Screening of novel β-carotene hydroxylases for the production of β-cryptoxanthin and zeaxanthin and the impact of enzyme localization and crowding on their production in Yarrowia lipolytica.

IF 4.3 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Microbial Cell Factories Pub Date : 2024-11-05 DOI:10.1186/s12934-024-02569-w
Mladen Soldat, Tadej Markuš, Vasilka Magdevska, Martin Kavšček, Aleksander Johannes Kruis, Jaka Horvat, Gregor Kosec, Štefan Fujs, Uroš Petrovič
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Abstract

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

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筛选新型β-胡萝卜素羟化酶以生产β-隐黄素和玉米黄质,以及酶定位和排挤对脂肪溶解亚罗维氏菌(Yarrowia lipolytica)生产β-隐黄素和玉米黄质的影响。
玉米黄质是一种重要的膳食类胡萝卜素,由植物、微藻和某些微生物自然合成。大规模生产玉米黄质可以通过植物提取、化学合成或微生物发酵来实现。前两种方法对环境和健康的影响使微生物发酵成为天然玉米黄质生产的一种有吸引力的替代方法,尽管在扩大生物工艺规模方面存在挑战。β-胡萝卜素和玉米黄质之间的中间体--β-隐黄素,只存在于特定的水果和蔬菜中,对人体健康有多种重要功能。这些来源中的β-隐黄素浓度较低,导致提取率较低,因此生物技术生产是提高提取率的一个很有前途的选择。目前,还没有与工业相关的微生物发酵工艺来生产 β-隐黄素,这主要是由于缺乏已确定的酶(能将 β-胡萝卜素特异性地转化为 β-隐黄素,而不会进一步转化为玉米黄质)。在这项研究中,我们利用基因工程技术,将含油酵母脂溶性亚罗酵母(Yarrowia lipolytica)作为生产玉米黄质和β-隐黄质的生物工厂。我们筛选了 22 种 β-胡萝卜素羟化酶,发现了 8 种具有 β-胡萝卜素羟化活性的新型酶:其中 6 种产生玉米黄质,2 种只产生 β-隐黄质。通过引入克氏软杆菌(Chondromyces crocatus,CcBCH)的β-胡萝卜素羟化酶,β-隐黄素的滴度达到了 24 ± 6 mg/L,这是迄今为止在脂溶性酵母菌中报告的唯一β-隐黄素的最高滴度。通过将产生玉米黄质的β-胡萝卜素羟化酶靶向到内质网和过氧物酶体,与未靶向的酶相比,我们将玉米黄质的产量分别提高了54%和66%。将β-胡萝卜素羟化酶靶向到过氧物酶体后,玉米黄质滴度最高,达到412 ± 34 mg/L。此外,通过构建带有短肽标签RIDD和RIAD的无支架多酶复合物,我们观察到与表达未修饰酶的菌株相比,玉米黄质滴度提高了39%,转化率提高了28%。本研究中获得的玉米黄质滴度并不是所报道的最高滴度;但是,我们的目标是证明特定的方法可以提高滴度和转化率,而不是达到最高滴度。这些发现强调了Y. lipolytica作为类胡萝卜素生产平台的潜力,并为未来的研究奠定了基础,未来的研究需要进一步优化,以最大限度地提高产量。
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来源期刊
Microbial Cell Factories
Microbial Cell Factories 工程技术-生物工程与应用微生物
CiteScore
9.30
自引率
4.70%
发文量
235
审稿时长
2.3 months
期刊介绍: Microbial Cell Factories is an open access peer-reviewed journal that covers any topic related to the development, use and investigation of microbial cells as producers of recombinant proteins and natural products, or as catalyzers of biological transformations of industrial interest. Microbial Cell Factories is the world leading, primary research journal fully focusing on Applied Microbiology. The journal is divided into the following editorial sections: -Metabolic engineering -Synthetic biology -Whole-cell biocatalysis -Microbial regulations -Recombinant protein production/bioprocessing -Production of natural compounds -Systems biology of cell factories -Microbial production processes -Cell-free systems
期刊最新文献
Combined metabolic engineering and lipid droplets degradation to increase vitamin A production in Saccharomyces cerevisiae. Increased cytoplasmic expression of PETase enzymes in E. coli. Optimized production of a truncated form of the recombinant neuraminidase of influenza virus in Escherichia coli as host with suitable functional activity. Retraction Note: Potential use of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition and prevention method in viral infection. Multiscale modelling of bioprocess dynamics and cellular growth.
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