摘要
虾青素（3,3’-二羟基-β,β’-胡萝卜素-4,4’-二酮）是从河螯虾外壳，牡蛎和鲑鱼中发现的一种红色类胡萝卜素，它具有抗氧化、抗衰老、抗肿瘤、预防心脑血管疾病作用。随着对虾青素需求量逐年加大，而化学方法合成的虾青素存在一定的安全性问题，所以从微生物（如红法夫酵母、雨生红球藻等）中提取天然虾青素是现今该领域的的热点研究问题。法夫酵母在发酵过程中需要消耗大量氧气用以维持细胞的正常代谢和虾青素的合成，如果要大幅提高法夫酵母发酵过程中的虾青素产量，需要重点解决发酵过程中酵母细胞对氧的摄取率问题。透明颤菌血红蛋白（VHb）是一种产自原核生物的氧结合蛋白，本研究尝试将透明颤菌血红蛋白基因（vgb）转入法夫酵母进行表达，试图借此提高酵母细胞对氧气的利用能力，进而达到促进菌体生长和提高产物合成的目的，而生产过程中降低搅拌速度和减少通气则降低了生产能耗，使得生产成本进一步降低。本研究还尝试在提取过程中添加适量的天然抗氧化剂（如生育酚）来提高虾青素的萃取得率，此外，在虾青素的提取过程中首次引入环境友好试剂如乳酸和乳酸乙酯。
（1）本研究利用RT-PCR技术分析了不同碳源对法夫酵母中虾青素合成途径中的关键酶的影响，发现不同碳源对细胞中虾青素合成途径的各关键酶的诱导作用不同，从而在分子水平上优化出了最佳碳源组合。最适碳源条件下，分析了不同溶氧对法夫酵母中关键酶表达的影响，发现25%溶氧最适合虾青素的合成。同时，这些现象也在分子层面上解释了细胞产色素能力和碳源、溶氧等培养条件之间的相互联系。
（2）本研究构建了包含cpc-1启动子、vgb基因和nos终止子的整合型表达载体pBC-Pcpc-vgb-Tnos，利用该载体，将vgb基因整合到法夫酵母的染色体上。摇瓶发酵实验结果显示：在含vgb基因的转化子中，其各项指标与对照菌株相比，以色素产量最高的一株B1为例，生物量、虾青素产量和虾青素含量分别提高了21%、37%和13%。将获得的色素高产菌株B1和C2通过斜面连续传五代后，发现两株菌产色素能力变化不大，最大降幅只有3.2%，说明该菌株生产性能比较稳定，此外，将高产菌株B1和C2在不含潮霉素的斜面上连续传五代后提取基因组DNA，通过PCR扩增vbg基因进行验证，B1和C2的传代菌株都能扩增出大约570bp大小的vgb片段，说明该质粒已经稳定的整合到法夫酵母的基因组DNA上，转化菌株的稳定性良好。
（3）本研究在酵母细胞破壁和虾青素萃取过程中加入适量的天然抗氧化剂（抗坏血酸和生育酚），发现随着温度的升高和时间的延长，色素得率比对照组有明显的提高。而环境友好试剂乳酸和乳酸乙酯分别进行破壁和提取实验后，结果显示破壁效率和萃取效率均高于传统试剂，而且安全无毒。通过设计正交试验对虾青素提取过程进行优化，在最佳条件下：提取时间30min，破壁温度65℃，破壁时间60min，提取试剂为乙酸乙酯︰乙醇=1︰1，色素得率达到1493.80±45.31μg g-1。
The red yeast Xanthophyllomyces dendrorhous (formerly known as Phaffiarhodozyma) is one of the most promising microorganisms for biotechnological productionof dietary astaxanthin. In the process of X. dendrorhous fermentation，large amount ofoxygen was consumed to maintain cell metabolic level. Vitreosilla Hemoglobin (VHb) is akind of oxygen-binding protein of prokaryotic origin. The aim of this study is to expressvgb gene in X. dendrorhous, and to improve cell growth and astaxanthin production byenhancing the oxygen usage ability of cell. In this study, we also reported that theapplication of lactic acid and ethyl lactate to astaxanthin extraction from X. dendrorhous,and efficacy of antioxidants addition in protecting against oxidative degradation during theextraction process.
(1) This report gives an insight into the specific changes in the transcription of fourkey carotenogenic genes (encoding geranylgeranyl diphosphate synthase (crtE), phytoenedesaturase (crtI), phytoene synthase lycopene cyclase (crtYB), and astaxanthin synthase(ast), respectively) in X. dendrorhous cultures, with regard to various dissolved oxygen(DO) contents and carbon sources. Our results suggested that a correlation betweenbiomass, astaxanthin production, and the transcripts of carotenogenic genes was evident inthe cells cultured with various substrates and DO concentration.
(2) The recombinant vector pBC-Pcpc-vgb-Tnos contain vgb gene which waspromoted by the promoter of cpc-1was constructed, and the Recombinant vector wasintegrated in the chromosome of X. dendrorhous by electroporation. We confirmedrecombinants by Hygromycin B selection and PCR, and screened out twoastaxanthin-overproducing strains from the recombinants. The biomass, astaxanthin yieldand astaxanthin content of engineering strains were increased by21%,37%and13%,respectively than the original strain. PCR analysis indicated vgb had been integrated in tothe chromosome of X. dendrorhous, and CO binding difference speetrum analysis ofrecombinant strain showed that active VHb protein has been expressed in X. dendrorhous.At the same time the recombinant strain was stable at the generation and yield, andsuitable for industrial application.
(3) The optimum extraction conditions were as follows: the yeast cell wall wasdisrupted by lactic acid at65oC for1h and then extracted with ethyl lactate: ethanol (1:1,v/v) for0.5h. The results showed that lactic acid and ethyl lactate has an obviouspredominance for the extraction of food-grade astaxanthin, and TOC was proved to be ahigh efficient natural antioxidant in our tests.
引文
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