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Establishing heterologous betaxanthin pigment biosynthesis in cyanobacteria

ABSTRACT:

Betalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants. But due to low concentrations of native pigments, the process is inherently inefficient. Now, an increase in consumer demand calls for the development of scalable and sustainable betalain production routes. To address this challenge, we introduced a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of an engineered variant of the cytochrome P450 CYP76AD1 (W13L, F309L) and the l‐DOPA 4,5‐dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroGfbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of low levels of phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO2-enriched air) increased titers approximately 165 times and led to the production of phenylalanine-betaxanthin with a final titer of 18.2 ± 5.1 mg L–1. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.

INSTRUMENT(S): Liquid Chromatography MS -

PROVIDER: MTBLS12639 | MetaboLights | 2026-01-06

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS12639_LC-MS___metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS12639_LC-MS___metabolite_profiling_v2_maf.tsv	Tabular
	s_MTBLS12639.txt	Txt

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Project description:Its characteristic rose-like aroma makes phenylethanol a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbor an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source such as glucose provides an economically attractive alternative for phenylalanine bioconversion. In this study, a Synthetic Genetic Array screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to cause a transcriptional upregulation of ARO10 during growth with ammonium sulfate as the sole nitrogen source. Physiological characterization revealed that the aro8M-oM-^AM-^D mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8 mutation also stimulated phenylethanol production when combined with other, previously documented mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced over 3 mM of phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. The goal of the present study was to identify genes that influence the transcriptional (de)repression of the Ehrlich pathway during growth with ammonium as the nitrogen source. With the aid of Synthetic Genetic Array technology, we constructed a strain collection in which deletions in the non-essential genes in the S. cerevisiae genome were combined with a reporter plasmid comprising the ARO10 promoter fused to a reporter gene (egfp) encoding a fluorescent reporter protein. After screening by flow cytometry, deletion of ARO8 led to a deregulated expression from the ARO10 promoter. The impact of this deletion was further studied by transcriptome and intracellular metabolite analyses. Furthermore, phenylethanol production was measured in strains that combined the aro8 mutation with mutations that were previously shown to deregulate aromatic amino acid biosynthesis.

Project description:Background: Anthocyanins are the most important compounds for nutritional quality and economic values of blood orange. However, there are few reports on the pre-harvest treatment accelerate the accumulation of anthocyanins in postharvest blood orange fruit. Here, we performed a comparative Transcriptome and metabolomics analysis to elucidate the underlying mechanism involved in seasonal drought (SD) treatment during fruit expansion stage on anthocyanin accumulation in postharvest ‘Tarocco’ blood orange fruit. Results: Our results showed that SD treatment slowed down the fruit enlargement and increased the sugar accumulation during fruit development and matured period. Obviously, under SD treatment, the accumulation of anthocyanin in blood orange fruit during postharvest storage was significantly accelerated and markedly higher than that in CK. Meanwhile, the total flavonoids and phenols contents and antioxidant activity in SD treatment fruit were also sensibly increased during postharvest storage. Based on metabolome, we found that substrates required for anthocyanin biosynthesis, such as amino acids and their derivatives, and phenolic acids, have significantly accumulated and higher in SD treated mature fruit compared with that of CK. Further according to the results of transcriptome data and weighted gene coexpression correlation network analysis (WGCNA) analysis, phenylalanine ammonia-lyase (PAL3) was considered key structural gene. qRT-PCR analysis verified that the PAL3 was highly expressed in SD treated postharvest stored fruit and was significantly positively correlated with the anthocyanin content. Moreover, we found that other structural genes in anthocyanin biosynthesis pathway were also upregulated under SD treatment through transcriptome data and qRT-PCR analysis. Conclusions: The findings suggest that SD treatment promotes the accumulation of substrates necessary for anthocyanin biosynthesis during fruit ripening process, and activates the expression of anthocyanin biosynthesis pathway genes during postharvest storage period, especially PAL3, co-contributed to the rapid accumulation of anthocyanin. The present study provides a theoretical basis for postharvest quality control and water-saving utilization of blood orange fruit.

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Establishing heterologous betaxanthin pigment biosynthesis in cyanobacteria

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