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用于……的分子遗传工具箱

A molecular genetic toolbox for .

作者信息

Bredeweg Erin L, Pomraning Kyle R, Dai Ziyu, Nielsen Jens, Kerkhoven Eduard J, Baker Scott E

机构信息

Earth and Biological Sciences Directorate, Environmental Molecular Sciences Laboratory, Richland, WA 99354 USA ; Department of Energy, Battelle EMSL, 3335 Innovation Blvd, Richland, WA 99354 USA.

Chemical & Biological Process Development Group, Energy and Environment Directorate, Pacific Northwest National Laboratories, Richland, WA 99354 USA.

出版信息

Biotechnol Biofuels. 2017 Jan 3;10:2. doi: 10.1186/s13068-016-0687-7. eCollection 2017.

DOI:10.1186/s13068-016-0687-7
PMID:28066508
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5210315/
Abstract

BACKGROUND

is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain.

RESULTS

We have developed a set of genetic and molecular tools in order to expand capabilities of for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in . To complement these strains, we constructed plasmids with -optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the " Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells.

CONCLUSIONS

These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in . This work provides the Yarrowia community with tools for cell biology and metabolism research in for further development of biofuels and natural products.

摘要

背景

是一种子囊菌酵母，因其能够分泌高浓度蛋白质和积累脂质而被用于生物技术研究。已经在与全面测序菌株具有不同相似性的多种背景下开发了遗传工具。

结果

我们开发了一套遗传和分子工具，以扩展其在生物学研究和工业生物工程应用方面的能力。在这项工作中，我们生成了一组同源营养缺陷型菌株，其非同源末端连接减少，用于靶向DNA整合。对该遗传背景进行基因组测序、组装和注释，发现了中以前未鉴定的基因。为了补充这些菌株，我们构建了带有优化的超级折叠绿色荧光蛋白的质粒，用于靶向过表达和荧光标记。我们使用这些工具构建了“细胞图谱”，这是一组在相同遗传背景下具有内源性荧光标记细胞器的菌株，以便定义活细胞中的细胞器形态。

结论

这些分子和同源工具可用于细胞器特异性蛋白质表达的实时评估，以及脂质生物合成酶或其他蛋白质在中的定位。这项工作为解脂耶氏酵母群体提供了用于细胞生物学和代谢研究的工具，以进一步开发生物燃料和天然产物。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/7ebf41790ef9/13068_2016_687_Fig12_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/d129499ebb5e/13068_2016_687_Fig1_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/6b498855c8ef/13068_2016_687_Fig11_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/7ebf41790ef9/13068_2016_687_Fig12_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/d129499ebb5e/13068_2016_687_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/7d5a172242b8/13068_2016_687_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/1966e7600461/13068_2016_687_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/b70cd19721e8/13068_2016_687_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/f9eb274d8355/13068_2016_687_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/d396035da2d7/13068_2016_687_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/2f2a80cd45af/13068_2016_687_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/37576b7ccc1b/13068_2016_687_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/234ff281711f/13068_2016_687_Fig9_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/84925b625b5c/13068_2016_687_Fig10_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/6b498855c8ef/13068_2016_687_Fig11_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7335/5210315/7ebf41790ef9/13068_2016_687_Fig12_HTML.jpg

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