一种用于微生物发酵培养原料的菊芋预处理方法

本发明涉及菊芋原料的预处理方法，具体地说是一种处理菊芋块茎，获取微生物发酵培养原料的方法。干菊芋粉或鲜菊芋块茎与水以一定质量比混合，匀浆，加入酸调节pH至1～4，于101℃～140℃处理2分钟～20分钟，得到水解液。水解液用碱中和，调节pH至5～8，得到用于微生物发酵培养的原料。本发明原料处理时间短、简便易行、能耗小、成本低，并且可使原料处理和培养基灭菌同时进行，水解液单糖浓度高，用于微生物发酵菌体生长快，目标产物产量高，发酵废水中有机物含量低，为高效利用菊芋原料进行微生物培养和生物炼制提供了经济可行的新技术。带填

一种产油酵母脂肪酸合酶及其编码基因与应用

本发明公开了一种产油酵母脂肪酸合酶及其编码基因与应用。该脂肪酸合酶由SrFAS1亚基和SrFAS2亚基组成，所述SrFAS1亚基是如下(a)或(b)的蛋白质：(a)由SEQ ID NO：1所示的氨基酸序列组成的蛋白质；(b)将SEQ ID NO：1的氨基酸经过一个或几个氨基酸的取代和/或缺失和/或添加且与具有脂肪酸合酶亚基1功能的由SEQ ID NO：1所衍生的蛋白质；所述SrFAS2亚基是如下(c)或(d)的蛋白质：(c)由SEQ ID NO：2所示的氨基酸序列组成的蛋白质；(d)将SEQ ID NO：2的氨基酸经过一个或几个氨基酸的取代和/或缺失和/或添加且与具有脂肪酸合酶亚基2功能的由SEQ ID NO：2所衍生的蛋白质

乳清酸磷酸核糖转移酶启动子及应用和构建体与载体

通过扩增圆红冬孢酵母乳清酸磷酸核糖转移酶基因组DNA上下游序列，进行生物学信息分析和功能验证，获得可有效表达目的基因于圆红冬孢酵母，并因此能够用于圆红冬孢酵母遗传工程操作和菌株改良的启动子和终止子。本发明还涉及包含这些元件的DNA构建体和载体。待填

Cloning and characterization of the isocitrate dehydrogenase gene from oleaginous yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids over 65% of its cell dry weight [1]. Mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrare to α-ketoglutarate in eukaryotic cells, an essential and rate-limiting step in citric acid cycle. Yeast S. cerevisiae IDH is composed of an octamer of four IDH1 and four IDH2 subunits [2]. Early biochemical study showed that IDH activity correlates with the lipid storage capacity [3]. In this work we cloned the full length cDNA sequence of the mitochondrial IDH1 and IDH2 gene using methods of degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends from L. starkeyi AS 2.1560, a useful oil-producing yeast. We further determined the expression level of both lsidh1 and lsidh2 by real time PCR during the lipid storage process. Our results showed the encoded products, LsIDH1 and LsIDH2, are similar in size with a molecular weight of 40,277 and 40,708, respectively, and have high similarity with IDH sequences from other yeast species. More importantly, putative mitochondrial transit peptides have been identified at the N-terminus of each sequence, suggesting that both proteins are likely located within the mitochondria. Further gene expression analysis by real time PCR showed that both idh1 and idh2 expression decreased concurrently with the evolution of cellular lipids. Our data may be valuable for further engineering oleaginous yeasts

转化N-乙酰-D-葡糖胺产油真菌的筛选

对21株真菌利用甲壳素解聚产物N-乙酰-D-葡糖胺（NAG）为碳源积累油脂的能力进行了筛选。碳源同化实验得到可同化NAG的真菌7株，进一步筛选出能利用NAG积累油脂的酵母3株。摇瓶实验表明，C．albidus ATCC 56298和T.fermentans CICC1368利用NAG发酵菌体油脂含量可分别达到67％和48％。气相色谱分析表明菌油富含棕榈酸、硬脂酸和油酸，与常规植物油脂的脂肪酸组成相似。研究结果拓宽了微生物油脂发酵的原料

Construction of E. coli NAD+ auxotrophic strains and the biotechnological application thereof

NAD+ and its reduced form NADH are essential cofactors in biological systems. They function as cofactors in over 300 redox reactions in vivo1. The level of NAD+ and NADH is tightly controlled by a variety of mechanisms including their biosynthesis and salvage. Therefore, it is difficult to answer some fundamental questions such as the minimal NAD+ level for cell growth and the biological consequences of abnormal activity of a specific NAD+-dependent enzyme. We expressed the NTT4 gene from Chlamydiae UWE25 in Escherichia coli BW25113, as the NTT4 protein was reported as a NAD+ transporter that can specifically transport intact NAD+ across cytoplasmic membrane2. We knocked out the nadC gene responsible for de novo biosynthesis of NAD+ and constructed the strain E. coli BW25113 (ΔnadC, NTT4). It was found that NAD+ in the culture media could significantly promote the growth of BW25113 (ΔnadC, NTT4), suggesting that the NTT4 protein was functional in E. coli (Fig A, B). We then disrupted the other two genes, nadD and nadE, and obtained the strains E. coli BW25113 (ΔnadD, NTT4) and BW25113 (ΔnadE, NTT4). Cell growth of these two strains are depending on exogenous NAD+ supplemented in the media, suggesting that we have successfully engineered E. coli to hold an NAD+ auxotrophic phenotype. We are carrying out a number of experiments using these NAD+ auxotrophic strains to address some interesting questions which may not be able to do otherwise. Results will be discussed during the conference.NAD+ and its reduced form NADH are essential cofactors in biological systems. They function as cofactors in over 300 redox reactions in vivo1. The level of NAD+ and NADH is tightly controlled by a variety of mechanisms including their biosynthesis and salvage. Therefore, it is difficult to answer some fundamental questions such as the minimal NAD+ level for cell growth and the biological consequences of abnormal activity of a specific NAD+-dependent enzyme. We expressed the NTT4 gene from Chlamydiae UWE25 in Escherichia coli BW25113, as the NTT4 protein was reported as a NAD+ transporter that can specifically transport intact NAD+ across cytoplasmic membrane2. We knocked out the nadC gene responsible for de novo biosynthesis of NAD+ and constructed the strain E. coli BW25113 (ΔnadC, NTT4). It was found that NAD+ in the culture media could significantly promote the growth of BW25113 (ΔnadC, NTT4), suggesting that the NTT4 protein was functional in E. coli (Fig A, B). We then disrupted the other two genes, nadD and nadE, and obtained the strains E. coli BW25113 (ΔnadD, NTT4) and BW25113 (ΔnadE, NTT4). Cell growth of these two strains are depending on exogenous NAD+ supplemented in the media, suggesting that we have successfully engineered E. coli to hold an NAD+ auxotrophic phenotype. We are carrying out a number of experiments using these NAD+ auxotrophic strains to address some interesting questions which may not be able to do otherwise. Results will be discussed during the conference

一种酵母菌落PCR菌体前处理的方法

本发明公开了一种酵母菌落PCR菌体前处理的方法。该方法为：菌体经过灭活，两步酶解，最后煮沸的处理后，上清可直接用于PCR反应。本方法制备的样品可有效地扩增基因组上单拷贝、长片段基因。此方法为大规模的酵母筛选和鉴定提供了新的途径