Dai Ziyu, Deng Shuang, Culley David E, Bruno Kenneth S, Magnuson Jon K
Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.
Zymergen, 6121 Hollis Street Suite 700, Emeryville, CA, 94608, USA.
Appl Microbiol Biotechnol. 2017 Aug;101(15):6099-6110. doi: 10.1007/s00253-017-8357-7. Epub 2017 Jun 19.
Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.
利用可再生碳源，特别是木质纤维素生物质来生产碳氢燃料和化学品的兴趣，激发了人们探索各种能够生产碳氢生物燃料、化学品或其前体的生物体的兴趣。产油酵母斯达氏油脂酵母（Lipomyces starkeyi）是利用各种碳水化合物和营养源生产甘油三酯作为碳氢燃料前体的积极研究对象。斯达氏油脂酵母的基因组已公布，这为通过开发和使用合成生物学工具来改良该产油物种的生产菌株打开了大门。为生物体建立合成生物学工具的第一步是开发具有合适选择标记基因的有效且可靠的转化方法，并证明引入基因表达或内源基因缺失所需的遗传元件的效用。基于化学的转化方法已经公布，但效率较低。为了解决这些问题，研究了农杆菌介导的转化作为斯达氏油脂酵母和其他油脂酵母属物种的替代方法。在本研究中，证明了农杆菌介导的转化对斯达氏油脂酵母和其他油脂酵母属物种的转化是有效的。还在斯达氏油脂酵母中证明了过氧化物酶体生物发生因子10基因的缺失。除了细菌抗生素选择标记基因潮霉素B磷酸转移酶外，在斯达氏油脂酵母翻译延伸因子1α启动子控制下的细菌β-葡萄糖醛酸酶报告基因也在六种不同的油脂酵母属物种中稳定表达。本研究结果表明，农杆菌介导的转化是在斯达氏油脂酵母和其他油脂酵母属物种中进行同源重组和异源基因表达的可靠且有效的遗传工具。
