,,

(1.齊魯工業(yè)大學(xué)食品科學(xué)與工程學(xué)院,山東濟(jì)南 250353;2.中國(guó)科學(xué)院過(guò)程工程研究所生化工程國(guó)家重點(diǎn)實(shí)驗(yàn)室,北京 100190)

短梗霉生產(chǎn)聚蘋(píng)果酸合成機(jī)制研究進(jìn)展

劉濱1,2,曹偉鋒2,于海峰1,*

(1.齊魯工業(yè)大學(xué)食品科學(xué)與工程學(xué)院,山東濟(jì)南 250353;2.中國(guó)科學(xué)院過(guò)程工程研究所生化工程國(guó)家重點(diǎn)實(shí)驗(yàn)室,北京 100190)

聚蘋(píng)果酸是一種可完全生物降解的高分子材料,具有高度水溶性、無(wú)免疫原性等優(yōu)良特性,在生物醫(yī)藥及生物材料領(lǐng)域具有廣泛應(yīng)用前景。文章介紹了微生物法合成聚蘋(píng)果酸的代謝途徑、關(guān)鍵酶系及其代謝調(diào)控機(jī)制,展望了聚蘋(píng)果酸合成代謝的研究方向。

聚蘋(píng)果酸,代謝途徑,關(guān)鍵酶系,代謝調(diào)控

聚蘋(píng)果酸(Poly malic acid,PMLA)是以蘋(píng)果酸為唯一單體通過(guò)酯鍵連接而成的高分子聚合物,有α型、β型和γ型三種結(jié)構(gòu)形態(tài)[1](圖1),在生物體內(nèi)存在的只有β-PMLA。因L-蘋(píng)果酸參與生物體內(nèi)的三羧酸循環(huán),因此β-PMLA容易在生物體內(nèi)通過(guò)三羧酸循環(huán)代謝途徑除去。由于β-PMLA具有懸掛羧基,因此可通過(guò)與其它官能團(tuán)、功能基團(tuán)或小分子藥物反應(yīng)而制得許多具有特殊功能的衍生物,如β-PMLA可通過(guò)共價(jià)鍵和非共價(jià)鍵與藥物結(jié)合,將藥物特異性靶向釋放至細(xì)胞質(zhì)內(nèi)[2]。由于β-PMLA具有良好的水溶性,當(dāng)水不溶性藥物與其連接后,可以有效地改善藥物的水溶性。目前,β-PMLA正被開(kāi)發(fā)應(yīng)用于藥物載體、微膠囊材料、生物醫(yī)學(xué)材料、化妝品、食品包裝材料及表面活性劑等諸多領(lǐng)域[3-5]。

生物發(fā)酵法是獲得β-PMLA的主要來(lái)源,通過(guò)微生物發(fā)酵法生產(chǎn)的聚蘋(píng)果酸分子量高,反應(yīng)條件溫和,但發(fā)酵法獲得的聚蘋(píng)果酸產(chǎn)量低,因此限制了β-PMLA大規(guī)模商業(yè)化應(yīng)用。為實(shí)現(xiàn)聚蘋(píng)果酸規(guī)?；l(fā)酵生產(chǎn),須對(duì)微生物代謝生成聚蘋(píng)果酸的代謝途徑有充分的認(rèn)知,從而通過(guò)對(duì)特定位點(diǎn)的代謝調(diào)控,實(shí)現(xiàn)提高聚蘋(píng)果酸產(chǎn)量的目的。因此,本文將詳述微生物法合成聚蘋(píng)果酸的代謝途徑、關(guān)鍵酶系及其代謝調(diào)控機(jī)制,并展望聚蘋(píng)果酸合成代謝的研究方向。

圖1 PMLA的結(jié)構(gòu)[1]Fig.1 Structure of polymalicacid[1]

1 β-聚蘋(píng)果酸的產(chǎn)生菌

β-PMLA作為酸性蛋白酶抑制劑在青霉菌(Penicilliumcyclopium)中首先發(fā)現(xiàn)[6],之后發(fā)現(xiàn)尋多頭絨泡菌(Physarumpolycephalum)[7-9]、短梗霉(Aureobasidiumspp.)[10-11]、出芽短梗霉(Aureobasidiumpullulans)[12-15]和其他黏菌及分絲孢子真菌都能合成β-PMLA。P.polycephalum分泌的β-PMLA最高濃度僅有2.7 g/L[7],且分子量范圍廣(從10～300 kDa不等)[16-17]。從不同環(huán)境中分離篩選的Aureobasidiumspp.具有合成大量的β-PMLA的能力[18],濃度可達(dá)152.5 g/L[19]。因此,Aureobasidiumspp.是有望工業(yè)化生產(chǎn)β-PMLA的菌株,已報(bào)道高產(chǎn)β-PMLA的產(chǎn)生菌如見(jiàn)表1所示。

表1 β-聚蘋(píng)果酸產(chǎn)生菌Table 1 β-PMLA production by different microorganisms

2 短梗霉合成β-PMLA的代謝途徑及關(guān)鍵酶系

2.1Aureobasidiumspp.合成β-PMLA的代謝途徑

合成β-PMLA代謝途徑的研究是基于L-蘋(píng)果酸是β-PMLA的前體物,通過(guò)添加代謝中間體及相關(guān)酶的抑制劑進(jìn)行。

Liu等[13]發(fā)現(xiàn)三氟乙酸抑制β-PMLA的合成,而丙二酸、馬來(lái)酸、丁二酸和蘋(píng)果酸促進(jìn)其合成,同時(shí)補(bǔ)充三氟乙酸與丁二酸或蘋(píng)果酸鹽能夠解除三氟乙酸的抑制作用,證明β-PMLA的合成涉及三羧酸循環(huán)途徑,異檸檬酸裂解酶和蘋(píng)果酸合成酶參與β-PMLA的合成。同時(shí)Liu等[13]依據(jù)丙二酸(琥珀酸脫氫酶的競(jìng)爭(zhēng)性抑制劑)對(duì)β-PMLA產(chǎn)生的促進(jìn)作用,證明乙醛酸代謝途徑是其產(chǎn)生的另一途徑。靳挺等[30]通過(guò)添加代謝中間體及抑制劑的實(shí)驗(yàn),推測(cè)β-PMLA的合成主要涉及TCA循環(huán)及乙醛酸循環(huán)。程若東等[31]通過(guò)向發(fā)酵培養(yǎng)基中添加TCA循環(huán)的抑制劑丙二酸和順丁烯二酸及酶活分析發(fā)現(xiàn)A.pullulansBS24產(chǎn)β-PMLA的合成途徑除了TCA循環(huán)外還存在乙醛酸途徑。Zou等[32]通過(guò)補(bǔ)充涉及TCA循環(huán)還原途徑的外源輔助因子及CO2供體提高了β-PMLA濃度。Cao等[33]發(fā)現(xiàn)丙酮酸強(qiáng)烈抑制β-PMLA的合成,通過(guò)在β-PMLA發(fā)酵過(guò)程中添加外源的中間代謝物和酶活抑制物,證明菌株A.Pullulansipe-1合成β-PMLA的主要代謝途徑為還原途徑,即磷酸烯醇式丙酮酸(PEP)在PEP羧化酶催化下經(jīng)由草酰乙酸合成β-PMLA。喬長(zhǎng)晟等[34]通過(guò)代謝通量分析及關(guān)鍵酶活測(cè)定發(fā)現(xiàn),丙酮酸羧化途徑及乙醛酸途徑是β-PMLA合成的主要途徑,TCA循環(huán)途徑在發(fā)酵后期代謝較弱,酶活分析證明了丙酮酸羧化途徑的加強(qiáng)是高產(chǎn)菌株比出發(fā)菌株的β-PMLA合成能力強(qiáng)的主要原因。綜合目前對(duì)A.pullulans合成β-PMLA研究成果,可將A.pullulans利用葡萄糖生成聚蘋(píng)果酸的途徑歸納為三條,即:草酰乙酸還原反應(yīng),TCA循環(huán),乙醛酸循環(huán)(如圖2所示)。

圖2 β-聚蘋(píng)果酸合成路徑Fig.2 Pathways for β-PMLA biosynthesis

2.2Aureobasidiumspp.合成β-PMLA的關(guān)鍵酶系及基因

3 Aureobasidium spp.合成β-PMLA的代謝調(diào)控

大多遺傳系統(tǒng)發(fā)育分支的Aureobasidiumspp.能夠大量合成β-PMLA[41],產(chǎn)量從9.8 g/L[12]到152.5 g/L[19]不等。因此,Aureobasidiumspp.在β-PMLA工業(yè)化生產(chǎn)中具有極大的潛力,通過(guò)對(duì)Aureobasidiumspp.合成β-PMLA的代謝過(guò)程調(diào)控,以達(dá)到提高β-PMLA產(chǎn)量的目的。目前出芽短梗霉發(fā)酵合成聚蘋(píng)果酸的調(diào)控研究主要集中在培養(yǎng)基優(yōu)化和發(fā)酵過(guò)程的調(diào)控。

3.1Aureobasidiumspp.合成β-PMLA培養(yǎng)基優(yōu)化

3.1.1 碳源、氮源的影響Aureobasidiumspp.發(fā)酵合成聚蘋(píng)果酸時(shí),可利用的碳源有葡萄糖、果糖、蔗糖、木糖、麥芽糖等,此外,一些生物質(zhì)也被用于聚蘋(píng)果酸的發(fā)酵生產(chǎn)。Zan等[29]以生甘薯為原料,將甘薯經(jīng)均質(zhì)、酸解、液化、糖化處理作為碳源,通過(guò)有氧纖維素床生物反應(yīng)器(AFBB)發(fā)酵,β-PMLA的產(chǎn)量可達(dá)57.5 g/L。Timothy等[26]對(duì)比了經(jīng)預(yù)堿性H2O2處理的玉米秸稈和小麥秸稈對(duì)出芽短梗霉發(fā)酵生產(chǎn)聚蘋(píng)果酸的影響,結(jié)果表明,經(jīng)過(guò)預(yù)處理的小麥秸稈的發(fā)酵性能優(yōu)于玉米秸稈,A.pullulansNRRL 50383在含有5%小麥秸稈,3% CaCO3培養(yǎng)基中可產(chǎn)生23.5 g/L的β-PMLA。Zhou等[27]考察了用玉米芯粉作為碳源對(duì)A.pullulanYJ 6-11產(chǎn)聚蘋(píng)果酸的影響,玉米芯水解液中糖類主要包含木糖、葡萄糖和少量的阿拉伯糖,結(jié)果發(fā)現(xiàn)用玉米芯水解液發(fā)酵產(chǎn)生聚蘋(píng)果酸的產(chǎn)量與用木糖和葡萄糖混合發(fā)酵時(shí)聚蘋(píng)果酸產(chǎn)量相近。Wang等[19]采用玉米漿與葡萄糖混合發(fā)酵,經(jīng)10L發(fā)酵罐發(fā)酵,最終獲得152.52 g/L的Ca2+-PMLA。Amartey等[42]報(bào)道玉米漿中含有豐富的營(yíng)養(yǎng)元素,包括生物素、氨基酸、礦物質(zhì)和其他營(yíng)養(yǎng)元素,玉米漿中所含有的生物素激活了丙酮酸羧化酶的活性,提高了β-PMLA的產(chǎn)量。玉米漿還可以提供氮源,氨基酸等滿足細(xì)胞生長(zhǎng)代謝所需的營(yíng)養(yǎng)成分。Cheng等[22]報(bào)道了采用大豆制品加工的副產(chǎn)物大豆皮和大豆糖蜜發(fā)酵生產(chǎn)聚蘋(píng)果酸,大豆糖蜜可無(wú)需經(jīng)過(guò)酶解預(yù)處理步驟而直接被A.pullulansZX-10利用,原因是A.pullulans中含有編碼α-半乳糖苷酶和β-呋喃糖苷酶的基因并能表達(dá),從而使A.pullulans直接分解利用大豆低聚糖。Wei等[23]研究了甘蔗糖蜜對(duì)發(fā)酵產(chǎn)生β-PMLA的影響,結(jié)果發(fā)現(xiàn)采用甘蔗糖蜜發(fā)酵時(shí)無(wú)需添加額外氮源即可滿足發(fā)酵需要,補(bǔ)加氮源反而會(huì)抑制β-PMLA的生成。

當(dāng)培養(yǎng)基中含有高濃度的氮源時(shí),聚蘋(píng)果酸的合成受到抑制[19]。Knuf等[43]發(fā)現(xiàn),在氮限制的發(fā)酵培養(yǎng)基中,Aspergillusoryzae胞內(nèi)大部分的糖酵解基因和與胞質(zhì)L-蘋(píng)果酸生產(chǎn)途徑相關(guān)的所有基因的表達(dá)都高度上調(diào)。R?dkr等[44]認(rèn)為在氮限制條件下,許多鋅指蛋白如Msn2/4、Gat1、Gln3和AreA參與了有機(jī)酸、胞外多糖、抗生素等的合成。殷海松等[45]報(bào)道了氨基酸對(duì)出芽短梗霉發(fā)酵生產(chǎn)聚蘋(píng)果酸的影響,發(fā)現(xiàn)隨著氨基酸添加量的增加,β-PMLA的產(chǎn)量先增加后減小,天冬氨酸、亮氨酸、纈氨酸和蘇氨酸對(duì)出芽短梗霉生長(zhǎng)和β-PMLA合成最有利。天冬氨酸對(duì)產(chǎn)物的合成影響最明顯,原因可能是因?yàn)槠渫ㄟ^(guò)轉(zhuǎn)氨作用形成草酰乙酸,草酰乙酸再還原為蘋(píng)果酸最終合成β-PMLA。王麗燕等[46]考察了NaNO3、蛋白胨、尿素、NH4Cl,丁二酸銨、(NH4)2SO4對(duì)β-PMLA產(chǎn)量的影響,結(jié)果發(fā)現(xiàn),丁二酸銨作為氮源時(shí)β-PMLA的產(chǎn)量最高。Wang等[47]研究了不同濃度的NH4NO3對(duì)細(xì)胞的生長(zhǎng)和β-PMLA產(chǎn)量的影響。發(fā)現(xiàn)在最低濃度(0.1 g/L)時(shí)在發(fā)酵前期無(wú)法滿足細(xì)胞生長(zhǎng)。在最高濃度(10 g/L),細(xì)胞生長(zhǎng)迅速,但發(fā)酵結(jié)束時(shí)仍有未被消耗的過(guò)量的氮源不利于β-PMLA的生成。當(dāng)在氮限制條件下(2 g/L)時(shí),β-PMLA產(chǎn)量最高。在發(fā)酵36 h后消耗完畢,發(fā)酵前期適量的氮源維持細(xì)胞生長(zhǎng),在發(fā)酵后期低濃度的氮有利于β-PMLA的合成。在氮限制條件下,參與聚蘋(píng)果酸合成(GLK、CS、FUM、DAT、MCL)和TOR信號(hào)通路(GS、TOR1、Tap42、Gat1)相關(guān)的基因表達(dá)水平明顯上調(diào)。在整個(gè)發(fā)酵過(guò)程中,細(xì)胞內(nèi)ATP/ADP的整體水平都高于氮飽和條件,更高水平的ATP可以提供聚合形成β-PMLA所需的能量。

Tu等[52]研究了吐溫80對(duì)β-PMLA發(fā)酵的影響,結(jié)果表明培養(yǎng)基中添加0.05%吐溫80,β-PMLA產(chǎn)量與對(duì)照組相比增加了75.08%。其原因是吐溫80在發(fā)酵前期能夠調(diào)控和增加氧吸收速率和二氧化碳釋放速率,改變細(xì)胞的形態(tài)有利于細(xì)胞與底物接觸。同時(shí)還發(fā)現(xiàn)線粒體二羧酸轉(zhuǎn)運(yùn)蛋白和跨膜轉(zhuǎn)運(yùn)蛋白的轉(zhuǎn)錄水平顯著上調(diào)。

3.2Aureobasidiumspp.合成β-PMLA發(fā)酵過(guò)程調(diào)控

3.2.1 溶氧、pH及氧化還原電位的影響 Cao等[24]研究了溶氧與轉(zhuǎn)速對(duì)A.pullulansipe-1發(fā)酵的影響,認(rèn)為發(fā)酵過(guò)程分為三個(gè)階段,第一階段,在高溶氧(DO>70%)下使細(xì)胞大量生長(zhǎng),此階段轉(zhuǎn)速維持在800 r/min。第二階段,通過(guò)自動(dòng)調(diào)節(jié)轉(zhuǎn)速,使溶氧維持在70%以促進(jìn)菌體大量合成β-PMLA。第三階段通過(guò)采用低轉(zhuǎn)速控制細(xì)胞的生長(zhǎng),將溶氧維持在70%以提高β-PMLA產(chǎn)量。此外研究還發(fā)現(xiàn)在發(fā)酵后期缺乏還原力是β-PMLA合成效率降低的因素之一,控制發(fā)酵過(guò)程氧化還原電位(ORP)低于70 mV,β-PMLA的產(chǎn)量提高15.6%。Xia等[53]報(bào)道采用分階段溶氧控制策略能同時(shí)提高β-PMLA和普魯蘭多糖的產(chǎn)量。第一階段,將溶氧控制在30%以提高普魯蘭多糖的產(chǎn)量,在發(fā)酵72 h之后,將溶氧提高到70%以促進(jìn)β-PMLA的積累,經(jīng)過(guò)兩個(gè)階段的發(fā)酵過(guò)程,最終獲得118.6 g/L的β-PMLA和27.2 g/L普魯蘭多糖。

控制pH6.0及溶解氧濃度大于70%,能顯著促進(jìn)A.pullulans酵母形態(tài)細(xì)胞的比例,并提高β-PMLA的濃度[15]。發(fā)酵48 h后,合成的β-PMLA分子量開(kāi)始下降[33],說(shuō)明合成的高分子量β-PMLA被降解或者新合成的β-PMLA分子量較小,可能原因是在該階段β-PMLA降解酶的活力增強(qiáng),致使高分子量β-PMLA發(fā)生降解。發(fā)酵培養(yǎng)基在低pH條件下(pH<5),會(huì)造成β-PMLA水解,多糖含量的增加。在pH為3的條件下,A. pullulans會(huì)形成厚垣孢子,抑制β-PMLA的產(chǎn)生[54]。

3.2.2 代謝抑制物及中間體的調(diào)控 Liu等[13]報(bào)道外源三氟乙酸抑制β-PMLA的生成,當(dāng)培養(yǎng)基中添加琥珀酸鹽或蘋(píng)果酸鹽時(shí),這種抑制作用被解除。程若東等[31]報(bào)道培養(yǎng)基中添加不同濃度的富馬酸和L-蘋(píng)果酸對(duì)菌體生長(zhǎng)影響不大,但都可明顯促進(jìn)β-PMLA的累積。通過(guò)調(diào)節(jié)代謝流在TCA循環(huán)和乙醛酸途徑之間的分布,調(diào)節(jié)代謝中間體的量,可促使菌株能夠高效地利用碳源合成β-PMLA。Zou等[32]發(fā)現(xiàn)通過(guò)補(bǔ)充TCA還原途徑中的輔助因子生物素及CO2供體提高了β-PMLA濃度。Cao等[33]發(fā)現(xiàn)添加外源丙酮酸抑制了β-PMLA的合成及細(xì)胞的生長(zhǎng)。

4 展望

目前,關(guān)于β-PMLA的生物合成途徑的報(bào)道主要是基于其單體L-蘋(píng)果酸的合成途徑分析的,涉及了L-蘋(píng)果酸生物合成的所有已知途徑,但對(duì)β-PMLA合成的關(guān)鍵途徑和酶尚沒(méi)有一致的結(jié)論。Wittmann-Liebold等[55]和B?hmer等[56]報(bào)道蛋白質(zhì)組學(xué)可以定性和定量測(cè)量大量直接影響細(xì)胞的形態(tài)學(xué)和生物化學(xué)過(guò)程的蛋白質(zhì),從而準(zhǔn)確提供細(xì)胞生長(zhǎng)、分化以及響應(yīng)環(huán)境因素過(guò)程中的蛋白質(zhì)狀態(tài)變化的分析。然而Aureobasidiumspp.合成β-PMLA關(guān)鍵基因還存在爭(zhēng)議,因此合成β-PMLA的關(guān)鍵蛋白質(zhì)與關(guān)鍵基因還有待進(jìn)一步深入研究,而采用蛋白質(zhì)組學(xué)技術(shù)篩選并分析β-PMLA合成相關(guān)差異蛋白質(zhì),進(jìn)而采用合成生物學(xué)手段構(gòu)建調(diào)控關(guān)鍵蛋白質(zhì)基因的工程菌,為研究β-PMLA的合成途徑提供了另一思路。

[1]Nagata N,Nakahara T,Tabuchi T,et al. Characterization of Poly(β-L-malic acid)Produced byAureobasidiumsp. A-91[J]. Polymer Journal,1993,25(6):585-592.

[2]Ding H,Inoue S,Ljubimov A V,et al. Inhibition of brain tumor growth by intravenous poly(β-l-malic acid)nanobioconjugate with pH-dependent drug release[J]. Proceedings of the National Academy of Sciences of the United States of America,2010,107(42):18143-18148.

[3]Ding H,Portilla-Arias J,Patil R,et al. The optimization of polymalic acid peptide copolymers for endosomolytic drug delivery[J]. Biomaterials,2011,32(22):5269-5278.

[4]Huang Z W,Laurent V,Chetouani G,et al. New functional degradable and bio-compatible nanoparticles based on poly(malic acid)derivatives for site-specific anti-cancer drug delivery[J]. International Journal of Pharmaceutics,2012,423(1):84-92.

[5]Fujita M,Lee B,Khazenzon N,et al. Brain tumor tandem targeting using a combination of monoclonal antibodies attached to biopoly(β-L-malic acid)[J]. Journal of Controlled Release,2007,122(3):356-363.

[6]Shimada K,Matsushima K,Fukumoto J,et al. Poly-(L)-malic acid:a new protease inhibitor fromPenicilliumcyclopium[J]. Biochemical and biophysical research communications,1969,35(5):619-624.

[7]Lee B,Holler E. Effects of culture conditions onβ-poly(L-malate)production byPhysarumpolycephalum[J]. Applied Microbiology and Biotechnology,1999,51(5):647-652.

[8]Fischer H,Erdmann S,Holler E. An unusual polyanion fromPhysarumpolycephalumthat inhibits homologous DNA polymerase alphainvitro[J]. Biochemistry,1989,28(12):5219-5226.

[9]Lee B,Holler E.β-poly(L-malate)production by non-growing microplasmodia ofPhysarumpolycephalum:Effects of metabolic intermediates and inhibitors[J]. FEMS microbiology letters,2000,193:69-74.

[10]Nagata N,Nakahara T,Tabuchi T. Fermentative production of poly(β-L-malic acid),a polyelectrolytic biopolyester,byAureobasidiumsp.[J]. Bioscience,Biotechnology,and Biochemistry,1993,57(4):638-642.

[11]Nakajima-Kambe T,Hirotani N,Nakahara T. Poly(β-malic acid)production by the non-growing cells ofAureobasidiumsp. strain A-91[J]. Journal of Fermentation and Bioengineering,1996,82(4):411-413.

[12]Liu S,Steinbüchel A. Investigation of poly(β-L-malic acid)production by strains ofAureobasidiumpullulans[J]. Applied Microbiology and Biotechnology,1996,46(3):273-278.

[13]Liu S,Steinbüchel A. Production of poly(malic acid)from different carbon sources and its regulation inAureobasidiumpullulans[J]. Biotechnology Letters,1997,19(1):11-14.

[14]Zhang H,Cai J,Dong J,et al. High-level production of poly(β-L-malic acid)with a new isolatedAureobasidiumpullulansstrain. Appl. Microbiol. Biotechnol.,2011,92(2):295-303.

[15]Cao W,Qi B,Zhao J,et al. Control strategy of pH,dissolved oxygen concentration and stirring speed for enhancingβ-poly(malic acid)production byAureobasidiumpullulansipe-1[J]. Journal of Chemical Technology and Biotechnology,2013,88(5):808-817.

[16]Holler E,Angerer B,Achhammer G,et al. Biological and biosynthetic properties of poly-L-malate[J]. Fems Microbiology Letters,1992,103(2-4):109-118.

[17]Lee B,Maurer T,Kalbitzer H,et al.β-Poly(L-malate)production by Physarum polycephalum[J]. Applied Microbiology and Biotechnology,1999,52(3):415-420.

[18]Rathberger K,Reisner H,Willibald B,et al. Comparative synthesis and hydrolytic degradation of poly(L-malate)by myxomycetes and fungi[J]. Mycological Research,1999,103(5):513-520.

[19]Wang Y K,Chi Z,Zhou H X,et al. Enhanced production of Ca2+-polymalate(PMA)with high molecular mass byAureobasidiumpullulansvar.pullulansMCW[J]. Microbial Cell Factories,2015,14(1):1-11.

[20]Manitchotpisit P,Leathers T D,Peterson S W,et al. Multilocus phylogenetic analyses,pullulan production and xylanase activity of tropical isolates ofAureobasidiumpullulans[J].Mycological Research,2009,113(10):1107-1120.

[22]Chenga C,Zhouab Y,Lin M,et al. Polymalic acid fermentation by Aureobasidium pullulans for malic acid production from soybean hull and soy molasses:Fermentation kinetics and economic analysis[J]. Bioresource Technology,2017,223:166-174.

[23]Wei P,Chi C,Meng L,et al. Production of poly(malic acid)from sugarcane juice in fermentation byAureobasidiumpullulans:Kinetics and process economics[J]. Bioresource Technology,2017,224:581-589.

[24]Cao W,Luo J,Zhao J,et al. Intensification ofβ-poly(l-malic acid)production byAureobasidiumpullulansipe-1 in the late exponential growth phase[J]. Journal of Industrial Microbiology & Biotechnology,2012,39(7):1073-1080.

[25]P M,CD S,SW P,et al. Poly(β-L-malic acid)production by diverse phylogenetic clades ofAureobasidiumpullulans[J]. Journal of Industrial Microbiology & Biotechnology,2012,39(1):125-132.

[26]Leathers T D,Manitchotpisit P. Production of poly(β-l-malic acid)(PMA)from agricultural biomass substrates byAureobasidiumpullulans[J]. Biotechnology Letters,2013,35(1):83-89.

[27]Zou X,Yang J,Tian X,et al. Production of polymalic acid and malic acid from xylose and corncob hydrolysate by a novelAureobasidiumpullulansYJ 6-11 strain[J]. Process Biochemistry,2016,51(1):16-23.

[28]Xia J,Xu J,Hu L,et al. Enhanced poly(L-malic acid)production from pretreated cane molasses byAureobasidiumpullulansin fed-batch fermentation[J]. Preparative Biochemistry & Biotechnology,2016,46(8):798-802.

[29]Zan Z Q,Zou X. Efficient production of polymalic acid from raw sweet potato hydrolysate with immobilized cells ofAureobasidiumpullulansCCTCC M2012223 in aerobic fibrous bed bioreactor[J]. Journal of Chemical Technology & Biotechnology,2013,88(10):1822-1827.

[30]靳挺,武玉學(xué),陳真,等. 出芽短梗霉發(fā)酵生產(chǎn)聚蘋(píng)果酸的研究[J]. 中國(guó)食品學(xué)報(bào),2012,12(10):125-130.

[31]程若東,王浩,周華,等. 出芽短梗霉積累聚蘋(píng)果酸途徑及調(diào)控研究[J]. 化工學(xué)報(bào),2012,63(11):3639-3644.

[32]Zou X,Tu G,Zan Z. Cofactor and CO2donor regulation involved in reductive routes for polymalic acid production byAureobasidiumpullulansCCTCC M2012223[J]. Bioprocess and biosystems engineering,2014,37(10):2131-2136.

[33]Cao W,Luo J,Qi B,et al.β-poly(L-malic acid)production by fed-batch culture ofAureobasidiumpullulansipe-1 with mixed sugars[J]. Engineering in Life Sciences,2014,14(2):180-189.

[34]喬長(zhǎng)晟,鄭志達(dá),孟迪,等. 出芽短梗霉發(fā)酵生產(chǎn)聚蘋(píng)果酸的代謝通量及關(guān)鍵酶活性分析[J]. 現(xiàn)代食品科技,2014,30(7):74-80.

[35]Li Y,Chi Z,Wang G Y,et al. Taxonomy ofAureobasidiumspp. and biosynthesis and regulation of their extracellular polymers[J]. Critical Reviews in Microbiology,2015,41(2):228-237.

[37]Olickal T. Search for malic acid activating enzyme involved in the synthesis of polymalic acid from plasmodia of Physarum polycephalum[D]. 2005.

[38]Willibald B,Bildl W,Lee B S,et al. Isβ-poly(L-malate)synthesis catalysed by a combination ofβ-L-malyl-AMP-ligase andβ-poly(L-malate)polymerase?[J]. European Journal of Biochemistry,1999,265(3):1085-1090.

[39]Feng J,Jing Y,Li X,et al. Reconstruction of a genome-scale metabolic model and in silico analysis of the polymalic acid producerAureobasidiumpullulansCCTCC M2012223[J]. Gene,2016.

[40]吳小燕,周峰,涂光偉,等. 聚蘋(píng)果酸聚合途徑中蘋(píng)果酰輔酶A連接酶基因的克隆、表達(dá)及酶學(xué)性質(zhì)[J]. 微生物學(xué)報(bào),2014,(08):919-925.

[41]Leathers T D,Manitchotpisit P. Production of poly(β-l-malic acid)(PMA)from agricultural biomass substrates byAureobasidiumpullulans[J]. Biotechnology Letters,2013,35(1):83-89.

[42]Amartey S,Jeffries T W. Comparison of corn steep liquor with other nutrients in the fermentation of D-Xylose by Pichia stipitis CBS 6054[J]. Biotechnology Letters,1994,16(2):211-214.

[43]Knuf C,Nookaew I,Brown S H,et al. Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions[J]. Applied & Environmental Microbiology,2013,79(19):6050-6058.

[45]殷海松,范栩嘉,湯衛(wèi)華,等. 氨基酸對(duì)出芽短梗霉發(fā)酵生產(chǎn)蘋(píng)果酸的影響[J]. 食品工業(yè)科技,2016,37(8).

[46]王麗燕,鄭誼豐,劉婷婷,等. 聚蘋(píng)果酸的發(fā)酵培養(yǎng)條件優(yōu)化[J]. 生物加工過(guò)程,2010,8(2):41-45.

[47]Wang Y,Song X,Zhang Y,et al. Effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungusAureobasidiumpullulans[J]. Microbial Cell Factories,2016,15(1):146.

[48]Chi Z,Wang X X,Geng Q,et al. Role of a GATA-type transcriptional repressor Sre1 in regulation of siderophore biosynthesis in the marine-derivedAureobasidiumpullulansHN6.2[J]. 2013,26(6):955-967.

[49]Zelle R M,Hulster E D,Kloezen W,et al. Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an EngineeredSaccharomycescerevisiaeStrain[J]. Applied & Environmental Microbiology,2010,76(3):744-750.

[50]李睿穎,喬長(zhǎng)晟,國(guó)華,等. 出芽短梗霉發(fā)酵合成聚蘋(píng)果酸的研究[J]. 食品與發(fā)酵科技,2011,47(5):68-71.

[51]Cao W,Chen X,Luo J,et al. High molecular weightβ-poly(l-malic acid)produced byA.pullulanswith Ca2+added repeated batch culture[J]. International journal of biological macromolecules,2016,85:192-199.

[52]Tu G,Wang Y,Ji Y,et al. The effect of Tween 80 on the polymalic acid and pullulan production byAureobasidiumpullulansCCTCC M2012223[J]. World Journal of Microbiology and Biotechnology,2015,31(1):219-226.

[53]Xia J,Liu X,Xu J,et al. Simultaneously enhanced production of poly(β-malic acid)and pullulan using a dissolved oxygen shift(DO-shift)control strategy[J]. Journal of Chemical Technology & Biotechnology,2016.

[54]Li B X,Ning Z,Peng Q,et al. Production of pigment-free pullulan by swollen cell inAureobasidiumpullulansNG which cell differentiation was affected by pH and nutrition[J]. Applied Microbiology and Biotechnology,2009,84(2):293-300.

[55]Wittmann-Liebold B,Graack H R,Pohl T. Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry[J]. Proteomics,2006,6(17):4688-4703.

[56]B?hmer M,Colby T,B?hmer C,et al. Proteomic analysis of dimorphic transition in the phytopathogenic fungusUstilagomaydis[J]. Proteomics,2007,7(5):675-685.

ResearchadvancesonmetabolismandregulationofpolymalicacidproductionbystrainsofAureobasidiumspp.

LIUBin1,2,CAOWei-feng2,YUHai-feng1，*

(1.College of Food Science and Engineering,Qilu University of Technology,Jinan 250353,China;2.State Key Laboratory of Biochemical Engineering,Institute of Process Engineering, Chinese Academy of Sciences,Beijing 100190,China)

Poly(malic acid)is a fully biodegradable polymer material with advantages of high water-solubility,biodegradability and non-immunogenicity,which has wide application prospects in biomedicine and biomaterials industries.The metabolic pathway,the key enzyme systemsand the regulation metabolismin the biosynthesis of polymalic acid were summarized in this review. And the research tendency of poly-malic acid biosynthesis metabolism was also proposed.

polymalic acid;metabolic pathway;key enzyme system;metabolic regulation

2017-03-03

劉濱(1992-)，男，碩士研究生，研究方向：食品生物技術(shù)，E-mail:liubin761@163.com。

*通訊作者:于海峰(1975-)，女，博士，教授， 研究方向：食品生物技術(shù)，E-mail:yhfdzz@126.com。

國(guó)家自然科學(xué)基金資助項(xiàng)目(21406240)。

TS201.3

:A

:1002-0306(2017)17-0347-06

10.13386/j.issn1002-0306.2017.17.067