WO2023000243A1 - 重组酵母生物转化生产葡萄糖的方法 - Google Patents

重组酵母生物转化生产葡萄糖的方法 Download PDF

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WO2023000243A1
WO2023000243A1 PCT/CN2021/107787 CN2021107787W WO2023000243A1 WO 2023000243 A1 WO2023000243 A1 WO 2023000243A1 CN 2021107787 W CN2021107787 W CN 2021107787W WO 2023000243 A1 WO2023000243 A1 WO 2023000243A1
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glucose
recombinant yeast
recombinant
yeast strain
saccharomyces cerevisiae
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于涛
吴良焕
郭姝媛
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深圳先进技术研究院
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Priority to PCT/CN2022/107431 priority patent/WO2023001294A1/zh
Priority to EP22845457.5A priority patent/EP4375366A1/en
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  • the invention belongs to the technical field of synthetic biology, and in particular relates to a method for producing glucose through biotransformation of recombinant yeast.
  • Glucose with the chemical formula C 6 H 12 O 6 , is the most widely distributed and most important hexose sugar in nature.
  • glucose is widely used in the fields of food, fermentation and medicine.
  • glucose preparation mainly uses starch extracted from corn, potato and other food crops as raw materials.
  • the main production processes include: acid method, acid enzyme method and double enzyme method, among which the double enzyme method is currently the most ideal and widely used.
  • Sugar method Starch undergoes steps such as gelatinization, liquefaction, and saccharification, and is hydrolyzed to form glucose.
  • the production process is complicated, the energy consumption is large, and a large amount of food crops are consumed. my country is a country with a large population.
  • the object of the present invention is to provide a method for recombinant yeast biotransformation to produce glucose.
  • the technical solution adopted in the present invention is as follows: On the one hand, the present invention provides a recombinant yeast strain, which obtains glucose utilization defects and has Recombinant yeast strains capable of secreting glucose.
  • the recombinant yeast strain that is deficient in glucose utilization and capable of secreting glucose is overexpressed with glucose phosphatase and HAD4 of Escherichia coli, or HAD4 of Escherichia coli, to obtain a recombinant yeast strain that increases the production of glucose synthesis.
  • glucose phosphatase has glucose-1-phosphate and/or glucose-6-phosphate activity
  • the source of the glucose phosphatase is heterologous enzyme or specific modification of yeast itself or other eukaryotic or prokaryotic organisms enzyme.
  • yeast includes Saccharomyces cerevisiae, methanolic yeast, oleaginous yeast and other microorganisms recognized as biosafety.
  • yeast is Saccharomyces cerevisiae
  • glucokinase gene glk1 of glucokinase activity in Saccharomyces cerevisiae and the two isoenzyme genes hxk1 and hxk2 of hexokinase glucose utilization deficiency and glucose secretion are obtained.
  • HAD4 haloacid dehalogenase-like phosphatase 4
  • HAD4 haloacid dehalogenase-like phosphatase 4
  • the HAD4 (haloacid dehalogenase-like phosphatase 4) gene yhix was used to obtain a recombinant Saccharomyces cerevisiae that is putatively deficient in glucose utilization and capable of secreting glucose, while overexpressing the glucose phosphatase gene agpP from Pantoea and Escherichia coli at the YLR446W and emi2 loci, respectively HAD4 (haloacid dehalogenase-like phosphatase 4) gene yhix, or knock out the hexokinase genes emi2 and YLR446W in the recombinant Saccharomyces cerevisiae that is assumed to be deficient in glucose utilization and have the ability to secrete glucose, and over
  • nucleotide sequence of the agpP is shown in SEQ ID NO: 4, or a sequence having at least 70% homology therewith;
  • nucleotide sequence of said yhix is shown in SEQ ID NO: 5, or a sequence having at least 70% homology therewith.
  • the present invention provides an application of any one of the above-mentioned recombinant yeast strains in biotransformation to produce glucose, glucose analogues or derivatives.
  • analogs of glucose are isomers of glucose
  • the analogs of glucose include fructose, galactose and the like.
  • the derivatives of glucose include derivatives of glucose alcohols, amino groups, oligosaccharides and polysaccharides;
  • the derivatives of glucose include sugar alcohol, ammonia sugar, disaccharide sucrose, polysaccharide starch and the like.
  • the present invention provides a method for biotransforming glucose production using any of the above-mentioned recombinant yeast strains, using any of the above-mentioned recombinant yeast strains in a culture medium with a carbon source as a substrate. Glucose is obtained by fermentation.
  • the carbon source is a non-grain carbon source, including acetic acid, methanol, ethanol, propanol, glycerin, etc., which can be rapidly prepared from fossil raw materials such as petroleum, coal, natural gas, or renewable energy technology by using photoelectrocatalytic CO2 reduction.
  • the beneficial effects of the present invention are: due to the characteristics of microorganisms using glucose, there is currently no industrial utilization of microbial fermentation to produce glucose. Grain carbon sources quickly prepare food raw material glucose, and then produce food needed by human life.
  • Figure 1 is a schematic diagram of the construction of recombinant Saccharomyces cerevisiae strains in the embodiment of the present invention
  • Fig. 2 is a graph showing the verification result of glucose deficiency by the recombinant Saccharomyces cerevisiae strain in Example 1 of the present invention
  • Fig. 3 is the detection result figure of the content of glucose produced by the biotransformation of the recombinant Saccharomyces cerevisiae strain in Example 1 of the present invention
  • Fig. 4 is the detection result figure of the content of glucose produced by the biotransformation of the recombinant Saccharomyces cerevisiae strain in Example 2 of the present invention
  • Fig. 5 is a graph showing the content detection results of glucose produced by fermentation of LY031 using electrocatalytically synthesized acetic acid as a substrate in Example 3 of the present invention.
  • the embodiment of the present application takes Saccharomyces cerevisiae as an example to describe the method of recombinant yeast biotransformation to produce glucose in detail.
  • the information of the Saccharomyces cerevisiae strain used is: MATa ura3-52 can1 ⁇ ::cas9-natNT2 TRP1 LEU2 HIS3.
  • the glucokinase gene glk1 (the nucleotide sequence of the glk1 gene is shown in SEQ ID NO: 1) and the isoenzyme gene hxk1 (hxk1) of two hexokinases with glucokinase activity in Saccharomyces cerevisiae are first knocked out
  • the nucleotide sequence of the gene is shown in SEQ ID NO: 2)
  • hxk2 the nucleotide sequence of the hxk2 gene is shown in SEQ ID NO: 3
  • a recombinant Saccharomyces cerevisiae strain with glucose utilization deficiency and ability to secrete glucose was obtained Bacterial strain
  • emi2 the nucleotide sequence of emi2 gene is as shown in SEQ ID NO: 17
  • YLR446W the nucleus of YLR446W gene
  • the knockout method used in the experiment can also be other technologies that can achieve the same effect, such as RNA interference, reduction of enzyme activity, low-strength promoter replacement or knockout.
  • Example 1 Preparation of recombinant Saccharomyces cerevisiae strains deficient in glucose utilization and capable of secreting glucose
  • glk1 knockout gRNA primers (gRNA primer 1 SEQ ID NO: 6 and gRNA primer 2 SEQ ID NO: 7) on the website (http://yeastriction.tnw.tudelft.nl/#!/) to obtain primers for amplification 2 ⁇ m fragments.
  • the reaction system is as follows:
  • primers Glk1-DN-F SEQ ID NO: 11
  • primer Glk1-DN-R SEQ ID NO: 12
  • primer Glk1-UP-F SEQ ID NO: 9
  • primer Glk1 -DN-R SEQ ID NO: 12
  • the obtained repair fragment and knockout plasmid are transferred into cells.
  • the transformation system is as follows:
  • the starting strain and the engineered strains were subjected to a spot plate test.
  • the specific steps are as follows: Configure an SC agarose plate with glucose as the single carbon source, serially dilute fresh Saccharomyces cerevisiae cells to OD 600 of 1, 0.1, 0.01, 0.001, 0.0001, take 5 ⁇ L for spot plate test, The test results are shown in Figure 2.
  • the engineered strains were deficient in glucose utilization after deletion of glk1, hxk1 and hxk2.
  • the high-performance liquid chromatography analysis method is as follows: column: Aminex HPX-87H; mobile phase: 5mM H 2 SO 4 ; column temperature: 50°C; injection volume 5 ⁇ L, detection time for each sample is 30min; glucose detector is a differential detector .
  • the results are shown in Figure 3. Glucose production was detected in the fermentation liquid of the glk1, hxk1 and hxk2 three-deficiency engineered strains, with a content of 1.7 g/L.
  • construct emi2 and YLR446W double knockout plasmids use Saccharomyces cerevisiae genome as a template to construct emi2 UP-emi2 DN repair fragments, YLR446W-UP-YLR446W-DN repair fragments; chemical synthesis Gene agpP, construct YLR446W UP-CCW12p-agpP-PYK1t-YLR446W DN repair fragment, CCW12p is the Saccharomyces cerevisiae CCW12 promoter (SEQ ID NO: 13), PYK1t is the Saccharomyces cerevisiae PYK1 terminator (SEQ ID NO: 14); chemical synthesis Gene yhix, construct EMI2 UP-TEF1p-yhix-DIT1t-EMI2 DN repair fragment, TEF1p is Saccharomyces cerevisiae TEF1 promoter (SEQ ID NO: 15), D
  • Example 1 using the LY027 engineering strain as the starting strain, the engineering strains LY028, LY029, LY030, and LY031 were constructed respectively, and the genotypes of the strains are shown in Figure 1; the obtained Saccharomyces cerevisiae strains were fermented and analyzed, and the results were shown in Figure 4 , after knocking out two putative hexokinase genes in Saccharomyces cerevisiae and knocking out two putative hexokinase genes, introducing the glucose phosphatase gene agpP from Pantoea alone did not significantly improve the glucose production, while in The Saccharomyces cerevisiae strains introduced with the HAD4 (haloacid dehalogenase-like phosphatase 4) gene yhix of Escherichia coli significantly increased the glucose production, and the glucose production in the LY031 strain reached 2.2g/L, an increase of 30%.
  • HAD4 haloa
  • Example 3 The method for producing glucose by biotransformation of recombinant Saccharomyces cerevisiae

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Abstract

本发明公开了一种重组酵母生物转化生产葡萄糖的方法，使用的重组酵母菌株，通过敲除酵母中葡萄糖激酶及相关己糖激酶的同工酶，获得葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酵母菌株。由于微生物利用葡萄糖的特性，目前暂无微生物发酵生产葡萄糖的工业利用，本发明利用光电催化或传统化工合成的非粮碳源为底物，通过重组酵母细胞，实现非粮碳源快速制备食品原料葡萄糖。

Description

重组酵母生物转化生产葡萄糖的方法 技术领域
本发明属于合成生物学技术领域，具体涉及一种重组酵母生物转化生产葡萄糖的方法。
背景技术
葡萄糖，化学式为C 6H 12O 6，是自然界分布最广、最重要的一种己糖。葡萄糖作为重要的食品、化工原料，在食品、发酵和医药等领域有着广泛的利用。目前，葡萄糖制备主要以玉米、薯类等粮食作物提取而来的淀粉为原料，主要生产工艺有：酸法、酸酶法和双酶法，其中双酶法是目前最为理想、运用最广的制糖方法。淀粉经糊化、液化、糖化等步骤，水解形成葡萄糖，生产工艺复杂、能耗较大、且消耗大量粮食作物。我国是人口大国，截至2020年，全国人口已达14亿，占全球人口20％。但是总耕地面积仅占全球7％，人均耕地面积不足世界人均耕地面积的四成，耕地生产能力已近极限，且粮食生产过程中时间周期长，容易受地域，气候，战争的因素的影响，粮食危机日趋严重。因此，急需开发一种新的生产葡萄糖的方法，以有效避免以上问题，实现大规模，快速的制备食品原料葡萄糖。
发明内容
为了解决上述背景技术中所提出的问题，本发明的目的在于提供一种重组酵母生物转化生产葡萄糖的方法。
为了达到上述目的，本发明所采用的技术方案为：一方面，本发明提供了一种重组酵母菌株，通过敲除酵母中葡萄糖激酶及相关己糖激酶的同工酶，获得葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酵母菌株。
进一步地，将所述葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酵母菌株过表达葡萄糖磷酸酶和大肠杆菌的HAD4，或，大肠杆菌的HAD4，得到提高葡萄糖合成产量的重组酵母菌株。
进一步地，所述葡萄糖磷酸酶具备葡萄糖-1-磷酸和/或葡萄糖-6-磷酸活性，所述葡萄糖磷酸酶的来源为酵母自身或者其他真核、原核生物的异源酶或特异性的改造酶。
进一步地，所述酵母包括酿酒酵母、甲醇酵母、产油酵母等被认定为生物安全的微生物。
进一步地，当所述酵母为酿酒酵母时，通过敲除酿酒酵母中葡萄糖激酶活性的葡萄糖激酶基因glk1及两个己糖激酶的同工酶基因hxk1、hxk2，获得葡萄糖利用缺陷且具备分泌葡糖能力的重组酿酒酵母菌株；
敲除假定的所述葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酿酒酵母中己糖激酶基因emi2和YLR446W，同时在YLR446W和emi2位点分别过表达来自泛菌属的葡萄糖磷酸酶基因agpP和大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix，或，敲除假定的所述葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酿酒酵母中己糖激酶基因emi2和YLR446W，在emi2位点过表达大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix，得到提高葡萄糖合成产量的重组酵母菌株。
进一步地，所述agpP的核苷酸序列如SEQ ID NO：4所示，或与之具有至少70％同源性的序列；
优选地，所述yhix的核苷酸序列如SEQ ID NO：5所示，或与之具有至少70％同源性的序列。
另一方面，本发明提供了一种上述任一所述的重组酵母菌株在生物转化生产葡萄糖、葡萄糖的类似物或衍生物中的应用。
进一步地，所述葡萄糖的类似物为葡萄糖的异构体；
优选地，所述葡萄糖的类似物包括果糖、半乳糖等。
进一步地，所述葡萄糖的衍生物包括葡萄糖的醇类、氨基类、寡糖类以及多糖类的衍生物；
优选地，所述葡萄糖的衍生物包括糖醇、氨糖、二糖蔗糖、多糖淀粉等。
另一方面，本发明提供了一种利用上述任一所述的重组酵母菌株生物转化生产葡萄糖的方法，利用上述任一所述的重组酵母菌株，在培养基中，以碳源为底物进行发酵得到葡萄糖。
进一步地，所述碳源为非粮碳源，包括乙酸、甲醇、乙醇、丙醇、甘油等可由石油、煤炭、天然气等化石原料或者可再生能源技术利用光电催化CO 2还原快速制备。
本发明的有益效果是：由于微生物利用葡萄糖的特性，目前暂无微生物发酵生产葡萄糖的工业利用，本发明利用光电催化或传统化工合成的非粮碳源为底物，通过重组酵母细胞，实现非粮碳源快速制备食品原料葡萄糖，进而生产人类生活所需要的食品等。
附图说明
图1为本发明实施例中重组酿酒酵母菌株构建示意图；
图2为本发明实施例1中重组酿酒酵母菌株利用葡萄糖缺失验证结果图；
图3为本发明实施例1中重组酿酒酵母菌株生物转化生产葡萄糖的含量检测结果图；
图4为本发明实施例2中重组酿酒酵母菌株生物转化生产葡萄糖的含量检测结果图；
图5为本发明实施例3中LY031利用电催化合成乙酸为底物发酵生产葡萄糖的含量检测结果图。
具体实施方式
为了更好地理解本发明的内容，下面结合具体实施方法对本发明内容作进一步说明，但本发明的保护内容不局限以下实施例。
本申请实施例以酿酒酵母为例来详细说明重组酵母生物转化生产葡萄糖的方法，所用酿酒酵母菌株信息为：MATa ura3-52 can1Δ::cas9-natNT2 TRP1 LEU2 HIS3。
本申请实施例所构建菌株均采用CRISPR/CAS9方法，参照文献(Robert M,Van R H M,Melanie W,et al.CRISPR/Cas9:a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae[J].Fems Yeast Research,2015(2):2.)。酿酒酵母转化方法参照文献(Gietz R D,Woods R A.Transformation of yeast by the LiAc/ss carrier DNA/PEG method[J].Methods in Molecular Biology,2006,313:107-120.)。其余实施例中未注明具体条件的实验方法，按照常规条件，《分子克隆：实验室指南》(New York:Cold Spring Harbor laboratory Press,2001)中所述的条件进行。
以下实施例中首先敲除酿酒酵母中具有葡萄糖激酶活性的葡萄糖激酶基因glk1(glk1基因的核苷酸序列如SEQ ID NO：1所示)和两个己糖激酶的同工酶基因hxk1(hxk1基因的核苷酸序列如SEQ ID NO：2所示)、hxk2(hxk2基因的核苷酸序列如SEQ ID NO：3所示)，获得一株葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酿酒酵母菌株；其次敲除假定的葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酿酒酵母菌株中己糖激酶基因emi2(emi2基因的核苷酸序列如SEQ ID NO：17所示)和YLR446W(YLR446W基因的核苷酸序列如SEQ ID NO：18所示)，同时在YLR446W和emi2位点分别过表达来自泛菌属的葡萄糖磷酸酶基因agpP(agpP基因的核苷酸序列如SEQ ID NO：4所示)(该酶具备葡萄糖-1-磷酸和葡萄糖-6-磷酸活性)和大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix(yhix基因的核苷酸序列如SEQ ID NO：5所示)(该基因编码产生的酶特异性水解葡萄糖-1-磷酸产生葡萄糖)，有效的增加了30％的葡萄糖分泌产量。以电催化合成乙酸为碳源进行发酵，合成葡萄糖产量达1.81g/L。
实验中所采用的敲除手段，也可以是RNA干扰，酶活性降低，低强度启动子置换或敲除等能达到相同效果的其他技术。
实施例1 葡萄糖利用缺陷且具备分泌葡糖能力的重组酿酒酵母菌株的制备
构建glk1敲除菌株，具体步骤如下：
在网站(http://yeastriction.tnw.tudelft.nl/#！/)设计glk1敲除gRNA引物(gRNA引物1 SEQ ID  NO：6和gRNA引物2SEQ ID NO：7)，获得引物用于扩增2μm片段。反应体系如下：
Figure PCTCN2021107787-appb-000001
将2μm片段和质粒骨架通过Gibson Assembly方法组装构建glk1敲除质粒。以酿酒酵母基因组为模板，引物Glk1-UP-F(SEQ ID NO：9)和引物Glk1-UP-R(SEQ ID NO：10)扩增glk1敲除修复上游片段；引物Glk1-DN-F(SEQ ID NO：11)和引物Glk1-DN-R(SEQ ID NO：12)扩增glk1敲除修复下游片段；获得片段后，以引物Glk1-UP-F(SEQ ID NO：9)和引物Glk1-DN-R(SEQ ID NO：12)进行融合PCR，获得修复片段Glk1-UP-Glk1-DN。将获得修复片段和敲除质粒转入细胞。具体步骤如下：挑取新鲜酵母克隆至1mL YPE培养基(Yeast Extract Peptone Ethanol Medium)，培养过夜，取适量菌液转接至20mL YPE中，使起始OD 600＝0.1，30℃培养至OD 600＝0.6，4200rpm离心去除培养基，用1mL无菌水重悬细胞沉淀，4200rpm离心去除上清后加入1mL 0.1M醋酸锂重悬，4200rpm离心去除上清，加入200μL 0.1M醋酸锂制得酿酒细胞感受态细胞，将获得酵母感受态通过醋酸锂/聚乙二醇转化法进行重组酿酒酵母构造，转化体系如下：
Figure PCTCN2021107787-appb-000002
同时分别构建hxk1、hxk2缺陷型酿酒酵母菌株，和glk1和hxk1的双基因缺陷型菌株；以glk1 和hxk1的双基因缺陷型菌株为底盘，构建glk1、hxk1和hxk2的三基因缺陷型菌株，菌株构建如图1所示。
为了验证工程菌株葡萄糖利用能力，分别将出发菌株和工程菌株进行点板测试。具体步骤如下：配置以葡萄糖为单一碳源的SC琼脂糖平板，将新鲜的酿酒酵母细胞进行系列稀释，分别稀释至OD 600为1、0.1、0.01、0.001、0.0001，取5μL进行点板测试，测试结果如图2所示。如图所示，在缺失glk1、hxk1和hxk2后工程菌株出现葡萄糖利用能力缺陷。
同时进行HPLC分析葡萄糖的产生，具体步骤如下：将新鲜酵母细胞接种至Delft培养基(minimal medium。参考文献见Verduyn,C.,Postma,E.,Scheffers,W.A.,Van Dijken,J.P.,1992.Effect of benzoic acid on metabolic fluxes in yeasts:A continuous-culture study on the regulation of respiration and alcoholic fermentation.Yeast 8,501–517.doi:10.1002/yea.320080703)中，乙醇为唯一碳源，初始OD 600＝0.1，发酵6天后取1mL发酵液，12000rpm离心10min，取上清经0.22μm滤膜过滤，进行高效液相色谱分析。高效液相色谱分析方法如下：柱子：Aminex HPX-87H；流动相为5mM H 2SO 4；柱温：50℃；进样量5μL，每个样品检测时长为30min；葡萄糖检测器为示差检测器。结果如图3所示，glk1、hxk1和hxk2三缺工程菌株发酵液中检测出葡萄糖产生，含量为1.7g/L。
实施例2 提高葡萄糖合成产量的重组酿酒酵母菌株的制备
以glk1、hxk1和hxk2三缺工程菌株为出发菌株构建emi2、YLR446W双缺菌株，在网站(http://yeastriction.tnw.tudelft.nl/#！/)设计emi2、YLR446W敲除gRNA引物，获得引物用于扩增2μm片段，参照实施例1，构建emi2、YLR446W双敲质粒；以酿酒酵母基因组为模板，分别构建emi2 UP-emi2 DN修复片段、YLR446W-UP-YLR446W-DN修复片段；化学合成基因agpP，构建YLR446W UP-CCW12p-agpP-PYK1t-YLR446W DN修复片段，CCW12p为酿酒酵母CCW12启动子(SEQ ID NO：13)，PYK1t为酿酒酵母PYK1终止子(SEQ ID NO：14)；化学合成基因yhix，构建EMI2 UP-TEF1p-yhix-DIT1t-EMI2 DN修复片段，TEF1p为酿酒酵母TEF1启动子(SEQ ID NO：15)，DIT1t为酿酒酵母DIT1终止子(SEQ ID NO：16)。参考实施案例1，以LY027工程菌株为出发菌株，分别构建获得工程菌株LY028、LY029、LY030、LY031，菌株基因型如图1所示；对获得酿酒酵母菌株进行发酵分析，结果如图4所示，在敲除酿酒酵母的两个假定的己糖激酶基因和敲除两个假定的己糖激酶基因后单独引入来自泛菌属的葡萄糖磷酸酶基因agpP对葡糖糖产量无明显提升，而在引入大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix的酿酒酵母菌株，葡萄糖产量显著提高，且在LY031菌株葡萄糖产量达2.2g/L，产量提高30％。
实施例3 重组酿酒酵母生物转化生产葡萄糖的方法
以电催化合成乙酸为碳源生物转化合成葡萄糖，将实施例2中制备得到的同时过表达来自泛菌属的葡萄糖磷酸酶基因agpP和大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix的重组酿酒酵母菌株(LY031)在新鲜YPE固体平板上划线，于30℃培养5天，挑取单克隆转接至1mL YPE液体培养基中，于30℃，200rpm的条件下培养至OD 600＝8，得到培养液；4200rpm离心去上清，加入1mL无菌水，离心去上清，重复一次；用1mL无菌水重悬后，转接至20mL Delft E(2％v/v)的摇瓶中30℃条件下培养4天；将全部菌液转移至50mL离心管中，离心去上清；加入20mL无菌水重悬，离心去上清，重复一次。将剩余细胞用20mL Delft以乙酸(质量百分数1％，光电催化合成)为碳源的培养基进行重悬，转移至摇瓶，30℃，200rpm培养；每两天取400μL发酵液进行葡萄糖含量检测，且在第二天，第四天分别补充乙酸(光电催化合成)至终浓度为1％继续培养。葡萄糖产量检测如图5所示，乙酸添加总量为29.52g/L，葡萄糖产量为1.81g/L。
以上所述仅为本发明的具体实施方式，不是全部的实施方式，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。

Claims (10)

  1. 一种重组酵母菌株，其特征在于，通过敲除酵母中葡萄糖激酶及相关己糖激酶的同工酶，获得葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酵母菌株。
  2. 根据权利要求1所述的重组酵母菌株，其特征在于，将所述葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酵母菌株过表达葡萄糖磷酸酶和大肠杆菌的HAD4，或，大肠杆菌的HAD4，得到提高葡萄糖合成产量的重组酵母菌株。
  3. 根据权利要求2所述的重组酵母菌株，其特征在于，所述葡萄糖磷酸酶具备葡萄糖-1-磷酸和/或葡萄糖-6-磷酸活性，所述葡萄糖磷酸酶的来源为酵母自身或者其他真核、原核生物的异源酶或特异性的改造酶。
  4. 根据权利要求1-3任一项所述的重组酵母菌株，其特征在于，所述酵母包括酿酒酵母、甲醇酵母、产油酵母。
  5. 根据权利要求4所述的重组酵母菌株，其特征在于，当所述酵母为酿酒酵母时，通过敲除酿酒酵母中葡萄糖激酶活性的葡萄糖激酶基因glk1及两个己糖激酶的同工酶基因hxk1、hxk2，获得葡萄糖利用缺陷且具备分泌葡糖能力的重组酿酒酵母菌株；
    敲除假定的所述葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酿酒酵母中己糖激酶基因emi2和YLR446W，同时在YLR446W和emi2位点分别过表达来自泛菌属的葡萄糖磷酸酶基因agpP和大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix，或，敲除假定的所述葡萄糖利用缺陷且具备分泌葡萄糖能力的重组酿酒酵母中己糖激酶基因emi2和YLR446W，在emi2位点过表达大肠杆菌的HAD4(haloacid dehalogenase-like phosphatase 4)基因yhix，得到提高葡萄糖合成产量的重组酵母菌株。
  6. 根据权利要求5所述的重组酵母菌株，其特征在于，所述agpP的核苷酸序列如SEQ ID NO：4所示，或与之具有至少70％同源性的序列；
    优选地，所述yhix的核苷酸序列如SEQ ID NO：5所示，或与之具有至少70％同源性的序列。
  7. 权利要求1-6任一项所述的重组酵母菌株在生物转化生产葡萄糖、葡萄糖的类似物或衍生物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述葡萄糖的类似物为葡萄糖的异构体；优选地，所述葡萄糖的类似物包括果糖、半乳糖；
    优选地，所述葡萄糖的衍生物包括葡萄糖的醇类、氨基类、寡糖类以及多糖类的衍生物；更优选地，所述葡萄糖的衍生物包括糖醇、氨糖、二糖蔗糖、多糖淀粉。
  9. 一种利用权利要求1-6任一项所述的重组酵母菌株生物转化生产葡萄糖的方法，其特征在于，利用权利要求1-6任一项所述的重组酵母菌株，在培养基中，以碳源为底物进行发酵得到葡萄糖。
  10. 根据权利要求9所述的方法，其特征在于，所述碳源为非粮碳源，包括乙酸、甲醇、乙醇、丙醇、甘油。
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