鈕亭亭,孫茜萍,吳 濤

(1.上海體育學(xué)院,上海 200438;2.浙江工業(yè)大學(xué) 海洋學(xué)院,浙江 杭州 310014)

酵母生產(chǎn)類胡蘿卜素的研究進(jìn)展

鈕亭亭1,孫茜萍2,吳 濤2

(1.上海體育學(xué)院,上海 200438;2.浙江工業(yè)大學(xué) 海洋學(xué)院,浙江 杭州 310014)

類胡蘿卜素是醫(yī)藥、化學(xué)、食品和飼料產(chǎn)業(yè)中具有重要價(jià)值的產(chǎn)品,具有著色、抗氧化和防癌等功能.動(dòng)物不能合成類胡蘿卜素,因此必須從食物中進(jìn)行攝取.作為潛在色素物質(zhì)的來(lái)源,紅酵母和法夫酵母是微生物法生產(chǎn)類胡蘿卜素的重要菌種.闡述了紅酵母和法夫酵母生物合成類胡蘿卜素的途徑、影響發(fā)酵產(chǎn)量的因素及廉價(jià)培養(yǎng)基的應(yīng)用,進(jìn)一步介紹了誘變育種方法及代謝工程技術(shù)在產(chǎn)類胡蘿卜素酵母菌株改良上的應(yīng)用.

類胡蘿卜素;紅酵母;法夫酵母;廉價(jià)培養(yǎng)基

類胡蘿卜素對(duì)動(dòng)物和人類具有重要意義,包括增強(qiáng)免疫、作為維生素A前體和淬滅氧自由基的組分.食用類胡蘿卜素有益于預(yù)防多種疾病,如動(dòng)脈硬化、白內(nèi)障、多硬化和癌癥.動(dòng)物體內(nèi)不能自發(fā)合成類胡蘿卜素,必須從含類胡蘿卜素的飼料中攝取.相比從蔬菜中提取或者化學(xué)合成類胡蘿卜素,微生物發(fā)酵生產(chǎn)類胡蘿卜素只需要低成本天然底物作為碳源,具備明顯的經(jīng)濟(jì)優(yōu)勢(shì).紅酵母和法夫酵母[1]具有合成各種天然類胡蘿卜素(β-胡蘿卜素、紅酵母烯、紅酵母紅素和蝦青素等)的能力,比藻類和霉菌更適于大規(guī)模發(fā)酵生產(chǎn).另外以代謝工程方法構(gòu)建的釀酒酵母也表現(xiàn)出產(chǎn)類胡蘿卜素的良好優(yōu)勢(shì).

1 紅酵母、法夫酵母的類胡蘿卜素合成途徑

紅酵母(Rhodotorula)和法夫酵母(Phaffia)是產(chǎn)類胡蘿卜素的常見(jiàn)酵母種屬.由紅酵母生產(chǎn)的類胡蘿卜素種類主要為β-胡蘿卜素、紅酵母烯(3′,4′-雙脫氫-β-ψ-胡蘿卜素)和紅酵母紅素(3′,4′-雙脫氫-β-ψ-胡蘿卜素-16′-甲酸),三種主要的類胡蘿卜素占總量的相對(duì)比例不確定,這與菌株及培養(yǎng)條件相關(guān).Rhodotorulaglutinis和Rhodotorulagraminis生產(chǎn)的γ-胡蘿卜素量(β-γ-胡蘿卜素)占類胡蘿卜素總量11%～15%,而法夫酵母主要生產(chǎn)蝦青素(3,3-脫氫-β-β-胡蘿卜素-4,4-二酮).

1964年,Simpson[2]及Goodwin[3]重新審視了酵母合成類胡蘿卜素的一般途徑:1) 由HMG-COA合酶催化乙酰輔酶A向3-羥基-3-甲基戊二酸單酰輔酶A(HMG-COA)轉(zhuǎn)化,HMG-COA再轉(zhuǎn)化為一個(gè)C6化合物甲羥戊酸(MVA),MVA通過(guò)MVA激酶磷酸化、脫羧等一系列反應(yīng)進(jìn)一步轉(zhuǎn)化為異戊二烯基焦磷酸(IPP);2) IPP與依次加入的三個(gè)IPP分子異構(gòu)化得到二甲基烯丙基焦磷酸(DMAPP),上述反應(yīng)產(chǎn)物由異戊烯基轉(zhuǎn)移酶催化得到C20化合物香葉基焦磷酸(GGPP),兩分子GGPP縮合得到八氫番茄紅素,脫氫后得到番茄紅素;3) 番茄紅素異構(gòu)化同時(shí)發(fā)生在八氫番茄紅素脫氫形成第一或第二個(gè)雙鍵過(guò)程中,番茄紅素作為環(huán)狀結(jié)構(gòu)類胡蘿卜素前體,經(jīng)一系列代謝反應(yīng)形成β-胡蘿卜素、γ-胡蘿卜素、紅酵母烯、紅酵母紅素和蝦青素.

2 影響酵母發(fā)酵生產(chǎn)類胡蘿卜素的因素

在工業(yè)規(guī)模下利用微生物生產(chǎn)類胡蘿卜素必須尋求工藝成本低、產(chǎn)率高以及環(huán)境友好的技術(shù).然而,類胡蘿卜素的生物合成受諸多因素影響,例如光照、溫度、通氣和金屬離子都有可能對(duì)類胡蘿卜素發(fā)酵產(chǎn)量產(chǎn)生影響.

光照被認(rèn)為是微生物生產(chǎn)類胡蘿卜素的一個(gè)重要因素.微生物需要防止自身由于光照而受到損傷,類胡蘿卜素生物合成機(jī)制就是一個(gè)光保護(hù)機(jī)制.類胡蘿卜素生產(chǎn)受到白光的積極影響.Moliné等[4]研究紅酵母菌株中類胡蘿卜素和麥角固醇以及細(xì)胞紫外光抗性之間的關(guān)系,結(jié)果表明超著色菌株存活率增大(250%).他們還指出,紅酵母紅素的高產(chǎn)量可以提高紫外光下紅酵母的存活率.另一方面,Yen等[5]計(jì)算了在具有兩個(gè)LED(發(fā)光二極管)燈的間歇反應(yīng)器中β-胡蘿卜素的生產(chǎn)效率,結(jié)果β-胡蘿卜素濃度達(dá)24.6 μg/g,而無(wú)光照條件下β-胡蘿卜素濃度只有14.69 μg/g.

溫度是酵母生產(chǎn)類胡蘿卜素過(guò)程中需要考慮到的另一個(gè)因素.溫度會(huì)影響細(xì)胞的生長(zhǎng)以及代謝產(chǎn)物的生產(chǎn),主要通過(guò)改變生物合成途徑包括類胡蘿卜素合成途徑來(lái)發(fā)揮作用.溫度的影響效果取決于微生物的種類以及產(chǎn)品產(chǎn)量.根據(jù)Hayman等[6]的研究,溫度影響了涉及類胡蘿卜素生產(chǎn)的酶濃度的調(diào)節(jié)水平.

通氣影響類胡蘿卜素生產(chǎn)的種類和總產(chǎn)量.這是因?yàn)轭惡}卜素生物合成是一個(gè)需氧過(guò)程,氧氣影響著基質(zhì)同化的速率、生物量的增長(zhǎng)以及類胡蘿卜素的生物合成.通氣的影響取決于微生物的種類.Saenge等[7]研究了通氣量對(duì)細(xì)胞生長(zhǎng)、脂質(zhì)產(chǎn)量、類胡蘿卜素產(chǎn)量及甘油消耗量的影響,當(dāng)通氣速率從0 vvm增加到2 vvm時(shí),生物量和脂質(zhì)產(chǎn)量均達(dá)到最高,分別為8.17, 4.32 g/L.

金屬離子(鋇、鐵、鎂、鈣、鋅和鈷)也已經(jīng)被證明是R.Glutinis生產(chǎn)類胡蘿卜素的促進(jìn)劑[8].除此以外,Buzzini等[9]報(bào)道了在R.Graminis中某些微量元素表現(xiàn)出對(duì)類胡蘿卜素的結(jié)構(gòu)具有選擇性的影響,比如Zn2+對(duì)β胡蘿卜素和γ-胡蘿卜素生產(chǎn)具有促進(jìn)作用,而對(duì)紅酵母烯和紅酵母紅素生產(chǎn)具有抑制作用.

3 廉價(jià)培養(yǎng)基的應(yīng)用

絕大多數(shù)酵母的類胡蘿卜素在菌種對(duì)數(shù)生長(zhǎng)后期積累,在穩(wěn)定期持續(xù)增加.在含有多種精細(xì)碳源(如葡萄糖、木糖、纖維二糖、蔗糖、甘油和山梨糖醇)的合成培養(yǎng)基中培養(yǎng)時(shí),酵母菌能合成類胡蘿卜素.以天然底物(如葡萄汁、未發(fā)酵葡萄汁、棗汁、芥末廢棄物水解液提取物、半纖維素水解液、水解綠豆廢渣、甘蔗汁、甘蔗、玉米糖漿、玉米水解液、乳清等)為碳源的類胡蘿卜素合成研究已成為近些年的熱點(diǎn).工農(nóng)業(yè)原產(chǎn)地的原料和副產(chǎn)物被認(rèn)為是廉價(jià)微生物發(fā)酵生產(chǎn)過(guò)程中所需碳水化合物的可替代來(lái)源,這也同時(shí)減輕了因廢棄物造成的環(huán)境問(wèn)題.雞毛和甜馬鈴薯已作為氮源和碳源[10]來(lái)生產(chǎn)類胡蘿卜素,同時(shí)解決了雞毛和甜馬鈴薯廢棄物環(huán)保處理的能耗問(wèn)題,最大限度地減輕了這些廢棄物對(duì)環(huán)境的壓力.表1列出了最新的工農(nóng)業(yè)廢渣在類胡蘿卜素發(fā)酵生產(chǎn)中的應(yīng)用實(shí)例.用工農(nóng)業(yè)廢棄物生產(chǎn)類胡蘿卜素,其生產(chǎn)能力取決于碳源和氮源的種類、礦物質(zhì)和其他成分的配比.這些營(yíng)養(yǎng)物質(zhì)的種類和配比對(duì)確定發(fā)酵培養(yǎng)基制定方法、提高微生物的類胡蘿卜素生物合成能力非常重要.

表1 酵母以工農(nóng)業(yè)廢棄物作為基質(zhì) 發(fā)酵生產(chǎn)類胡蘿卜素的研究

Table 1 Researches of using agro-industrial wastes as substrates to yeasts carotenoid production

基質(zhì)酵母產(chǎn)量乳清R．glutinis46．00mg/Lβ?胡蘿卜素[11]馬鈴薯培養(yǎng)基R．mucilaginosa56．00mg/Lβ?胡蘿卜素[11]粗甘油R．glutinis135．20mg/L類胡蘿卜素[7]雞毛R．glutinis92．00mg/L類胡蘿卜素[12]乳清超濾液R．a(chǎn)cheniorum262．00mg/Lβ?胡蘿卜素[13]乳清S．salmonicolor590．40μg/L類胡蘿卜素[14]發(fā)酵蘿卜鹽水R．glutinis19．00μg/(L·h)β?胡蘿卜素[15]發(fā)酵蘿卜鹽水R．glutinis201．00μg/Lβ?胡蘿卜素[16]綠豆廢棄面粉和紅薯提取物R．glutinis3．48mg/L類胡蘿卜素[17]超濾乳清R．glutinis10．20mg/L類胡蘿卜素[18?19]

4 產(chǎn)類胡蘿卜素酵母菌選育策略

4.1 誘 變

Vijayalaksmi等[20]以紫外線誘變R.gracilis菌株,其類胡蘿卜素合成能力較原始菌株提高了約1.8倍.粉色酵母株R.glutinis經(jīng)紫外誘變得到黃色誘變株32,其類胡蘿卜素產(chǎn)量比野生株高出24倍.Frengova等[21]發(fā)現(xiàn)NTG誘變菌R.rubra56-13也表現(xiàn)出了更高的類胡蘿卜素和β-胡蘿卜素生產(chǎn)能力.Wang等[22]用高靜水壓(300 MPa)重復(fù)5次處理后得到誘變株,其β-胡蘿卜素產(chǎn)量增加了57.89%.

暗紅色酵母菌株P(guān).rhodozyma經(jīng)甲基磺酸乙酯處理后其β-類胡蘿卜素產(chǎn)量是之前的5倍[23].NTG誘變XanthophyllomycesDendrourhous后分離得到兩種產(chǎn)類胡蘿卜素強(qiáng)化菌株X.DendrourhousJH1和JH2[24].蝦青素高產(chǎn)突變株JH1產(chǎn)蝦青素約為野生型的15倍多.產(chǎn)β-胡蘿卜素突變株JH2的β-胡蘿卜素產(chǎn)量比野生型增產(chǎn)約4倍.An等[25]發(fā)現(xiàn)抗霉素突變株和亞硝基胍衍生突變株產(chǎn)蝦青素能力比原始菌株高出很多.Schroeder等[26]還發(fā)現(xiàn)游離氧自由基能夠通過(guò)激活基因誘發(fā)P.rhodozyma體內(nèi)類胡蘿卜素的合成.Fleno等[27]用EMS及紫外線處理得到高產(chǎn)類胡蘿卜素和蝦青素的突變株.

4.2 產(chǎn)類胡蘿卜素酵母的代謝工程

代謝工程通過(guò)具體的生化反應(yīng)或利用重組DNA技術(shù)引進(jìn)新的基因來(lái)改進(jìn)細(xì)胞性能和提高目標(biāo)產(chǎn)物的產(chǎn)量[28].非類胡蘿卜素生物合成微生物的改造工程是類胡蘿卜素生產(chǎn)非常有用的工具,如釀酒酵母被認(rèn)為是一種安全的酵母并具有容易進(jìn)行基因操作等優(yōu)點(diǎn).雖然自然條件下釀酒酵母不產(chǎn)生類胡蘿卜素,但它產(chǎn)生香葉基焦磷酸,如果此酵母集成了源于法夫酵母的兩個(gè)主要的類胡蘿卜素合成酶的基因(即編碼番茄紅素合成酶(crtYB)和番茄紅素脫氫酶(CTRI)的基因)就可以生產(chǎn)類胡蘿卜素[29].Yamano等[30]早期嘗試把細(xì)菌基因?qū)脶劸平湍钢猩a(chǎn)β-胡蘿卜素,雖然在工程上取得了成功,但是生產(chǎn)水平低下,產(chǎn)量?jī)H103 μg/g.畢赤酵母也是進(jìn)行類胡蘿卜素生產(chǎn)研究的非類胡蘿卜素生物合成酵母,主要優(yōu)勢(shì)在于它可以在甲醇中生長(zhǎng).Araya-Garay等[31]設(shè)計(jì)并構(gòu)建了兩個(gè)含有編碼番茄紅素和β胡蘿卜素的基因的質(zhì)粒并轉(zhuǎn)入畢赤酵母中,結(jié)果表明得到的重組菌株番茄紅素和β胡蘿卜素產(chǎn)量分別達(dá)到1.141,339 μg/g.

5 結(jié) 論

類胡蘿卜素在人類健康領(lǐng)域發(fā)揮著重要作用.化學(xué)合成類胡蘿卜素雖能滿足市場(chǎng)的部分需求,但其生產(chǎn)過(guò)程不夠環(huán)保高效,產(chǎn)品結(jié)構(gòu)和天然類胡蘿卜素也有差異.目前,對(duì)微生物生產(chǎn)類胡蘿卜素已經(jīng)進(jìn)行了廣泛研究,微生物發(fā)酵生產(chǎn)類胡蘿卜素不存在由于季節(jié)性和地域性變化而導(dǎo)致的產(chǎn)品和市場(chǎng)問(wèn)題,從而具有更大的經(jīng)濟(jì)效益.目前,紅酵母和法夫酵母是類胡蘿卜素的潛在來(lái)源,而以代謝工程方法改造釀酒酵母生產(chǎn)類胡蘿卜素也具有良好前景.生產(chǎn)成本高是大規(guī)模發(fā)酵生產(chǎn)類胡蘿卜素的最大限制因素,采用工農(nóng)業(yè)廢棄物等廉價(jià)底物可以降低生產(chǎn)成本,并且有助于減少?gòu)U棄物本身對(duì)環(huán)境的污染.通過(guò)酵母菌種的基因改造和發(fā)酵工藝的改進(jìn)等各種方法提高產(chǎn)量,將有助于實(shí)現(xiàn)微生物法發(fā)酵生產(chǎn)類胡蘿卜素產(chǎn)品的工業(yè)化.

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(責(zé)任編輯：朱小惠)

The research development of carotenoids production by yeast

NIU Tingting1, SUN Xiping2, WU Tao2

(1. Shanghai University of Sport, Shanghai 200438, China; 2. Ocean College, Zhejiang University of Technology, Hangzhou 310014, China)

Carotenoids represent a valuable product for the pharmaceutical, chemical, food and feed industries for their coloring, antioxidant and possible tumor-inhibiting activity. Animals cannot synthesize carotenoids, and these pigments must therefore be obtained from food. As the potential pigment sources, the generaRhodotorulaandPhaffiawere important strains for carotenoids production by microorganism method. In this review, the carotenoids biosynthesis pathway inRhodotorulaandPhaffiawas interpreted and the influence factors and low-cost media were reviewed. It was further introduced that mutation breeding and metabolic engineering technology was applied in yeast strain improvement for the carotenoid production.

carotenoids;Rhodotorula;Phaffia; low-cost media

2016-12-29

鈕亭亭(1985—),女,浙江湖州人,講師,碩士,研究方向?yàn)闋I(yíng)養(yǎng)學(xué),E-mail:niutingting@sus.edu.cn.通信作者:吳濤副教授,E-mail:wt_hz@zjut.edu.cn.

TQ92

A

1674-2214(2017)01-0050-04