CN114480232A - 生产丙二酸的工程菌及其构建方法和应用 - Google Patents
生产丙二酸的工程菌及其构建方法和应用 Download PDFInfo
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- CN114480232A CN114480232A CN202011154682.3A CN202011154682A CN114480232A CN 114480232 A CN114480232 A CN 114480232A CN 202011154682 A CN202011154682 A CN 202011154682A CN 114480232 A CN114480232 A CN 114480232A
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- malonic acid
- producing
- bacterium
- engineering
- xylose
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Abstract
本发明提供了一种生产丙二酸的工程菌,包括宿主菌和转入所述宿主菌的质粒载体,其中,所述质粒载体中导入了编码木糖脱氢酶、木糖酸脱水酶、醛缩酶、丙二酸单酰辅酶A还原酶和醛脱氢酶的基因。上述工程菌还包括过表达来自大肠杆菌的乙酰辅酶A羧化酶,敲除大肠杆菌体内木糖本身的代谢途径以及副产物途径中的醛还原酶基因来实现丙二酸的增产。本发明还提供一种上述生产丙二酸的工程菌的构建方法和应用。利用上述生产丙二酸的工程菌,以木糖为碳源来生产丙二酸,实现了丙二酸的从头合成,扩大了木糖的工业化应用。
Description
技术领域
本发明涉及生物工程技术领域,尤其涉及一种生产丙二酸的工程菌及其构建方法和应用。
背景技术
丙二酸又称缩苹果酸、胡萝卜酸或甜菜酸,是一种有机酸。目前主要用于香料和医药中间体,尤其是在医药工业中,用于生产巴比妥酸、维生素B1,维生素B2等。另外还可以应用于胶粘剂和树脂添加剂等的生产,也可用于皮革制品或铝制品表面处理剂、泡沫塑料发泡剂和核反应器的化学清洗剂。此外,丙二酸还是铝表面处理剂,处理时加热分解只生成二氧化碳和水,不会造成污染,比过去采用的甲酸等酸型处理剂具有很大的优点。其下游产品覆盖面非常大,涉及到塑料、染料、医药、农药、电镀等行业。丙二酸目前已经成为非常重要的中间体。目前丙二酸主要是通过化学合成,合成方法主要有:氰乙酸钠法,氰乙酸水解法和丙二酸酯水解法。目前国内工业化生产基本都是采用的丙二酸酯水解法。丙二酸生物合成的其中一种方法是通过β-丙氨酸途径,经过天冬氨酸酶、天冬氨酸-α-脱羧酶、β-丙氨酸丙酮酸转氨酶和琥珀酸半醛脱氢酶作用最终得到丙二酸,但是这种方法过程繁杂,得率较低,不利于丙二酸的工业化生产。
随着自然环境中能源的过度开采,人类社会的生存和发展受到了巨大的挑战,所以迫切需要寻找可再生的生物质能源。可再生生物质能源分为三部分:纤维素、半纤维素和木质素。其中,半纤维素在生物质能源中占25%-30%,但是它的利用却远不足木质纤维素,半纤维素主要包括以下几种:木糖、葡萄糖、甘露糖和阿拉伯糖等,D-木糖作为半纤维素含量最为丰富的五碳糖,它的利用率却远不及葡萄糖。而且目前以木糖为源头合成丙二酸的相关报道较少。
发明内容
有鉴于此,本发明的第一个目的是从大量生物或微生物体内能够合成丙二酸的酶中筛选出在体外依然具有催化效率的酶来实现以木糖为源头的丙二酸的异源合成。为此,本发明优选了来源于细菌、真菌或蛋白质工程改造的酶的相关基因,通过这些酶的表达,实现了利用D-木糖生物合成丙二酸,扩大了木糖的利用,利用丙二酸的工业化生产。
本发明的第二个目的在于通过增强上游途径、抑制竞争途径等一系列代谢调控方式来提高丙二酸的产量,实验结果表明,在正常条件下,代谢工程改造菌利用木糖简单碳源来生产丙二酸,能达到108±6 mg/L的产量,而在经过优化后,利用简单碳源的代谢工程菌能达到1039±15 mg/L的最终产量。
为了实现上述目的,本发明提供一种生产丙二酸的工程菌,包括宿主菌和转入所述宿主菌的质粒载体,其中,所述质粒载体中导入了编码木糖脱氢酶、木糖酸脱水酶、醛缩酶、丙二酸单酰辅酶A还原酶和醛脱氢酶的基因。
其中,优选地,所述木糖脱氢酶(xylBC)包括xylB和xylC,所述木糖酸脱水酶优选为xylD,所述醛缩酶为yagE。所述丙二酸单酰辅酶A还原酶具有双功能,一方面可以将丙二酸单酰辅酶A还原为中间体丙二酸半醛,另一方面可以将丙二酸半醛还原为3-羟基丙酸。优选地,所述丙二酸单酰辅酶A还原酶为ChaMCR (Chloroflexus aurantiacus)、CaMCR (C. aggregans)或RcMCR(Roseiflexus castenholzii)。所述醛脱氢酶来源于乙醛脱氢酶ALDH(Saccharomyces cerevisiae)、琥珀酸半醛脱氢酶GabD (Pseudomonas sp)或YneI(E.coli)。
所述宿主菌为细菌、酵母或真菌,其中,所述细菌或真菌为原始的或改造过的。优选地,所述宿主菌为大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌、酿酒酵母或黑曲霉。
基于上述生产丙二酸的工程菌,所述宿主菌过表达编码乙酰辅酶A羧化酶的基因。优选地,所述乙酰辅酶A羧化酶(AccADBC)包括大肠杆菌内的生物素羧基载体蛋白(accB)、生物素羧化酶(accC)和羧基转移酶基因(accA和accD)。所述宿主菌内过表达编码乙酰辅酶A羧化酶的基因,主要是为了提高由乙酰辅酶A合成丙二酰辅酶A的产量,进而实现增强上游途径的方式提高上述生产丙二酸的工程菌生产丙二酸的产量。
基于上述生产丙二酸的工程菌,所述宿主菌敲除了编码木糖异构酶(xylA)的基因。所述工程菌的宿主菌内敲除编码木糖异构酶(xylA)的基因的主要目的是抑制宿主菌体内木糖本身代谢途径,以减少因宿主菌造成的木糖消耗,实现通过抑制竞争途径的方式提高上述生产丙二酸的工程菌生产丙二酸的产量。
基于上述生产丙二酸的工程菌,所述宿主菌还敲除了编码yqhD、adhP、eutG、yiaR、yjgB和fucO的基因。所述宿主菌内敲除了该6种基因主要是抑制3-羟基丙酸的合成,减少丙二酸半醛的消耗,进而实现通过抑制竞争途径的方式提高利用上述生产丙二酸的工程菌合成丙二酸的产量。
基于上述生产丙二酸的工程菌,所述宿主菌进一步敲除了编码betA、eutE、yahK、yqhE、gldA、ybbO、yqhA的基因。所述宿主菌内进一步敲除了该7种基因主要是抑制3-羟基丙酸的合成,减少丙二酸半醛的消耗,进而实现通过抑制竞争途径的方式提高利用上述生产丙二酸的工程菌合成丙二酸的产量。
本发明还提供了一种上述生产丙二酸的工程菌的构建方法,包括步骤:
重组表达质粒 将编码所述木糖脱氢酶、木糖酸脱水酶、醛缩酶、丙二酸单酰辅酶A还原酶和醛还原酶的基因连接至表达质粒上,获得重组质粒载体;
构建工程菌 将所述重组质粒载体转化到所述宿主菌中,得到生产丙二酸的工程菌。
其中,所述表达质粒可以为pZE12-luc、pCS27或pSA74。
其中,所述生产丙二酸的工程菌的构建方法还包括采用Red重组的方法敲除所述宿主菌内编码木糖异构酶(xylA)的基因,获得敲除木糖在大肠杆菌体内代谢的工程菌株;该生产丙二酸的工程菌的构建方法还进一步包括采用Red重组的方法敲除所述宿主菌内编码yqhD、adhP、eutG、yiaR、yjgB和fucO的基因;该生产丙二酸的工程菌的构建方法还进一步包括采用Red重组的方法敲除所述宿主菌内编码betA、eutE、yahK、yqhE、gldA、ybbO和yqhA的基因。
基于上述生产丙二酸的工程菌的构建方法,还包括采用中拷贝质粒表达编码乙酰辅酶A羧化酶的基因。
本发明还提供一种生产丙二酸的工程菌的应用,其中,按照体积比1%~5%的接种量,将上述工程菌接种到培养基中,并加入诱导剂,在30℃~40℃进行发酵处理,制得丙二酸;所述培养基包括:1~5 g∙L‾1 MOPS,5~40 g∙L‾1木糖, 1~5 g∙L‾1酵母粉,5~8 g∙L‾1 NaHPO4,0.3~2 g∙L‾1 NaCl,3 g∙L‾1 KH2PO4,1~5 g∙L‾1 NH4Cl,240~250 mg∙L‾1 MgSO4,14~15.5 mg∙L‾1 CaCl2。
本发明提供的上述生产丙二酸的工程菌在以木糖为碳源生物合成丙二酸的途径如图1所示,D-木糖在木糖脱氢酶(xylB和xylC)的作用下得到D-木糖酸,木糖酸脱水酶作用于D-木糖酸得到2-酮基-3-脱氧-D-木糖酸,接着在醛缩酶的催化下得到产物丙酮酸,丙酮酸在大肠体内酶的作用下得到乙酰辅酶A,乙酰辅酶A在乙酰辅酶A羧化酶(AccADBC)的催化下生成丙二酰辅酶A,接着在丙二酰辅酶A还原酶的作用下生成丙二酸半醛,丙二酸被醛脱氢酶氧化生成最终产物丙二酸,从而实现由木糖从头合成丙二酸,也扩大了木糖的工业化利用。
因此,通过筛选出在体外具有活性的酶,获得本发明提供的上述生产丙二酸的工程菌;以该生产丙二酸的工程菌为基础,设计出如图1所示的丙二酸的生物合成途径,并且利用分子生物学以及代谢调控来优化合成路径,最后对发酵过程中的培养基及培养条件进行系统优化,从而实现木糖为源头采用生物方法高效合成丙二酸。
附图说明
图1是本发明提供的以木糖为源头生物合成丙二酸的路径。
图2是本发明实施例3利用工程菌BW1、BW2、BW3生产丙二酸的发酵结果图。
图3是本发明实施例3利用工程菌BW1、BW4、BW5生产丙二酸的发酵结果图。
图4是本发明实施例4提供的工程菌BW6生产丙二酸的发酵结果图。
图5是本发明实施例5采用模块优化方式提供的工程菌BW10、BW11和BW12分别生产丙二酸的发酵结果图。
具体实施方式
下面通过具体实施方式,对本发明的技术方案做进一步的详细描述。
本发明中,对表达质粒的种类没有特殊要求,可认为在大肠杆菌中表达目的基因的构建方法可以采用本领域常用的各种方法,如将目的基因经过酶切处理后连接至在载体中,之后不再赘述。
以下实施例中,大肠杆菌菌株JCL16,trans5α和BL21(DE3)均为常用大肠杆菌菌株,均可市售获得,其中trans5α用于载体构建,BL21(DE3)用于蛋白表达,BW25113作为发酵用菌株,其中,本发明各实施例中使用的质粒和菌株见后续表1所示。
实施例1
重组质粒pZE - xylD - yagE - ChaMCR - GabD
本实施例提供的重组质粒pZE - xylD - yagE - ChaMCR - GabD主要是将基因xylD、yagE、ChaMCR、GabD连接至大肠杆菌表达载体pZE12-luc获得。
本实施例提供的上述重组质粒的构建方法具体包括以下步骤:筛选来源于细菌,真菌或蛋白质工程改造的编码木糖酸脱水酶(xylD)、醛缩酶(yagE)、丙二酸单酰辅酶A还原酶(ChaMCR)和醛脱氢酶(GabD)基因。通过将编码木糖酸脱水酶(xylD)、醛缩酶(yagE)、丙二酸单酰辅酶A还原酶(ChaMCR)和醛脱氢酶(GabD)的目的基因进行PCR扩增获得目的片段后,接着用合适的酶对目的片段和载体进行酶切,将酶切后的片段进行回收,之后插入到表达质粒pZE12-luc(PLlacO1, colE ori, luc, Ampr)上,获得pZE -xylD -yagE - ChaMCR -GabD重组质粒(见表1)。本实施例中,所述木糖酸脱水酶(xylD)来源于C. crescentus,所述醛缩酶(yagE)来源于E.coli。
参照上述方法构建重组质粒pZE - xylD - yagE - CaMCR-GabD,pZE - xylD -yagE - RcMCR - GabD,pZE - xylD - yagE -ChaMCR - YneI, pZE - xylD - yagE –ChaMCR - ALDH,pCS - xylBC,其中,木糖脱氢酶xylBC中的xylB和、xylC均来源于C.crescentus。
实施例2
生产丙二酸的工程菌:重组大肠杆菌BW1、BW2、BW3、BW4、BW5(见表1)
本发明提供的生产丙二酸的工程菌对用于构建表达质粒的宿主菌菌株种类没有特殊要求,本发明实施例采用了BW25113菌株作为构建表达质粒的初始宿主菌。
首先挑取新鲜的BW25113菌落接种到4 mL LB培养基中,37℃培养8~12 h后,取1mL接种到100 mL LB培养基中,37℃培养至OD600长到0.6时,在4℃的温度下6000 rpm离心10min收集菌体,用10 mL 10%预冷的甘油洗涤,6000 rpm离心10 min,再次重复甘油洗涤步骤,离心后尽量倒干剩余甘油,最后加入适量10%甘油重悬细胞,制得感受态细胞。取90 μL感受态加入2 μL 重组质粒pCS - xylBC和pZE - xylD - yagE - ChaMCR - GabD ,冰上放置两分钟,电转后加入600 μL LB培养基,洗出电转后细胞,37 ℃复苏1 h,涂布到氨苄和卡那双抗性平板,于37℃恒温培养箱中过夜培养,待平板上长出菌落后,挑菌于含有氨苄和卡那抗性的4 mL LB培养基中37℃培养8~10 h,即可获得生产丙二酸的工程菌:含有重组质粒pCS - xylBC和pZE -xylD -yagE - ChaMCR - GabD 的大肠杆菌菌株,用重组大肠杆菌BW1表示。
利用上述相同方法构建:含有重组质粒pCS - xylBC和pZE - xylD -yagE -CaMCR - GabD的大肠杆菌菌株用BW2表示,pCS - xylBC和pZE - xylD – yagE - RcMCR -GabD的大肠杆菌菌株用BW3表示;含有重组质粒pCS-xylBC和pZE - xylD – yagE - ChaMCR- YneI的大肠杆菌菌株,用BW4表示,含有重组质粒pCS - xylBC和pZE - xylD - yagE -ChaMCR - ALDH的大肠杆菌菌株,用BW5表示。
实施例3
生产丙二酸的工程菌的应用:分别通过重组大肠杆菌BW1、BW2、BW3、BW4、BW5的发酵培养生产丙二酸
分别在平板上挑取新鲜的重组大肠杆菌BW1、BW2、BW3、BW4和BW5的工程单菌落分别接种到4 mL含有相应抗生素的 LB试管中,37℃培养8 h后,转入含有相应抗生素的50 mL培养基的摇瓶中进行发酵培养,体积比接种量为2%发酵温度30℃或37℃,转速200 rpm。其中,所述培养基:2 g∙L‾1 MOPS,20 g∙L‾1木糖,5 g∙L‾1酵母粉,6 g∙L‾1 NaHPO4,0.5 g∙L‾1NaCl,3 g∙L‾1 KH2PO4,2 g∙L‾1 NH4Cl,246.5 mg∙L‾1 MgSO4,14.7 mg∙L‾1 CaCl2,并根据实际情况加入相应抗生素。
发酵初始加入终浓度为0.5 mM的诱导剂IPTG,发酵12 h,24 h,36 h,48 h。取出部分发酵液用以测定菌体生长状况及目标产物丙二酸的产量,结果如图2和3所示。从图2和3中可以看出利用重组大肠杆菌BW1生产丙二酸的产量最好,而且该菌株的生长状况最好,所以,相对其它两种菌株,使用ChaMCR和GabD酶的重组大肠杆菌BW1生产丙二酸的能力最强。
实施例4
生产丙二酸的工程菌:重组大肠杆菌BW6和及其构建方法和应用
本实施例提供的重组大肠杆菌菌株BW6主要是进一步通过过表达上游途径中的基因方式获得。具体地,重组大肠杆菌菌株BW6相当于对于重组大肠杆菌菌株BW25113中的内源基因进行了优化,在菌株BW25113的基础上转入pCS – xylBC - AccADBC质粒。该pCS – xylBC- AccADBC质粒主要是将编码木糖脱氢酶xylB和xylC以及编码乙酰辅酶A羧化酶(AccADBC)生物素的accB、accC、accA和accD的基因连接至大肠杆菌表达载体pCS27获得。
参照实施例3提供的生产丙二酸的工程菌的应用,取适量分别含有上述重组大肠杆菌菌株BW6的菌液涂到含有相应抗生素的平板上,37℃过夜培养。挑取平板菌落并接到4mL的带有相应抗性的液体LB中,37℃下培养一定时间,最后将菌液转接到50 mL的所述培养基中,直接加入终浓度0.5 mM的IPTG进行诱导,在发酵12 h,24 h,36 h,48 h后取出部分发酵液用以测定目标产物丙二酸的产量,结果图4所示。
实施例5
生产丙二酸的工程菌-重组大肠杆菌BW10、BW11、BW12(见表1),构建方法和应用
重组大肠杆菌BW10、BW11、BW12通过抑制竞争途径获得,具体地,在经过系统的筛选后决定敲除来源于细菌,真菌或蛋白质工程改造的与丙二酸半醛利用途径竞争的13种醛还原酶和木糖体内代谢途径中的第一个基因,本发明采用Red重组的方法敲除宿主菌中的这些基因,制备敲除宿主菌BW7、BW8和BW9。
从宿主菌BW25113中敲除编码木糖异构酶xylA的基因,获得敲除宿主菌BW7,表示为BWΔxylA。从敲除宿主菌BW7中进一步敲除编码yqhD、adhP、eutG、yiaR、yjgB和fucO的基因,获得敲除宿主菌BW8,表示为BWΔxylAΔyqhDΔadhPΔeutGΔyiaRΔyjgBΔfucO。从敲除宿主菌BW8中进一步敲除编码betA、eutE、yahK、yqhE、gldA、ybbO、yqhA的基因,获得敲除宿主菌BW9,表示为BWΔxylAΔyqhDΔadhPΔeutGΔyiaRΔyjgBΔfucOΔbetAΔeutEΔyahKΔyqhEΔgldAΔybbOΔyqhA。
具体地,首先挑取新鲜的BW25113菌落接种到4 mL LB培养基中,37℃培养8-12 h后,取1 mL接种到100 mL LB培养基中,37℃培养至OD600长到0.6时,在4℃的温度下6000rpm离心10 min收集菌体,用10 mL 10%预冷的甘油洗涤,6000 rpm离心10 min,再次重复甘油洗涤步骤,离心后尽量倒干剩余甘油,最后加入适量10%甘油重悬细胞,制得感受态细胞。取90 μL感受态加入2 μL pKD46质粒,冰上放置两分钟,电转后加入600 μL LB培养基,洗出电转后细胞,30 ℃复苏1 h,涂布到氨苄抗性平板。之后以pKD4质粒为模板,PCR扩增得到带有同源臂的敲除片段,回收进行纯化。挑取单菌落接入氨苄抗性试管8~12 h后,转接100mL LB摇瓶培30 ℃培养;OD600长到0.2后,添加终浓度为100 mM的阿拉伯糖诱导;待摇瓶OD600D长到0.6时,开始制备电转感受态细胞,取90 μL感受态加入5 μL带同源臂的PCR片段,电转后,37℃复苏90 min,涂布于卡那抗性平板,过夜培养,待细胞长到足够大小,挑取单菌落于加入卡那抗性的试管中,37℃培养8~12 h后进行菌落PCR验证,确保kan片段替换掉目的基因。最后,向上一步正确的菌转入pCP20载体,涂布于氨苄和氯霉素混合的抗性平板中,30℃过夜培养,待细胞长到足够大小,挑取单菌落于加入氨苄和氯霉素混合的抗性的试管中30℃培养8~12 h,后转接到无抗性的LB试管中,42℃培养24 h左右进行消除卡那抗性,再划线到无抗性平板,挑取单菌落接种到无抗性LB试管中,再分别转接到无抗性,氨苄抗性,卡那抗性和氨苄和氯霉素混合的抗性试管中,用以验证pKD46、pCP20是否丢失,卡那抗性是否消除。确定在以上两种抗性中均不生长后,保存于冻存管,并进一步菌落PCR验证。此过程得到所述敲除宿主菌BW7:BWΔxylA,该菌株中敲除了xylA基因。同理其他基因也用类似的方法进行敲除,获得所述敲除宿主菌BW8和BW9。
之后将实施例1构建好的重组质粒pZE – xylD – yagE - ChaMCR - GabD和实施例4使用的重组质粒pZE - xylBC - AccADBC参照实施例2提供的方法转入所述敲除宿主菌BW7、BW8和BW9中,获得生产丙二酸的工程菌:重组大肠杆菌BW10、BW11和BW12。
参照实施例3提供的生产丙二酸的工程菌的应用,将上述重组大肠杆菌菌株BW10、BW11、BW12在所述培养基中发酵12 h,24 h,36 h,48 h后取出部分发酵液,该发酵液可以用以测定菌体生长状况及目标产物丙二酸的产量,结果如图5所示。
表1 本发明各实施例中采用的质粒和菌株列表
最后应当说明的是:以上实施例仅用以说明本发明的技术方案而非对其限制;尽管参照较佳实施例对本发明进行了详细的说明,所属领域的普通技术人员应当理解:依然可以对本发明的具体实施方式进行修改或者对部分技术特征进行等同替换;而不脱离本发明技术方案的精神,其均应涵盖在本发明请求保护的技术方案范围当中。
Claims (10)
1.一种生产丙二酸的工程菌,其特征在于:包括宿主菌和转入所述宿主菌的质粒载体,其中,所述质粒载体中导入了编码木糖脱氢酶、木糖酸脱水酶、醛缩酶、丙二酸单酰辅酶A还原酶和醛脱氢酶的基因。
2.根据权利要求1所述的生产丙二酸的工程菌,其特征在于:所述丙二酸单酰辅酶A还原酶为ChaMCR、CaMCR或RcMCR;所述醛脱氢酶来源于乙醛脱氢酶ALDH、琥珀酸半醛脱氢酶GabD或YneI。
3.根据权利要求1或2所述的生产丙二酸的工程菌,其特征在于:所述宿主菌过表达编码乙酰辅酶A羧化酶的基因。
4.根据权利要求3所述的生产丙二酸的工程菌,其特征在于:所述宿主菌还敲除了编码木糖异构酶的基因。
5.根据权利要求4所述的生产丙二酸的工程菌,其特征在于:所述宿主菌进一步敲除了编码yqhD、adhP、eutG、yiaR、yjgB和fucO的基因。
6.根据权利要求5所述的生产丙二酸的工程菌,其特征在于:所述宿主菌敲除了编码betA、eutE、yahK、yqhE、gldA、ybbO、yqhA的基因。
7.一种权利要求1所述的生产丙二酸的工程菌的构建方法,包括步骤:
重组表达质粒 将编码木糖脱氢酶、木糖酸脱水酶、醛缩酶、丙二酸单酰辅酶A还原酶的基因和编码醛脱氢酶的基因连接至表达质粒上,获得重组质粒载体;
构建工程菌 将所述重组质粒载体转化到所述宿主菌中,得到生产丙二酸的工程菌。
8.根据权利要求7所述的生产丙二酸的工程菌的构建方法,其特征在于:还包括采用中拷贝质粒表达编码乙酰辅酶A羧化酶的基因。
9.根据权利要求7或8所述的生产丙二酸的工程菌的构建方法,其特征在于:还包括采用Red重组的方法敲除所述宿主菌内编码木糖异构酶、yqhD、adhP、eutG、yiaR、yjgB、fucO、betA、eutE、yahK、yqhE、gldA、ybbO和yqhA的基因。
10.一种生产丙二酸的工程菌的应用,其特征在于:按照体积比1%~5%的接种量,将权利要求1~6任一项所述的生产丙二酸的工程菌接种到培养基中,并加入诱导剂,在30℃~40℃进行发酵处理,制得丙二酸;所述培养基包括1~5 g∙L‾1 MOPS,5~40 g∙L‾1木糖,1~5 g∙L‾1酵母粉,5~8 g∙L‾1 NaHPO4,0.3~2 g∙L‾1 NaCl,3 g∙L‾1 KH2PO4,1~5 g∙L‾1 NH4Cl,240~250 mg∙L‾1 MgSO4,14~15.5 mg∙L‾1 CaCl2。
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