of converting aldehydes to potentially less toxic compounds; pentose phosphate pathways enzymes, fumarase C and enzymes of the glyoxylate shunt; proteins involved in several key stress responses, in particular oxidative stress; proteins mediating uptake and processing of metal ions. 1H-NMR confirmed that E. coli detoxifies vanillin by reduction to vanillyl alcohol; the aldehyde reductases YqhD and DkgA were highly upregulated by vanillin and the purified enzymes reduced vanillinin an NADPH dependent manner. Vanillin caused accumulation of reactive oxygen species and activated an oxidative stress response through SoxRS, OxyR and MarA pathways. Slow vanillin dependent copper (II) to copper (I) reduction lead to upregulation of the copA gene and growth in the presence of vanillin was hypersensitive to inhibition by copper ions. RT-PCR and mutant growth data suggested AcrD and AaeAB as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct non-synonymous SNPs in the citrate synthase gene gltA, in addition to strain specific mutations in cpdA, rob and marC. One strain had a large ~10 kb deletion including the marRAB region. Purifed variant GltA enzymes all showed higher activity due to a lowered Km for oxaloacetate. Our data provide new understanding and novel gene targets for engineering vanillin-tolerant strains of E. coli, including deletion of the efflux pumps eamA, acrA, and acrB, enhancing oxidative stress defences and NADPH production, improving copper homeostasis and increasing citrate synthase activity using variant enzymes.

Dataset Information

Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production.

Background

Results

Conclusions

Publications

Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production.

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