Enzymatic production and in situ separation of natural β-ionone from β-carotene

Nacke, Christoph; Hüttmann, Sonja; Etschmann, M. M. W.; Schrader, Jens

doi:10.1007/s10295-012-1182-1

Cited by 29 publications

(24 citation statements)

References 19 publications

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“…For example, it demonstrated the ability to inhibit breast cancer cells in vivo [9] and to limit tumor incidence in a rat cancer model [10]. Commercial β-ionone can be chemically synthesized, but such a product is less valuable than plant derived β-ionone due to recent increases in consumer desire for natural food products (i.e., natural aroma compounds have a range of 10–100 fold price increase over the chemically synthetic alternative) [11]. Natural aroma molecules can be obtained via extraction from plants [12], but low biochemical abundance makes product isolation labor intensive and costly.…”

Section: Introductionmentioning

confidence: 99%

“…A second method for obtaining natural β-ionone involves using in vitro enzymatic processes (i.e., the biotransformation of β-carotene to β-ionone). However, the enzymatic bio-transformations often lead to a variety of undesirable byproducts [11, 14]. These specific limitations motivate research into new reliable production processes for obtaining natural β-ionone.…”

Section: Introductionmentioning

confidence: 99%

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Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push–pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0984-x) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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“…These aromas are the result of enzyme activities performing hydroxylations, methylations, or epoxidation reactions. Aroma production from carotenoids requires activities such as peroxidases (Scheibner et al, 2008;Zelena et al, 2009) and dioxygenases (Rubio et al, 2008;Nacke et al, 2012). Recently, these studies have focused on the gene cloning and expression of carotenoids-cleaving enzymes (García-Limones et al, 2008;Baldermann et al, 2010;Young et al, 2012;Adami et al, 2013;Lashbrooke et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Identification of Bioproducts Generated Enzymatically by Cell-Free Extracts ofSaccharomyces cerevisiaefrom Cells Grown in the Presence or Absence of Heteranthin

2015

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Ditaxis heterantha seeds are a source of the apocarotenoid heteranthin (methyl 3-oxo-12´-apo-ε-caroten-12´-oate). Saccharomyces cerevisiae can degrade, utilize this apocarotenoid, and produce seven aromas (3-oxo-α-ionone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionone, 3-hydroxy-β-cyclocitral, safranal, 4-oxo-isophorone, and isophorone). However, nothing is known about the enzymology of this process or the steps to produce them. In the present work, cell-free extracts of S. cerevisiae from cells grown in the presence or absence of heteranthin were evaluated for its degradation. When grown in heteranthin, cell-free extracts produced the same seven aromas, while in its absence only four aromas (3-hydroxy-β-cyclocitral, safranal, 4-oxo-isophorone, and isophorone) were detected. Polyacrylamide gel electrophoresis (PAGE) of cell extracts previously grown in heteranthin yielded a protein band of 27.6 kDa able to cleave this apocarotenoid. Band intensity was proportional to the heteranthin concentration and was not present in extracts from cultures grown in the absence of heteranthin. A purified sample of 27.6 kDa protein produced 3-oxo-α-ionone from heteranthin as the only reaction product, suggesting oxidation of the apocarotenoid at the 9-10 position of its moiety. Maximal enzymatic production of 3-oxo-α-ionone was observed at pH 5.5 and 35 • C with activation energy of 7.23 kJ mol −1 . The enzyme exhibited a K m value of 27 µM for heteranthin and a V max of 135 µmoles min −1 mg −1 . Our findings clearly supported the presence of at least two ways for heteranthin degradation in S. cerevisiae: one that is probably committed to the heteranthin-induced synthesis of 3-oxo-α-ionol, and the other, constitutive, likely committed to safranal production.

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“…Using a polyoctylmethylsiloxane membrane the process yielded higher concentrations of esters; using a polyetheramide-block-copolymer membrane the permeate contained higher concentrations of the terpenoids citronellol and geraniol. Another approach also used a flat-sheet organophilic pervaporation membrane to demonstrate proof-ofprinciple for in situ separation of the C13-norisoprenoid β-ionone from enzymatic β-carotene cleavage [59]. The biggest challenge still hampering a broader application of organophilic pervaporation for in situ volatiles separation is the limited transmembrane flux of the product.…”

Section: Stripping and Pervaporationmentioning

confidence: 99%

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

Schewe

Mirata²,

Schrader

2015

Biotechnology of Isoprenoids

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Isoprenoids represent a natural product class essential to living organisms. Moreover, industrially relevant isoprenoid molecules cover a wide range of products such as pharmaceuticals, flavors and fragrances, or even biofuels. Their often complex structure makes chemical synthesis a difficult and expensive task and extraction from natural sources is typically low yielding. This has led to intense research for biotechnological production of isoprenoids by microbial de novo synthesis or biotransformation. Here, metabolic engineering, including synthetic biology approaches, is the key technology to develop efficient production strains in the first place. Bioprocess engineering, particularly in situ product removal (ISPR), is the second essential technology for the development of industrial-scale bioprocesses. A number of elaborate bioreactor and ISPR designs have been published to target the problems of isoprenoid synthesis and conversion, such as toxicity and product inhibition. However, despite the many exciting applications of isoprenoids, research on isoprenoid-specific bioprocesses has mostly been, and still is, limited to small-scale proof-of-concept approaches. This review presents and categorizes different ISPR solutions for biotechnological isoprenoid production and also addresses the main challenges en route towards industrial application.

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Enzymatic production and in situ separation of natural β-ionone from β-carotene

Cited by 29 publications

References 19 publications

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

Identification of Bioproducts Generated Enzymatically by Cell-Free Extracts ofSaccharomyces cerevisiaefrom Cells Grown in the Presence or Absence of Heteranthin

Bioprocess Engineering for Microbial Synthesis and Conversion of Isoprenoids

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