Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2023-06-26 DOI:10.1016/j.mec.2023.e00226
Mark Seger , Fakhriyya Mammadova , Melany Villegas-Valencia , Bárbara Bastos de Freitas , Clarissa Chang , Iona Isachsen , Haley Hemstreet , Fatimah Abualsaud , Malia Boring , Peter J. Lammers , Kyle J. Lauersen
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引用次数: 2

Abstract

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal β-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

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工程化酮类胡萝卜素在多亲性红微藻Cyanidoschyzon merolae 10D中的生物合成
多亲蓝藻科是一种真核红色微藻,由于其低pH值和高温度要求,可以最大限度地减少大规模培养污染,因此具有良好的生物技术特性。Cyanidoschyzon merolae 10D是一种细胞壁缺陷物种,具有完全测序的基因组,可通过靶向同源重组进行核转基因整合。C.merolae保持着最小的类胡萝卜素谱,在这里,我们试图确定其通过异源表达绿藻β-胡萝卜素酮症酸酶(BKT)和羟化酶(CHYB)介导的酮类胡萝卜素积累的能力。为了实现这一点,建立了一个合成的转基因表达盒系统,通过将天然的C.merolae转录、翻译和叶绿体靶向信号融合到密码子优化的编码序列中,整合和表达来源于莱茵衣藻(Cr)的酶。使用氯霉素抗性来选择将合成的线性DNA整合到宿主基因组内的中性位点中。CrBKT的表达导致角黄素和阿多尼鲁宾作为主要类胡萝卜素的积累,而CrBKT与CrCHYB的共表达则产生虾青素作为主要的类胡萝卜素。与绿藻和植物不同,酮类胡萝卜素在C.merolae中的积累并没有降低类胡萝卜素的总含量,但观察到叶绿素a的减少。光照强度影响所有色素的整体比率,但不影响单个色素组成,并且藻蓝蛋白含量在亲本菌株和转化体之间没有显著差异。连续光照可以促进生物量的积累,所有菌株都可以在模拟的夏季条件下从两种不同的极端沙漠环境中培养。我们的发现提供了在红色真核微藻中进行类胡萝卜素代谢工程的第一个例子,并为使用C.merolae 10D同时生产藻蓝蛋白和酮类胡萝卜素色素开辟了可能性。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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