CN113563436A - Yh66-rs03880基因改造得到的工程菌及其在制备缬氨酸中的应用 - Google Patents
Yh66-rs03880基因改造得到的工程菌及其在制备缬氨酸中的应用 Download PDFInfo
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Abstract
本发明公开了一种YH66‑RS03880基因改造得到的工程菌及其在制备缬氨酸中的应用。本发明提供了YH66‑RS03880G160A蛋白,是将YH66‑RS03880蛋白的第54位氨基酸残基由E突变为其他氨基酸残基得到的。本发明发现YH66‑RS03880蛋白对细菌的缬氨酸产量存在正调控,即YH66‑RS03880蛋白含量增高、缬氨酸产量增高,YH66‑RS03880蛋白含量降低、缬氨酸产量降低。抑制YH66‑RS03880基因表达可以降低缬氨酸产量,过表达YH66‑RS03880基因提高缬氨酸产量。进一步的,本发明通过点突变,得到了YH66‑RS03880G160A蛋白,其功能优于YH66‑RS03880蛋白。本发明对于缬氨酸工业化生产,具有重大的应用价值。
Description
技术领域
本发明属于生物技术领域,涉及一种YH66-RS03880基因改造得到的工程菌及其在制备缬氨酸中的应用,具体的所述改造为G160A。
背景技术
缬氨酸是组成蛋白质的20种氨基酸之一,是人体必需的8种氨基酸和生糖氨基酸,它与其他两种高浓度氨基酸(异亮氨酸和亮氨酸)一起工作促进身体正常生长,修复组织,调节血糖,并提供需要的能量。在参加激烈体力活动时,缬氨酸可以给肌肉提供额外的能量产生葡萄糖,以防止肌肉衰弱。缬氨酸还帮助从肝脏清除多余的氮(潜在的毒素),并将身体需要的氮运输到各个部位。
缬氨酸是一种必需氨基酸,这意味着身体本身不能生产,必须通过膳食来源获得补充。它的天然食物来源包括谷物、奶制品、香菇、蘑菇、花生、大豆蛋白和肉类。尽管大多数人都可以从饮食中获得足够的数量,但是缬氨酸缺乏症的案例也屡见不鲜。当缬氨酸不足时,大脑中枢神经系统功能会发生紊乱,共济失调而出现四肢震颤。通过解剖切片脑组织,发现有红核细胞变性现象,晚期肝硬化病人因肝功能损害,易形成高胰岛素血症,致使血中支链氨基酸减少,支链氨基酸和芳香族氨基酸的比值由正常人的3.0-3.5降至1.0-1.5,故常用缬氨酸等支链氨基酸的注射液治疗肝功能衰竭以及酗酒和吸毒对这些器官造成的损害。此外,缬氨酸也可作为加快创伤愈合的治疗剂。L-缬氨酸,别名为2-氨基-3-甲基丁酸,CAS号为72-18-4,MDL号为MFCD00064220,EINECS号为200-773-6。目前制备L-缬氨酸主要是化学合成法。化学合成法的局限性:生产成本高,反应复杂,步骤多,且有许多副产物。
发明内容
本发明的目的是提供一种YH66-RS03880基因改造得到的工程菌及其在制备缬氨酸中的应用。
本发明提供了一种蛋白质(突变蛋白,命名为YH66-RS03880G160A蛋白),是将YH66-RS03880蛋白的第54位氨基酸残基由E突变为其他氨基酸残基得到的;
所述YH66-RS03880蛋白为如下(a1)或(a2)或(a3):
(a1)序列表的序列3所示的蛋白质;
(a2)来源于细菌且与(a1)具有95%以上同一性且与细菌产缬氨酸相关的蛋白质;
(a3)将(a1)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且与细菌产缬氨酸相关的由(a1)衍生的蛋白质。
这里使用的术语“同一性”指与天然氨基酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
所述95%以上同一性具体可为96%以上同一性或97%以上同一性或98%以上同一性或99%以上同一性。
所述其他氨基酸残基具体可为K。
具体的,所述YH66-RS03880G160A蛋白如序列表的序列1所示。
YH66-RS03880基因为编码所述YH66-RS03880蛋白的基因。
具体的,所述YH66-RS03880基因为如下(b1)或(b2)或(b3):
(b1)编码区如序列表的序列4所示的DNA分子;
(b2)来源于细菌且与(b1)具有95%以上同一性且编码所述蛋白质的DNA分子;
(b3)在严格条件下与(b1)杂交且编码所述蛋白质的DNA分子。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
所述95%以上同一性具体可为96%以上同一性或97%以上同一性或98%以上同一性或99%以上同一性。
所述严格条件可为在0.1×SSPE(或0.1×SSC),0.1%SDS的溶液中,在65℃条件下杂交并洗膜。
所述YH66-RS03880G160A蛋白的编码基因(命名为YH66-RS03880G160A基因)也属于本发明的保护范围。
具体的,YH66-RS03880G160A基因为如下(c1)或(c2)或(c3):
(c1)编码区如序列表的序列2所示的DNA分子;
(c2)来源于细菌且与(c1)具有95%以上同一性且编码所述蛋白质的DNA分子;
(c3)在严格条件下与(c1)杂交且编码所述蛋白质的DNA分子。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
所述95%以上同一性具体可为96%以上同一性或97%以上同一性或98%以上同一性或99%以上同一性。
所述严格条件可为在0.1×SSPE(或0.1×SSC),0.1%SDS的溶液中,在65℃条件下杂交并洗膜。
具有YH66-RS03880G160A基因的DNA分子、具有YH66-RS03880G160A基因的表达盒或具有YH66-RS03880G160A基因的重组载体或具有YH66-RS03880G160A基因的重组菌均属于本发明的保护范围。
示例性的,具有YH66-RS03880G160A基因的DNA分子可为序列表的序列6所示的DNA分子或序列表的序列7所示的DNA分子。
示例性的,具有YH66-RS03880G160A基因的重组载体可为具有序列表的序列6所示的DNA分子的质粒或具有序列表的序列7所示的DNA分子的质粒。
示例性的,具有YH66-RS03880G160A基因的重组载体可为实施例中的重组质粒003或重组质粒pXMJ19-YH66-RS03880G160A。
所述重组菌具体可为重组细菌。
示例性的,具有YH66-RS03880G160A基因的重组菌可为具有序列表的序列5所示的DNA分子的重组菌或具有序列表的序列6所示的DNA分子的重组菌或具有序列表的序列7所示的DNA分子的重组菌。
具有YH66-RS03880G160A基因的重组菌具体可采用如下方式制备:将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因。
示例性的,将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因的实现方式如下:将序列表的序列5所示的DNA分子导入细菌。
示例性的,将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因的实现方式如下:将具有序列表的序列5所示的DNA分子的质粒导入细菌。
示例性的,将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因的实现方式如下:将实施例中的重组质粒pK18-YH66-RS03880G160A导入细菌。
所述重组菌中,YH66-RS03880G160A基因可以整合至基因组DNA中进行表达也可以在质粒中进行表达。
示例性的,具有YH66-RS03880G160A基因的重组菌具体可采用如下方式制备:将序列表的序列6所示的DNA分子或序列表的序列7所示的DNA分子导入细菌。
示例性的,具有YH66-RS03880G160A基因的重组菌具体可采用如下方式制备:将具有序列表的序列6所示的DNA分子的质粒或具有序列表的序列7所示的DNA分子的质粒导入细菌。
示例性的,具有YH66-RS03880G160A基因的重组菌具体可采用如下方式制备:将实施例中的重组质粒003或重组质粒pXMJ19-YH66-RS03880G160A导入细菌。
本发明还保护YH66-RS03880G160A蛋白、YH66-RS03880G160A基因、具有YH66-RS03880G160A基因的表达盒或具有YH66-RS03880G160A基因的重组载体或具有YH66-RS03880G160A基因的重组菌的应用;
所述应用为如下(Ⅰ)或(Ⅱ)或(Ⅲ):
(Ⅰ)在提高细菌缬氨酸产量中的应用;
(Ⅱ)在生产缬氨酸中的应用;
(Ⅲ)在提高细菌菌量中的应用。
本发明还保护特定物质的应用;
所述应用为如下(Ⅰ)或(Ⅱ)或(Ⅲ):
(Ⅰ)在提高细菌缬氨酸产量中的应用;
(Ⅱ)在生产缬氨酸中的应用;
(Ⅲ)在提高细菌菌量中的应用。
所述特定物质为如下(d1)、(d2)、(d3)、(cd4)、(d5)或(d6):
(d1)用于提高YH66-RS03880G160A基因表达的物质;
(d2)用于提高YH66-RS03880G160A蛋白丰度的物质;
(d3)用于提高YH66-RS03880G160A蛋白活性的物质;
(d4)用于提高YH66-RS03880基因表达的物质;
(d5)用于提高YH66-RS03880蛋白丰度的物质;
(d6)用于提高YH66-RS03880蛋白活性的物质。
示例性的,所述用于提高YH66-RS03880G160A基因表达的物质具体可为YH66-RS03880G160A基因或具有YH66-RS03880G160A基因的重组质粒。示例性的,所述重组质粒可为实施例中的重组质粒003或重组质粒pXMJ19-YH66-RS03880G160A。
示例性的,所述用于提高YH66-RS03880基因表达的物质具体可为YH66-RS03880基因或具有YH66-RS03880基因的重组质粒。示例性的,所述重组质粒可为实施例中的重组质粒002或重组质粒pXMJ19-YH66-RS03880。
本发明还提供了一种重组菌,是在细菌中过表达YH66-RS03880G160A基因或YH66-RS03880基因得到的。
示例性的,过表达YH66-RS03880G160A基因的实现方式如下:在细菌中导入YH66-RS03880G160A基因或具有YH66-RS03880G160A基因的重组质粒。示例性的,所述重组质粒可为实施例中的重组质粒003或重组质粒pXMJ19-YH66-RS03880G160A。
示例性的,过表达YH66-RS03880基因的实现方式如下:在细菌中导入YH66-RS03880基因或具有YH66-RS03880基因的重组质粒。示例性的,所述重组质粒可为实施例中的重组质粒002或重组质粒pXMJ19-YH66-RS03880。
本发明还保护所述重组菌在制备缬氨酸中的应用。
本发明还保护一种提高细菌的缬氨酸产量的方法,包括如下步骤:将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因。
示例性的,将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因的实现方式如下:将实施例中的重组质粒pK18-YH66-RS03880G160A导入细菌。
本发明还提供了一种提高细菌的缬氨酸产量的方法,包括如下步骤:在细菌中过表达YH66-RS03880G160A基因或在细菌中过表达YH66-RS03880基因或提高细菌中YH66-RS03880G160A蛋白的丰度或提高细菌中YH66-RS03880蛋白的丰度或提高细菌中YH66-RS03880G160A蛋白的活性或提高细菌中YH66-RS03880蛋白的活性。
示例性的,过表达YH66-RS03880G160A基因的实现方式如下:在细菌中导入YH66-RS03880G160A基因或具有YH66-RS03880G160A基因的重组质粒。示例性的,所述重组质粒可为实施例中的重组质粒003或重组质粒pXMJ19-YH66-RS03880G160A。
示例性的,过表达YH66-RS03880基因的实现方式如下:在细菌中导入YH66-RS03880基因或具有YH66-RS03880基因的重组质粒。示例性的,所述重组质粒可为实施例中的重组质粒002或重组质粒pXMJ19-YH66-RS03880。
本发明还保护YH66-RS03880G160A蛋白或YH66-RS03880蛋白在调控细菌的缬氨酸产量中的应用。
所述调控为正调控,即YH66-RS03880G160A蛋白含量增高,缬氨酸产量增高。
所述调控为正调控,即YH66-RS03880G160A蛋白含量降低,缬氨酸产量降低。
所述调控为正调控,即YH66-RS03880蛋白含量增高,缬氨酸产量增高。
所述调控为正调控,即YH66-RS03880蛋白含量降低,缬氨酸产量降低。
本发明还保护YH66-RS03880G160A蛋白或YH66-RS03880蛋白在调控细菌的菌量中的应用。
所述调控为正调控,即YH66-RS03880G160A蛋白含量增高,细菌菌量增高。
所述调控为正调控,即YH66-RS03880G160A蛋白含量降低,细菌菌量降低。
所述调控为正调控,即YH66-RS03880蛋白含量增高,细菌菌量增高。
所述调控为正调控,即YH66-RS03880蛋白含量降低,细菌菌量降低。
应用所述重组菌制备缬氨酸时,具体方法包括如下步骤:发酵所述重组菌。
本领域技术人员可以采用现有技术中的发酵方法进行发酵。也可通过常规试验进行发酵方法的优化和改进。可以在本领域中已知的发酵条件下在合适的培养基中进行细菌的发酵。培养基可以包含:碳源、氮源、微量元素、及其组合。在培养中,可以调节培养物的pH。此外,培养时可以包括防止气泡产生,例如通过使用消泡剂进行气泡产生的防止。此外,培养时可以包括将气体注射入培养物中。气体可以包括能够维持培养物的需氧条件的任何气体。在培养中,培养物的温度可以是20至45℃。
所述方法还可包括如下步骤:从培养物中获得缬氨酸。从培养物中获得缬氨酸可通过各种方式实现,包括但不限于:用硫酸或氢氯酸等处理培养物,接着进行诸如阴离子交换层析、浓缩、结晶和等电点沉淀的方法的组合。
所述发酵中,示例性的发酵培养基的配方见表3,余量为水。
所述发酵中,示例性的发酵控制工艺见表4。
示例性的,所述发酵中,完成接种的初始时刻,体系OD值可为0.3-0.5。
示例性的,所述发酵的发酵过程中:用于调pH的为氨水;发酵体系中有泡沫时,加入适量消泡剂antifoam(CB-442);通过补加70%葡萄糖水溶液控制体系含糖量(残糖)。
以上任一所述细菌包括但不限于如下:棒杆菌属细菌,优选嗜乙酰棒杆菌(Corynebacterium acetoacidophilum)、醋谷棒杆菌(Corynebacteriumacetoglutamicum)、美棒杆菌(Corynebacterium callunae)、谷氨酸棒杆菌(Corynebacterium glutamicum)、黄色短杆菌(Brevibacterium flavum)、乳糖发酵短杆菌(Brevibacterium lactofermentum)、产氨棒杆菌(Corynebacterium ammoniagenes)、北京棒杆菌(Corynebacterium pekinense)、解糖短杆菌(Brevibacterium saccharolyticum)、玫瑰色短杆菌(Brevibacterium roseum)、生硫短杆菌(Brevibacterium thiogenitalis)。
以上任一所述细菌为具有生产缬氨酸的能力的细菌。
“具有生产缬氨酸的能力的细菌”是指细菌具有以下能力:在培养基和/或细菌的细胞中产生并累积缬氨酸的能力。从而,当细菌在培养基中培养时可以收集缬氨酸。
所述细菌可为自然采集的野生型细菌也可为修饰后的细菌。
“修饰后的细菌”指的是将自然采集的野生型细菌进行人工突变和/或诱变得到的改造后的细菌。
具体的,所述谷氨酸棒杆菌可为谷氨酸棒杆菌CGMCC21260。
谷氨酸棒杆菌(Corynebacterium glutamicum)YPFV1,已于2020年11月30日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC No.21260。谷氨酸棒杆菌(Corynebacterium glutamicum)YPFV1,又称为谷氨酸棒杆菌CGMCC21260。
以上任一所述缬氨酸的含义为广义的缬氨酸,包括游离形式缬氨酸、缬氨酸的盐或两者的混合物。
具体的,所述缬氨酸为L-缬氨酸。
以上任何方法或应用还可用于缬氨酸的下游产品的制备。
谷氨酸棒杆菌中的YH66-RS03880蛋白如序列表的序列3所示,其编码基因如序列表的序列4所示。本发明中通过引入点突变,得到了序列表的序列1所示的YH66-RS03880G160A蛋白,YH66-RS03880G160A蛋白的编码基因如序列表的序列2所示。与YH66-RS03880基因相比,YH66-RS03880G160A基因的差异在于第160位核苷酸由G突变为A。与YH66-RS03880蛋白相比,YH66-RS03880G160A蛋白的差异在于第54位氨基酸残基由E突变为K。
本发明发现YH66-RS03880蛋白对细菌的缬氨酸产量存在正调控,即YH66-RS03880蛋白含量增高、缬氨酸产量增高,YH66-RS03880蛋白含量降低、缬氨酸产量降低。抑制YH66-RS03880基因表达可以降低缬氨酸产量,过表达YH66-RS03880基因提高缬氨酸产量。进一步的,本发明通过点突变,得到了YH66-RS03880G160A蛋白,其功能优于YH66-RS03880蛋白。本发明对于缬氨酸工业化生产,具有重大的应用价值。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。pK18mobsacB质粒:Addgene公司;pK18mobsacB质粒中具有卡那霉素抗性基因作为筛选标记。pXMJ19质粒:BioVector质粒载体菌种细胞基因保藏中心;pXMJ19质粒中具有氯霉素抗性基因作为筛选标记。NEBuilder酶:NEB公司。如无特殊说明,实施例中的培养基为配方为表1的培养基(余量为水,pH为7.0)。不含卡那霉素培养基即表1所示的培养基。含卡那霉素的培养基由表1所示的培养基和卡那霉素组成,卡那霉素的含量为50μg/ml。如无特殊说明,实施例中的培养指的是32℃静置培养。实施例中的单链构象多态性聚丙烯酰胺凝胶电泳(sscp-PAGE):采用的胶浓度为8%,电泳胶的组成见表2;电泳条件为:使用1×TBE缓冲液,120V电压,电泳时间10h。
如无特殊说明,以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
表1
组分 | 在培养基中的浓度 |
蔗糖 | 10g/L |
多聚蛋白胨 | 10g/L |
牛肉膏 | 10g/L |
酵母粉 | 5g/L |
尿素 | 2g/L |
氯化钠 | 2.5g/L |
琼脂粉 | 20g/L |
表2
组分 | 加入量 |
40%丙烯酰胺 | 8mL |
ddH<sub>2</sub>O | 26mL |
甘油 | 4mL |
10×TBE | 2mL |
TEMED | 40μL |
10%AP | 600μL |
实施例1、谷氨酸棒杆菌CGMCC21260的获得
谷氨酸棒杆菌ATCC15168:ATCC中编号为15168的谷氨酸棒杆菌(Corynebacteriumglutamicum)。
将谷氨酸棒杆菌ATCC15168进行诱变,获得谷氨酸棒杆菌(Corynebacteriumglutamicum)YPFV1。
谷氨酸棒杆菌(Corynebacterium glutamicum)YPFV1,已于2020年11月30日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC No.21260。谷氨酸棒杆菌(Corynebacterium glutamicum)YPFV1,又称为谷氨酸棒杆菌CGMCC21260。
实施例2、构建重组菌YPV-001
一、构建重组质粒
P1:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTGGAGCTTTTTGAAACCCT-3';
P2:5'-CATTTTCCGGGGTGACTTTGACTTTGTTGTGCTG-3';
P3:5'-CAGCACAACAAAGTCAAAGTCACCCCGGAAAATG-3';
P4:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCAAATACGTCAAGATTAAAG-3'。
P5:5'-TCAGTCGCTCCAAAATCCGG-3';
P6:5'-TCAGCAAGGCAACGAGCGCC-3'。
1、以谷氨酸棒杆菌ATCC15168为模板,采用引物P1和引物P2组成的引物对进行PCR扩增,回收扩增产物(667bp)。
2、以谷氨酸棒杆菌ATCC15168为模板,采用引物P3和引物P4组成的引物对进行PCR扩增,回收扩增产物(701bp)。
3、同时将步骤1回收的扩增产物和步骤2回收的扩增产物作为模板,采用引物P1和引物P4组成的引物对进行PCR扩增(Overlap PCR),回收扩增产物(1334bp)。经测序,扩增产物如序列表的序列5所示。
4、取pK18mobsacB质粒,采用限制性内切酶Xba I进行单酶切,回收线性化质粒。
5、将步骤3回收的扩增产物与步骤4回收的线性化质粒共孵育(采用NEBuilder酶,50℃孵育30min),得到重组质粒pK18-YH66-RS03880G160A。经测序验证,重组质粒pK18-YH66-RS03880G160A中具有序列表的序列5所示的DNA分子。
二、构建重组菌YPV-001
1、采用重组质粒pK18-YH66-RS03880G160A对谷氨酸棒杆菌CGMCC21260进行电击转化,然后培养。
2、挑取步骤1中的菌株,采用含15%蔗糖的培养基培养,然后挑取单菌落,分别采用含卡那霉素的培养基和不含卡那霉素的培养基进行培养,筛选在含卡那霉素的培养基上不能生长且在不含卡那霉素的培养基上可以生长的菌株。
3、取步骤2筛选的菌株,采用引物P5和引物P6组成的引物对进行PCR扩增,然后回收扩增产物(271bp)。
4、取步骤3的扩增产物,先95℃变性10min再冰浴5min,然后进行sscp-PAGE。电泳时,采用重组质粒pK18-YH66-RS03880G160A的扩增片段(即以重组质粒pK18-YH66-RS03880G160A为模板,采用引物P5和引物P6组成的引物对进行PCR扩增的扩增产物)为阳性对照,采用谷氨酸棒杆菌CGMCC21260的扩增片段(即以谷氨酸棒杆菌CGMCC21260为模板,采用引物P5和引物P6组成的引物对进行PCR扩增的扩增产物)为阴性对照,水作为空白对照。由于片段结构不同,电泳位置不同,电泳位置与阴性对照不一致且与阳性对照一致的菌株为筛选的目的菌株(等位替换成功的重组菌株)。
5、根据步骤4的结果,将筛选得到的菌株的步骤3的扩增产物进行测序验证,得到重组菌YPV-001。与谷氨酸棒杆菌CGMCC21260相比,重组菌YPV-001的差异仅在于将谷氨酸棒杆菌CGMCC21260基因组中的序列表的序列4所示的YH66-RS03880基因取代为了序列表的序列2所示的YH66-RS03880G160A基因。序列2和序列4仅存在一个核苷酸差异,位于第160位。重组菌YPV-001即将谷氨酸棒杆菌CGMCC21260中的YH66-RS03880基因进行突变(单点突变)得到的工程菌株。
实施例2、构建重组菌YPV-003和重组菌YPV-002
P7:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCATGACGGCTGACTGGACTC-3';
P8:5'-TGCTAGCGCTCAGAAAAGACAATCGGACTCCTTAAATGGG-3';
P9:5'-CCCATTTAAGGAGTCCGATTGTCTTTTCTGAGCGCTAGCA-3';
P10:5'-CTATGTGAGTAGTCGATTTACTAAAGGTGTAGCTCTGTTC-3';
P11:5'-GAACAGAGCTACACCTTTAGTAAATCGACTACTCACATAG-3';
P12:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCATAAGAAACAACCACTT-3'。
P13:5'-GTCCGCTCTGTTGGTGTTCA-3';
P14:5'-AGTAATCAGCACATCGCCAT-3';
P15:5'-ATTTTGATGAACAGAATCTG-3';
P16:5'-TGGAGGAATATTCGGCCCAG-3'。
一、构建重组菌YPV-003
1、以重组菌YPV-001为模板,采用引物P7和引物P8组成的引物对进行PCR扩增,回收扩增产物(806bp)。
2、以重组菌YPV-001为模板,采用引物P9和引物P10组成的引物对进行PCR扩增,回收扩增产物(1966bp)。
3、以重组菌YPV-001为模板,采用引物P11和引物P12组成的引物对进行PCR扩增,回收扩增产物(783bp)。
4、取pK18mobsacB质粒,采用限制性内切酶Xba I进行单酶切,回收线性化质粒。
5、将步骤1回收的扩增产物、步骤2回收的扩增产物、步骤3回收的扩增产物与步骤4回收的线性化质粒共孵育(采用NEBuilder酶,50℃孵育30min),得到重组质粒003。经测序验证,重组质粒003中具有序列表的序列6所示的DNA分子。
6、采用重组质粒003对谷氨酸棒杆菌CGMCC21260进行电击转化,然后培养,然后对各个单菌落分别进行PCR鉴定(采用引物P13和引物P14组成的引物对),能扩增出1256bp条带的菌株为阳性菌株。
7、挑取步骤6中的阳性菌株,采用含15%蔗糖的培养基培养,然后挑取单菌落,分别采用含卡那霉素的培养基和不含卡那霉素的培养基进行培养,筛选在含卡那霉素的培养基上不能生长且在不含卡那霉素的培养基上可以生长的菌株。
8、取步骤7筛选的菌株,采用引物P15和引物P16组成的引物对进行PCR扩增,扩增出大小为1020bp的菌为YH66-RS03880G160A基因整合到CGMCC21260基因组上的阳性菌株,将其命名为重组菌YPV-003。重组菌YPV-003为基因组上过表达YH66-RS03880G160A基因的工程菌株。
二、构建重组菌YPV-002
将模板均由“YPV-001”替换为“谷氨酸棒杆菌ATCC15168”,其他同步骤一。
与重组质粒003相比,重组质粒002的差异仅在于:用序列4所示DNA分子取代了重组质粒中的序列2所示DNA分子。
得到YH66-RS03880基因整合到谷氨酸棒杆菌CGMCC21260基因组上的阳性菌株,将其命名为重组菌YPV-002。重组菌YPV-002为基因组上过表达YH66-RS03880基因的工程菌株。与重组菌YPV-003相比,重组菌YPV-002的差异仅在于:整合到谷氨酸棒杆菌CGMCC21260基因组的外源DNA的序列中,序列4取代了序列2。
实施例3、构建重组菌YPV-005和重组菌YPV-004
一、构建重组菌YPV-005
1、以重组菌YPV-001为模板,采用引物P17和引物P18组成的引物对进行PCR扩增,回收PCR扩增产物(1996bp)。经测序,扩增产物如序列表的序列7所示。
P17:5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGTCTTTTCTGAGCGCTAGCA-3';
P18:5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAAAGGTGTAGCTCTGTTC-3'。
2、取pXMJ19质粒,采用限制性内切酶EcoR I酶切进行单酶切,回收线性化质粒。
3、将步骤1回收的扩增产物与步骤2回收的线性化质粒共孵育(采用NEBuilder酶,50℃孵育30min),得到重组质粒pXMJ19-YH66-RS03880G160A。经测序验证,重组质粒pXMJ19-YH66-RS03880G160A中具有序列表的序列7所示的DNA分子。
4、将重组质粒pXMJ19-YH66-RS03880G160A电转导入谷氨酸棒杆菌CGMCC21260,得到重组菌YPV-005。重组菌YPV-005为通过质粒过表达YH66-RS03880G160A基因的工程菌株。
二、构建重组菌YPV-004
将模板由“重组菌YPV-001”替换为“谷氨酸棒杆菌ATCC15168”,其他同步骤一。
与重组质粒pXMJ19-YH66-RS03880G160A相比,重组质粒pXMJ19-YH66-RS03880的差异仅在于:用序列4所示DNA分子取代了重组质粒中的序列2所示DNA分子。
得到重组菌YPV-004。重组菌YPV-004为通过质粒过表达YH66-RS03880基因的工程菌株。与重组菌YPV-005相比,重组菌YPV-004的差异仅在于:通过质粒过表达的外源DNA的序列中,序列4取代了序列2。
实施例4、构建基因组上缺失YH66-RS03880基因的工程菌株
P19:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTTGCCCGCACCCCAAGTGG-3';
P20:5'-GTTGGCTAGCGCCTGCCTGAGCTCCTTTGAGTGGAGAAA-3';
P21:5'-TTTCTCCACTCAAAGGAGCTCAGGCAGGCGCTAGCCAAC-3';
P22:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTCATGTGCAGTCTTTGCGT-3'。
P23:5'-GTTGCCCGCACCCCAAGTGG-3';
P24:5'-CTCATGTGCAGTCTTTGCGT-3'。
一、构建重组质粒
1、以谷氨酸棒杆菌ATCC15168为模板,采用引物P19和引物P20组成的引物对进行PCR扩增,回收扩增产物(上游同源臂片段,682bp)。
2、以谷氨酸棒杆菌ATCC15168为模板,采用引物P21和引物P22组成的引物对进行PCR扩增,回收扩增产物(下游同源臂片段,658bp)。
3、同时将步骤1回收的扩增产物和步骤2回收的扩增产物作为模板,采用引物P19和引物P22组成的引物对进行PCR扩增(Overlap PCR),回收扩增产物(1300bp)。
4、取pK18mobsacB质粒,采用限制性内切酶Xba I进行单酶切,回收线性化质粒。
5、将步骤3回收的扩增产物与步骤4回收的线性化质粒共孵育(采用NEBuilder酶,50℃孵育30min),得到重组质粒pK18-ΔYH66-RS03880。
二、构建重组菌YPV-006
1、采用重组质粒pK18-ΔYH66-RS03880对谷氨酸棒杆菌CGMCC21260进行电击转化,然后培养,然后对各个单菌落分别进行PCR鉴定(采用引物P23和引物P24组成的引物对),能同时扩增出1214bp和3140bp条带的菌株为阳性菌株,只扩增出3140bp条带的菌株为转化失败的出发菌。
2、挑取步骤1中的阳性菌株,采用含15%蔗糖的培养基培养,然后挑取单菌落,分别采用含卡那霉素的培养基和不含卡那霉素的培养基进行培养,筛选在含卡那霉素的培养基上不能生长且在不含卡那霉素的培养基上可以生长的菌株。
3、取步骤2筛选的菌株,采用引物P23和引物P24组成的引物对进行PCR扩增,只显示单一扩增产物且大小为1214bp的菌株为YH66-RS03880基因编码区被敲除的阳性菌株。
4、将步骤3筛选得到的菌株,再次采用引物P23和引物P24组成的引物对进行PCR扩增并测序,将测序正确的菌株命名为重组菌YPV-006。与谷氨酸棒杆菌CGMCC21260的基因组DNA相比,重组菌YPV-006的差异仅在于缺失了序列表的序列4所示的DNA分子。
实施例5、发酵制备L-缬氨酸
供试菌株分别为:谷氨酸棒杆菌CGMCC21260、重组菌YPV-001、重组菌YPV-002、重组菌YPV-003、重组菌YPV-004、重组菌YPV-005和重组菌YPV-006。
发酵罐:BLBIO-5GC-4-H型号的发酵罐(上海百仑生物科技有限公司)。
发酵培养基的配方见表3,余量为水。
表3 发酵培养基配方
发酵控制工艺见表4。
完成接种的初始时刻,体系OD值为0.3-0.5。
发酵过程中:用于调pH的为氨水;发酵体系中有泡沫时,加入适量消泡剂antifoam(CB-442);通过补加70%葡萄糖水溶液控制体系含糖量(残糖)。
表4 发酵控制工艺
完成发酵后,收集上清,采用HPLC检测上清中的L-缬氨酸产量。
结果见表5。重组菌YPV-001的L-缬氨酸产量显著高于谷氨酸棒杆菌CGMCC21260。重组菌YPV-003的L-缬氨酸产量显著高于重组菌YPV-002。重组菌YPV-005的L-缬氨酸产量显著高于重组菌YPV-004。重组菌YPV-006的L-缬氨酸产量显著低于谷氨酸棒杆菌CGMCC21260。重组菌YPV-001、重组菌YPV-002、重组菌YPV-003、重组菌YPV-004和重组菌YPV-005的L-缬氨酸产量均高于谷氨酸棒杆菌CGMCC21260。结果表明,过表达YH66-RS03880基因提高L-缬氨酸产量,抑制YH66-RS03880基因表达降低L-缬氨酸产量。与YH66-RS03880蛋白相比,YH66-RS03880G160A蛋白的作用效果显著提高。
表5 L-缬氨酸发酵实验结果
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 宁夏伊品生物科技股份有限公司
<120> YH66-RS03880基因改造得到的工程菌及其在制备缬氨酸中的应用
<130> GNCYX211992
<160> 7
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Asp Ile Gly Thr Ser Pro Leu Tyr Ser Leu His Thr Ala Phe Ser Met
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Val Leu Leu Gly Ala Val Val Leu Thr Val Thr Gly Ala Glu Ala Leu
225 230 235 240
Tyr Ala Asp Met Gly His Phe Gly Ala Arg Pro Ile Arg Val Ala Trp
245 250 255
Phe Cys Val Val Met Pro Ala Leu Ile Leu Thr Tyr Leu Gly Gln Gly
260 265 270
Ala Leu Val Ile Asn Gln Pro Glu Ala Val Arg Asn Pro Met Phe Tyr
275 280 285
Leu Ala Pro Glu Gly Leu Arg Ile Pro Leu Val Ile Leu Ala Thr Ile
290 295 300
Ala Thr Val Ile Ala Ser Gln Ala Val Ile Ser Gly Ala Tyr Ser Leu
305 310 315 320
Thr Lys Gln Ala Val Asn Leu Lys Leu Leu Pro Arg Met Val Ile Arg
325 330 335
His Thr Ser Arg Lys Glu Glu Gly Gln Ile Tyr Met Pro Leu Val Asn
340 345 350
Gly Leu Leu Phe Val Ser Val Met Val Val Val Leu Val Phe Arg Ser
355 360 365
Ser Glu Ser Leu Ala Ser Ala Tyr Gly Leu Ala Val Thr Gly Thr Leu
370 375 380
Val Leu Val Ser Val Leu Tyr Leu Val Tyr Ala His Thr Thr Trp Trp
385 390 395 400
Lys Thr Ala Leu Phe Ile Val Phe Ile Gly Ile Pro Glu Val Leu Leu
405 410 415
Phe Ala Ser Asn Thr Thr Lys Ile His Asp Gly Gly Trp Leu Pro Leu
420 425 430
Leu Ile Ala Ala Val Leu Ile Val Val Met Arg Thr Trp Glu Trp Gly
435 440 445
Ser Asp Arg Val Asn Gln Glu Arg Ala Glu Leu Glu Leu Pro Met Asp
450 455 460
Lys Phe Leu Glu Lys Leu Asp Gln Pro His Asn Ile Gly Leu Arg Lys
465 470 475 480
Val Ala Glu Val Ala Val Phe Pro His Gly Thr Ser Asp Thr Val Pro
485 490 495
Leu Ser Leu Val Arg Cys Val Lys Asp Leu Lys Leu Leu Tyr Arg Glu
500 505 510
Ile Val Ile Val Arg Ile Val Gln Glu His Val Pro His Val Pro Pro
515 520 525
Glu Glu Arg Ala Glu Met Glu Val Leu His His Ala Pro Ile Arg Val
530 535 540
Val Arg Val Asp Leu His Leu Gly Tyr Phe Asp Glu Gln Asn Leu Pro
545 550 555 560
Glu Asn Leu His Ala Ile Asp Pro Thr Trp Asp Asn Ala Thr Tyr Phe
565 570 575
Leu Ser Ala Leu Thr Leu Arg Ser Arg Leu Pro Gly Lys Ile Ala Gly
580 585 590
Trp Arg Asp Arg Leu Tyr Leu Ser Met Glu Arg Asn Gln Ala Ser Arg
595 600 605
Thr Glu Ser Phe Lys Leu Gln Pro Ser Lys Thr Ile Thr Val Gly Thr
610 615 620
Glu Leu His Leu
625
<210> 4
<211> 1887
<212> DNA
<213> Corynebacterium glutamicum
<400> 4
atgcttaacc gcatgaaaag tgcgcggcca aaatcagtcg ctccaaaatc cggacaagct 60
ttactcactc ttggtgccct aggtgttgtg ttcggtgaca tcggcaccag ccccctgtat 120
tcacttcaca ctgcatttag catgcagcac aacaaagtcg aagtcacccc ggaaaatgtg 180
tacgggatca tctccatggt gctgtggaca atcaccttga tcgtcaccgt caaatacgtc 240
atgctggtca cccgagctga caaccaagga caaggtggca tcctggcgct cgttgccttg 300
ctgaaaaacc gtgggcactg gggaaaattc gtggcagtag ccggcatgtt gggtgcagcg 360
ctgttttatg gcgatgtgct gattactccg gcgatctctg tgcttagcgc gacggagggg 420
ttgacggtta tttccccaag ctttgagcgc ttcattctgc ccgtatctct cgcagttttg 480
atcgctattt ttgcaatcca accgctcggc acagaaaaag tcggcaaagc cttcggcccc 540
atcatgttgc tgtggtttgt cacccttgca ggattgggaa ttccgcaaat catcgtacat 600
ccagaaatct tgcagagctt gtctccacat tgggccctgc gcttgattgt ggctgagcct 660
ttccaagcat ttgtgctgct tggtgccgtt gtcctgacag taacgggtgc ggaagcgctc 720
tacgctgata tgggccattt tggggcgagg ccaattagag tggcgtggtt ttgcgtcgtc 780
atgcctgctt taatcttgac gtatttgggg cagggcgcct tggtgattaa ccagcctgaa 840
gcggtgcgca accccatgtt ttatctcgcg ccggaaggtc tgcggattcc gttggttatt 900
ttggcgacca tcgccacggt gatcgcatcg caggccgtga tttctggtgc gtattcattg 960
accaagcagg ccgtgaattt gaaactgctg ccacgcatgg tgatccggca tacctcccga 1020
aaagaggaag gccagatcta tatgccgctg gttaatggat tgctgtttgt atccgtgatg 1080
gtcgtggtgc tggtattccg atcctcagaa agcctcgcca gcgcgtatgg acttgctgtg 1140
accggaacat tggtgctggt cagcgtcttg tatctggtct acgcccacac cacatggtgg 1200
aaaacagcgt tgttcattgt gttcatcggt attccagaag tacttctatt cgcctcgaac 1260
accacgaaaa ttcacgacgg tggctggctt ccactactta ttgcagccgt gctcatcgtg 1320
gtgatgagga cctgggagtg gggaagtgac cgcgtcaatc aggaacgcgc agagctggaa 1380
cttcccatgg ataagttctt ggagaaactc gatcagccac acaatattgg gcttcgtaaa 1440
gttgccgaag tggcagtatt tccacatggc accagcgata ctgtcccgtt gtcattggtt 1500
cgctgcgtga aagacctcaa gcttttatac cgagagatcg tgatcgttcg aatcgtccaa 1560
gaacacgttc cgcacgtgcc accagaggaa cgcgcggaaa tggaagtgct ccatcacgcc 1620
ccgattaggg tggttcgggt tgatctgcac cttggttatt ttgatgaaca gaatctgcct 1680
gaaaatctcc acgccattga cccaacatgg gataacgcca cttacttcct gtctgcgctg 1740
acacttcgga gcaggttgcc tggaaagatc gctggctggc gtgatcgttt gtatctttcg 1800
atggaacgca atcaggcatc tcgaaccgag tctttcaaac tgcaaccaag caaaaccatc 1860
actgtcggaa cagagctaca cctttag 1887
<210> 5
<211> 1334
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctaggtgg agctttttga aacccttgac 60
cagaacagcc agccagagat ctttgtcaac gagctcgcca tgcgtccaca caacaccggc 120
cactggactc aagatggctg cgtgaccagc caattcgagc agcacctccg cgcagtcctc 180
gactacccac tgggtgctac cgacactttg gctgattaca ccgtgatggc caacgtgctc 240
ggtgctgaca ccgacccaga gatgcccatg gcaacccgca tggtggaagt atggcgcaaa 300
tacccagatg ccaagatcca cctctacggt aagggacatc gcccgggacg aaagattggc 360
cacgtcaaca tggtgggatc cgaccttgaa aagacccgaa ccgaagccct ggcctgcgca 420
tacttccttg tcaacgctcg ctgggattag gtcttttctg agcgctagca tttctccact 480
caaaggagca tgcttaaccg catgaaaagt gcgcggccaa aatcagtcgc tccaaaatcc 540
ggacaagctt tactcactct tggtgcccta ggtgttgtgt tcggtgacat cggcaccagc 600
cccctgtatt cacttcacac tgcatttagc atgcagcaca acaaagtcaa agtcaccccg 660
gaaaatgtgt acgggatcat ctccatggtg ctgtggacaa tcaccttgat cgtcaccgtc 720
aaatacgtca tgctggtcac ccgagctgac aaccaaggac aaggtggcat cctggcgctc 780
gttgccttgc tgaaaaaccg tgggcactgg ggaaaattcg tggcagtagc cggcatgttg 840
ggtgcagcgc tgttttatgg cgatgtgctg attactccgg cgatctctgt gcttagcgcg 900
acggaggggt tgacggttat ttccccaagc tttgagcgct tcattctgcc cgtatctctc 960
gcagttttga tcgctatttt tgcaatccaa ccgctcggca cagaaaaagt cggcaaagcc 1020
ttcggcccca tcatgttgct gtggtttgtc acccttgcag gattgggaat tccgcaaatc 1080
atcgtacatc cagaaatctt gcagagcttg tctccacatt gggccctgcg cttgattgtg 1140
gctgagcctt tccaagcatt tgtgctgctt ggtgccgttg tcctgacagt aacgggtgcg 1200
gaagcgctct acgctgatat gggccatttt ggggcgaggc caattagagt ggcgtggttt 1260
tgcgtcgtca tgcctgcttt aatcttgacg tatttggggt accgagctcg aattcgtaat 1320
catggtcata gctg 1334
<210> 6
<211> 3475
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cagtgccaag cttgcatgcc tgcaggtcga ctctagcatg acggctgact ggactcgact 60
tccatacgag gttctggaga agatctccac ccgcatcacc aacgaagttc cagatgtgaa 120
ccgcgtggtt ttggacgtaa cctccaagcc accaggaacc atcgaatggg agtaggcctt 180
aaatgagcct tcgttaagcg gcaatcacct tattggagat tgtcgctttt cccatttctc 240
cgggttttct ggaacttttt gggcgtatgc tgggaatgat tctattattg ccaaatcaga 300
aagcaggaga gacccgatga gcgaaatcct agaaacctat tgggcacccc actttggaaa 360
aaccgaagaa gccacagcac tcgtttcata cctggcacaa gcttccggcg atcccattga 420
ggttcacacc ctgttcgggg atttaggttt agacggactc tcgggaaact acaccgacac 480
tgagattgac ggctacggcg acgcattcct gctggttgca gcgctatccg tgttgatggc 540
tgaaaacaaa gcaacaggtg gcgtgaatct gggtgagctt gggggagctg ataaatcgat 600
ccggctgcat gttgaatcca aggagaacac ccaaatcaac accgcattga agtattttgc 660
gctctcccca gaagaccacg cagcagcaga tcgcttcgat gaggatgacc tgtctgagct 720
tgccaacttg agtgaagagc tgcgcggaca gctggactaa ttgtctccca tttaaggagt 780
ccgattgtct tttctgagcg ctagcatttc tccactcaaa ggagcatgct taaccgcatg 840
aaaagtgcgc ggccaaaatc agtcgctcca aaatccggac aagctttact cactcttggt 900
gccctaggtg ttgtgttcgg tgacatcggc accagccccc tgtattcact tcacactgca 960
tttagcatgc agcacaacaa agtcaaagtc accccggaaa atgtgtacgg gatcatctcc 1020
atggtgctgt ggacaatcac cttgatcgtc accgtcaaat acgtcatgct ggtcacccga 1080
gctgacaacc aaggacaagg tggcatcctg gcgctcgttg ccttgctgaa aaaccgtggg 1140
cactggggaa aattcgtggc agtagccggc atgttgggtg cagcgctgtt ttatggcgat 1200
gtgctgatta ctccggcgat ctctgtgctt agcgcgacgg aggggttgac ggttatttcc 1260
ccaagctttg agcgcttcat tctgcccgta tctctcgcag ttttgatcgc tatttttgca 1320
atccaaccgc tcggcacaga aaaagtcggc aaagccttcg gccccatcat gttgctgtgg 1380
tttgtcaccc ttgcaggatt gggaattccg caaatcatcg tacatccaga aatcttgcag 1440
agcttgtctc cacattgggc cctgcgcttg attgtggctg agcctttcca agcatttgtg 1500
ctgcttggtg ccgttgtcct gacagtaacg ggtgcggaag cgctctacgc tgatatgggc 1560
cattttgggg cgaggccaat tagagtggcg tggttttgcg tcgtcatgcc tgctttaatc 1620
ttgacgtatt tggggcaggg cgccttggtg attaaccagc ctgaagcggt gcgcaacccc 1680
atgttttatc tcgcgccgga aggtctgcgg attccgttgg ttattttggc gaccatcgcc 1740
acggtgatcg catcgcaggc cgtgatttct ggtgcgtatt cattgaccaa gcaggccgtg 1800
aatttgaaac tgctgccacg catggtgatc cggcatacct cccgaaaaga ggaaggccag 1860
atctatatgc cgctggttaa tggattgctg tttgtatccg tgatggtcgt ggtgctggta 1920
ttccgatcct cagaaagcct cgccagcgcg tatggacttg ctgtgaccgg aacattggtg 1980
ctggtcagcg tcttgtatct ggtctacgcc cacaccacat ggtggaaaac agcgttgttc 2040
attgtgttca tcggtattcc agaagtactt ctattcgcct cgaacaccac gaaaattcac 2100
gacggtggct ggcttccact acttattgca gccgtgctca tcgtggtgat gaggacctgg 2160
gagtggggaa gtgaccgcgt caatcaggaa cgcgcagagc tggaacttcc catggataag 2220
ttcttggaga aactcgatca gccacacaat attgggcttc gtaaagttgc cgaagtggca 2280
gtatttccac atggcaccag cgatactgtc ccgttgtcat tggttcgctg cgtgaaagac 2340
ctcaagcttt tataccgaga gatcgtgatc gttcgaatcg tccaagaaca cgttccgcac 2400
gtgccaccag aggaacgcgc ggaaatggaa gtgctccatc acgccccgat tagggtggtt 2460
cgggttgatc tgcaccttgg ttattttgat gaacagaatc tgcctgaaaa tctccacgcc 2520
attgacccaa catgggataa cgccacttac ttcctgtctg cgctgacact tcggagcagg 2580
ttgcctggaa agatcgctgg ctggcgtgat cgtttgtatc tttcgatgga acgcaatcag 2640
gcatctcgaa ccgagtcttt caaactgcaa ccaagcaaaa ccatcactgt cggaacagag 2700
ctacaccttt agtaaatcga ctactcacat agggtcgggc tagtcattct gatcagcgaa 2760
ttccacgttc acatcgccaa ttccagagtt cacaaccaga ttcagcattg gaccttctag 2820
atcagcattg tgggcggtga gatctccaac atcacagcgc gctgtgccca caccggcggt 2880
acaacttagg ctcacgggca catcatcggg cagggtgacc atgacttcgc cgatccctga 2940
ggtgatttgg atgttttgtt cctgatccaa ttgggtgagg tggctgaaat cgaggttcat 3000
ttcacccacg ccagaggtgt agctgctgag gagttcatcg ttggtgggga tgagattgac 3060
atcgccgatt ccagggtcgt cttcaaagta gatgggatcg atatttgaaa taaacaggcc 3120
tgcgagggcg ctcatgacaa ctccggtacc aactacaccg ccgacaatcc atggccacac 3180
atggcgcttt ttctgaggct tttgtggagg gacttgtaca tcccaggtgt tgtattggtt 3240
ttgggcaagt ggatcccaat gaggcgcttc gggggtttgt tgcgcgaagg gtgcatagta 3300
gccctcaacg ggggtgatag tgcttagatc tggttggggt tgtgggtaga gatcttcgtt 3360
tttcatggtg gcatcctcag aaacagtgaa ttcagtggtg agtagtccgc ggggtggaag 3420
tggttgtttc ttatgcaggg taccgagctc gaattcgtaa tcatggtcat agctg 3475
<210> 7
<211> 1996
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcttgcatgc ctgcaggtcg actctagagg atccccgtct tttctgagcg ctagcatttc 60
tccactcaaa ggagcatgct taaccgcatg aaaagtgcgc ggccaaaatc agtcgctcca 120
aaatccggac aagctttact cactcttggt gccctaggtg ttgtgttcgg tgacatcggc 180
accagccccc tgtattcact tcacactgca tttagcatgc agcacaacaa agtcaaagtc 240
accccggaaa atgtgtacgg gatcatctcc atggtgctgt ggacaatcac cttgatcgtc 300
accgtcaaat acgtcatgct ggtcacccga gctgacaacc aaggacaagg tggcatcctg 360
gcgctcgttg ccttgctgaa aaaccgtggg cactggggaa aattcgtggc agtagccggc 420
atgttgggtg cagcgctgtt ttatggcgat gtgctgatta ctccggcgat ctctgtgctt 480
agcgcgacgg aggggttgac ggttatttcc ccaagctttg agcgcttcat tctgcccgta 540
tctctcgcag ttttgatcgc tatttttgca atccaaccgc tcggcacaga aaaagtcggc 600
aaagccttcg gccccatcat gttgctgtgg tttgtcaccc ttgcaggatt gggaattccg 660
caaatcatcg tacatccaga aatcttgcag agcttgtctc cacattgggc cctgcgcttg 720
attgtggctg agcctttcca agcatttgtg ctgcttggtg ccgttgtcct gacagtaacg 780
ggtgcggaag cgctctacgc tgatatgggc cattttgggg cgaggccaat tagagtggcg 840
tggttttgcg tcgtcatgcc tgctttaatc ttgacgtatt tggggcaggg cgccttggtg 900
attaaccagc ctgaagcggt gcgcaacccc atgttttatc tcgcgccgga aggtctgcgg 960
attccgttgg ttattttggc gaccatcgcc acggtgatcg catcgcaggc cgtgatttct 1020
ggtgcgtatt cattgaccaa gcaggccgtg aatttgaaac tgctgccacg catggtgatc 1080
cggcatacct cccgaaaaga ggaaggccag atctatatgc cgctggttaa tggattgctg 1140
tttgtatccg tgatggtcgt ggtgctggta ttccgatcct cagaaagcct cgccagcgcg 1200
tatggacttg ctgtgaccgg aacattggtg ctggtcagcg tcttgtatct ggtctacgcc 1260
cacaccacat ggtggaaaac agcgttgttc attgtgttca tcggtattcc agaagtactt 1320
ctattcgcct cgaacaccac gaaaattcac gacggtggct ggcttccact acttattgca 1380
gccgtgctca tcgtggtgat gaggacctgg gagtggggaa gtgaccgcgt caatcaggaa 1440
cgcgcagagc tggaacttcc catggataag ttcttggaga aactcgatca gccacacaat 1500
attgggcttc gtaaagttgc cgaagtggca gtatttccac atggcaccag cgatactgtc 1560
ccgttgtcat tggttcgctg cgtgaaagac ctcaagcttt tataccgaga gatcgtgatc 1620
gttcgaatcg tccaagaaca cgttccgcac gtgccaccag aggaacgcgc ggaaatggaa 1680
gtgctccatc acgccccgat tagggtggtt cgggttgatc tgcaccttgg ttattttgat 1740
gaacagaatc tgcctgaaaa tctccacgcc attgacccaa catgggataa cgccacttac 1800
ttcctgtctg cgctgacact tcggagcagg ttgcctggaa agatcgctgg ctggcgtgat 1860
cgtttgtatc tttcgatgga acgcaatcag gcatctcgaa ccgagtcttt caaactgcaa 1920
ccaagcaaaa ccatcactgt cggaacagag ctacaccttt aggttttggc ggatgagaga 1980
agattttcag cctgat 1996
Claims (10)
1.一种蛋白质,命名为YH66-RS03880G160A蛋白,是将YH66-RS03880蛋白的第54位氨基酸残基由E突变为其他氨基酸残基得到的;
所述YH66-RS03880蛋白为如下(a1)或(a2)或(a3):
(a1)序列表的序列3所示的蛋白质;
(a2)来源于细菌且与(a1)具有95%以上同一性且与细菌产缬氨酸相关的蛋白质;
(a3)将(a1)所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且与细菌产缬氨酸相关的由(a1)衍生的蛋白质。
2.权利要求1所述YH66-RS03880G160A蛋白的编码基因。
3.具有权利要求2所述编码基因的表达盒或重组载体或重组菌。
4.权利要求1所述蛋白质、权利要求2所述编码基因、权利要求3所述表达盒、权利要求3所述重组载体或权利要求3所述重组菌的应用;
所述应用为如下(Ⅰ)或(Ⅱ)或(Ⅲ):
(Ⅰ)在提高细菌缬氨酸产量中的应用;
(Ⅱ)在生产缬氨酸中的应用;
(Ⅲ)在提高细菌菌量中的应用。
5.特定物质的应用;
所述应用为如下(Ⅰ)或(Ⅱ)或(Ⅲ):
(Ⅰ)在提高细菌缬氨酸产量中的应用;
(Ⅱ)在生产缬氨酸中的应用;
(Ⅲ)在提高细菌菌量中的应用;
所述特定物质为如下(d1)、(d2)、(d3)、(d4)、(d5)或(d6):
(d1)用于提高YH66-RS03880G160A基因表达的物质;
(d2)用于提高YH66-RS03880G160A蛋白丰度的物质;
(d3)用于提高YH66-RS03880G160A蛋白活性的物质;
(d4)用于提高YH66-RS03880基因表达的物质;
(d5)用于提高YH66-RS03880蛋白丰度的物质;
(d6)用于提高YH66-RS03880蛋白活性的物质;
所述YH66-RS03880G160A蛋白为权利要求1中所述的YH66-RS03880G160A蛋白;
所述YH66-RS03880G160A基因为编码所述YH66-RS03880G160A蛋白的基因;
所述YH66-RS03880蛋白为权利要求1中所述的YH66-RS03880蛋白;
所述YH66-RS03880基因为编码所述YH66-RS03880蛋白的基因。
6.一种重组菌,是在细菌中过表达YH66-RS03880G160A基因或YH66-RS03880基因得到的;所述YH66-RS03880G160A基因为权利要求5中所述的YH66-RS03880G160A基因;所述YH66-RS03880基因为权利要求5中所述的YH66-RS03880基因。
7.权利要求6所述重组菌在制备缬氨酸中的应用。
8.一种提高细菌的缬氨酸产量的方法,包括如下步骤:将细菌基因组中的YH66-RS03880基因替换为YH66-RS03880G160A基因;
所述YH66-RS03880G160A基因为权利要求5中所述的YH66-RS03880G160A基因;所述YH66-RS03880基因为权利要求5中所述的YH66-RS03880基因。
9.一种提高细菌的缬氨酸产量的方法,包括如下步骤:在细菌中过表达YH66-RS03880G160A基因或在细菌中过表达YH66-RS03880基因或提高细菌中YH66-RS03880G160A蛋白的丰度或提高细菌中YH66-RS03880蛋白的丰度或提高细菌中YH66-RS03880G160A蛋白的活性或提高细菌中YH66-RS03880蛋白的活性;
所述YH66-RS03880G160A蛋白为权利要求1中所述的YH66-RS03880G160A蛋白;
所述YH66-RS03880G160A基因为编码所述YH66-RS03880G160A蛋白的基因;
所述YH66-RS03880蛋白为权利要求1中所述的YH66-RS03880蛋白;
所述YH66-RS03880基因为编码所述YH66-RS03880蛋白的基因。
10.YH66-RS03880G160A蛋白或YH66-RS03880蛋白在调控细菌的缬氨酸产量中的应用或在调控细菌菌量中的应用;所述YH66-RS03880G160A蛋白为权利要求1中所述的YH66-RS03880G160A蛋白;所述YH66-RS03880蛋白为权利要求1中所述的YH66-RS03880蛋白。
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