CN101153295A - 一种辅因子调控碳代谢流实现α-酮戊二酸过量积累的方法 - Google Patents

一种辅因子调控碳代谢流实现α-酮戊二酸过量积累的方法 Download PDF

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CN101153295A
CN101153295A CNA2007101331552A CN200710133155A CN101153295A CN 101153295 A CN101153295 A CN 101153295A CN A2007101331552 A CNA2007101331552 A CN A2007101331552A CN 200710133155 A CN200710133155 A CN 200710133155A CN 101153295 A CN101153295 A CN 101153295A
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陈坚
张旦旦
刘立明
堵国成
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Jiangnan University
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Abstract

一种辅因子调控碳代谢流实现α-酮戊二酸(α-KG)过量积累的方法,属于辅因子代谢调控策略优化发酵过程技术领域。本发明采用在发酵过程中添加亚适量的维生素和金属离子等辅因子的方法,选择性的增加PDH和PC途径流量,达到过量积累α-KG的目的。在光滑球拟酵母(T.glabrata)发酵生产α-KG的过程中,增加维生素B1浓度,使α-KG积累量达到10.3g/L;增加生物素Bio浓度,使α-KG积累量达到14g/L;同时增加维生素B1和Bio浓度,使α-KG积累量达到20.8g/L;在同时增加维生素B1和Bio浓度的基础上,以60g/LCaCO3调节培养体系中pH,α-KG积累量达到43.7g/L,丙酮酸浓度则下降到21.8g/L。此策略对其它发酵产品的生产也有指导意义。

Description

一种辅因子调控碳代谢流实现α-酮戊二酸过量积累的方法
技术领域
本发明涉及一种辅因子调控碳代谢流实现α-酮戊二酸(α-KG)过量积累的方法。通过研究辅因子(维生素和金属离子)对代谢流流向及相应的代谢通量的影响,达到α-酮戊二酸的大量积累。
背景技术
α-酮戊二酸,又称α-胶酮酸,2-氧代戊二酸或α-羰基戊二酸,是三羧酸(TCA)循环中重要的中间产物之一,在微生物细胞的代谢中起着重要的作用,也是合成多种氨基酸、蛋白质的重要前体物质。其结构式为:
Figure A20071013315500031
α-酮戊二酸结构式
α-酮戊二酸在医药、有机合成、营养强化剂等领域有着重要的应用前景,目前主要应用领域为:作为运动营养饮料的成分;有机中间体;生化试剂和测肝功能的配套试剂;体格增强补剂;降低术后患者和长期病人的机体损耗;在脑部作为酪氨酸和谷氨酸的前体;同时研究还表明,α-酮戊二酸具有抗氰作用,与亚硝酸钠、硫代硫酸钠配合使用可提高抗氰能力,且有抗惊厥作用。α-酮戊二酸目前主要消费于医疗机构,用来诊断和对神经疾病进行治疗。
辅因子工程所涉及到的辅因子主要有:ATP/ADP/AMP、NADH/NAD+、NADPH/NADP+、辅酶A及其衍生物、维生素和微量元素。目前研究主要集中在调节ATP/ADP/AMP、NADH/NAD+、NADPH/NADP+和辅酶A及其衍生物等辅因子在细胞内的形式和含量对代谢途径及代谢流量的影响。然而,对维生素和金属离子如何影响工业微生物中碳代谢和碳代谢流流向分布的研究少见报道。
发明内容
本发明的目的是提供一种辅因子调控碳代谢流实现α-酮戊二酸(α-KG)过量积累的方法。利用多重营养缺陷型的光滑球拟酵母发酵生产α-KG,在发酵过程中添加亚适量的维生素和金属离子等辅因子,选择性的打开PDH和PC途径,达到大量积累α-KG的目的。
本发明的技术方案:一种辅因子调控碳代谢流实现α-酮戊二酸(α-KG)过量积累的方法,是利用多重维生素营养缺陷型的光滑球拟酵母(T.glabrata)CCTCCM202019为生产菌株,通过调节辅因子水平,在培养基中增加维生素B1浓度,增加生物素Bio浓度和/或以CaCO3调节培养体系中pH,选择性的打开PDH和/或PC途径,使碳流大量流向α-KG,实现α-酮戊二酸过量积累;调控方法为:
增加培养基中维生素B1浓度至0.04mg/L,选择性地打开PDH途径,培养体系中α-KG积累量达到10.3g/L;
或增加培养基中生物素Bio浓度至0.06mg/L,选择性地打开PC途径,培养体系中α-KG积累量达到14g/L;
或同时增加培养基中维生素B1至0.04mg/L和生物素Bio至0.06mg/L浓度,也即同时打开PDH和PC途径,培养体系中α-KG积累量达到20.8g/L;
或以CaCO3作为pH调节剂,当培养体系中碳酸钙浓度为60g/L时,α-KG积累量达到12.8g/L;
或在同时增加培养基中维生素B1至0.04mg/L和生物素Bio至0.06mg/L浓度的基础上,以60g/L浓度的CaCO3调节培养体系中pH,Ca2+可进一步提高PC活性,此时α-KG积累量达到43.7g/L,丙酮酸浓度则下降到21.8g/L。
1、菌株
光滑球拟酵母(T.glabrata)CCTCC M202019,烟酸(NA)、生物素(Bio)、硫胺素(B1)、吡哆醇(Pdx)等四种维生素营养缺陷型,且丙酮酸脱羧酶活性组成型降低,为本研究室选育菌株,该菌种已申请中国专利,申请号为02113142.2,公开号为CN1392246A。
2、培养基
种子和斜面培养基(g/L):葡萄糖30,蛋白胨10,磷酸二氢钾1,硫酸镁(七水)0.5;琼脂20(斜面培养基),pH 5.5。
基本发酵培养基(L):葡萄糖100g,NH4SO47g,MgSO4·7H2O 0.8g,KH2PO45g,KCl 5g,pH 5.0。
3、摇瓶培养:
将30℃、200rpm下培养24h的种子以10%的接种量分别转入发酵培养基于30℃、200rpm条件下培养48h,按实验要求添加不同浓度的维生素B1、生物素Bio和/或CaCO3
细胞干重的测定:取一定量的菌悬液置于10mL容量瓶中,加2mL盐酸溶解菌悬液中的碳酸钙,加入去离子水定容至10mL,摇匀,用UV 7500型可见分光光度计,于660nm处比色测OD值,用细胞干重标准曲线算得细胞干重。
丙酮酸和α-KG浓度的测定:高效液相色谱(HPLC)
仪器:Agilent 1100高效液相色谱仪(配紫外可见检测器、视差折光检测器和工作站),
色谱条件:
色谱柱:C18柱,5μm,4.6mm×250mm
流动相:0.1%H3PO4
流速:1mL/min
柱温:28℃
进样量:10μL
紫外检测器波长:215nm
样品制备:5mL发酵液在10000rpm下离心10min,取上清液移入试管中以备测α-KG和残余甘油时用。测α-KG时,取1mL上清液移入50mL容量瓶中,去离子水定容至刻线,经0.45μm滤膜过滤后,滤液供液相色谱分析用。
本发明的有益效果:利用本发明调控工业微生物中碳代谢和碳代谢流流向分布,使碳代谢流从积累丙酮酸转向大量积累,其中丙酮酸产量从69g/L降到21.8g/L,而α-KG产量从6.8g/L上升到了43.7g/L。
具体实施方式
实施例1提高维生素B1浓度增加丙酮酸脱氢酶(PDH)途径代谢通量
当培养基中维生素B1浓度增加到0.04mg/L时,α-KG产量达到最大值10.3g/L,丙酮酸产量降到了58.6g/L,丙酮酸与α-KG的碳摩尔比(CPYR/CKG)为3.9。继续提高培养基中的维生素B1的浓度时,α-KG的产量则会逐渐下降。
实施例2提高生物素Bio浓度增加丙酮酸羧化酶(PC)途径代谢通量
当培养基中生物素Bio浓度增加到0.06mg/L时,α-KG产量达到最大值14g/L,丙酮酸产量降到52.6g/L,丙酮酸与α-KG的碳摩尔比(CPYR/CKG)为3.74。
实施例3碳酸钙对碳代谢流流向和通量大小的影响
如下表所示,当摇瓶和发酵罐中不调节pH时,细胞生长很弱,产酸很少。在摇瓶中用CaCO3作为缓冲剂时,产生了较高浓度(6.8g/L)的α-KG。在发酵罐培养中,如果用NaOH调节pH,发酵液中α-KG浓度很低。但如果改用CaCO3调节pH,发酵液中α-KG的浓度则比用NaOH调节pH时增加了8倍。当培养体系中碳酸钙浓度为60g/L时,α-KG积累量达最大值(12.8g/L)。
摇瓶和发酵罐的发酵对照
  细胞/(g/L)   消耗葡萄糖/(g/L)   丙酮酸/(g/L)   α-酮戊二酸/(g/L) CPYR/CKG
 摇瓶(不添加CaCO3)摇瓶(添加CaCO3)发酵罐培养(不调节pH)发酵罐培养(NaOH调节pH)发酵罐培养(CaCO3调节pH)   2.810.94.914.816.7   5.684.814.390.789.7   2.337.85.169.462.3   06.801.311.5   ∞6.96∞67.26.84
说明:CPYR/CKG为丙酮酸的碳摩尔数与α-KG的碳摩尔数的比值。
实施例4同时增加PDH和PC途径代谢通量对代谢流分布的影响
在发酵培养基中同时添加0.06mg/L生物素Bio和0.04mg/L维生素B1,可使α-KG浓度达到最大值20.8g/L,丙酮酸产量降到了48.4g/L,丙酮酸与α-KG的碳摩尔比(CPYR/CKG)为2.31。在此基础上,在发酵培养体系中添加60g/L的碳酸钙,α-KG浓度达到最大值43.7g/L,而丙酮酸产量则下降到21.8g/L,此时丙酮酸与α-KG的碳摩尔比(CPYR/CKG)为0.49。

Claims (1)

1.一种辅因子调控碳代谢流实现α-酮戊二酸α-KG过量积累的方法,其特征是利用多重维生素营养缺陷型的光滑球拟酵母(T.glabrata)CCTCC M202019为生产菌株,通过调节辅因子水平,在培养基中增加维生素B1浓度,增加生物素Bio浓度和/或以CaCO3调节培养体系中pH,选择性的打开PDH和/或PC途径,使碳流大量流向α-KG,实现α-酮戊二酸过量积累;调控方法为:
增加培养基中维生素B1浓度至0.04mg/L,选择性地打开PDH途径,培养体系中α-KG积累量达到10.3g/L;
或增加培养基中生物素Bio浓度至0.06mg/L,选择性地打开PC途径,培养体系中α-KG积累量达到14g/L;
或同时增加培养基中维生素B1至0.04mg/L和生物素Bio至0.06mg/L浓度,也即同时打开PDH和PC途径,培养体系中α-KG积累量达到20.8g/L;
或以CaCO3作为pH调节剂,当培养体系中碳酸钙浓度为60g/L时,α-KG积累量达到12.8g/L;
或在同时增加培养基中维生素B1至0.04mg/L和生物素Bio至0.06mg/L浓度的基础上,以60g/L浓度的CaCO3调节培养体系中pH,Ca2+可进一步提高PC活性,此时α-KG积累量达到43.7g/L,丙酮酸浓度则下降到21.8g/L。
CNA2007101331552A 2007-09-20 2007-09-20 一种辅因子调控碳代谢流实现α-酮戊二酸过量积累的方法 Pending CN101153295A (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586347A (zh) * 2012-03-06 2012-07-18 江南大学 一种两阶段pH控制高产a-酮戊二酸的方法
CN102586128A (zh) * 2012-03-14 2012-07-18 江南大学 一种高产α-酮戊二酸酵母工程菌及其应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586347A (zh) * 2012-03-06 2012-07-18 江南大学 一种两阶段pH控制高产a-酮戊二酸的方法
CN102586128A (zh) * 2012-03-14 2012-07-18 江南大学 一种高产α-酮戊二酸酵母工程菌及其应用
CN102586128B (zh) * 2012-03-14 2014-04-09 江南大学 一种高产α-酮戊二酸酵母工程菌及其应用

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