Lee Heeseok, Han Changpyo, Lee Hyeok-Won, Park Gyuyeon, Jeon Wooyoung, Ahn Jungoh, Lee Hongweon
Biotechnology Process Engineering Center, Korean Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do 28116 Republic of Korea.
2Department of Bioprocess Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113 Republic of Korea.
Biotechnol Biofuels. 2018 Nov 9;11:310. doi: 10.1186/s13068-018-1310-x. eCollection 2018.
As a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast has been used as a host for DCA production, additional strains are continually investigated to meet productivity thresholds and industrial needs. In this regard, the yeast , a potential candidate strain, has been screened. However, the lack of genetic and physiological information for this uncommon strain is an obstacle that merits further research. To overcome this limitation, we attempted to develop a method to facilitate genetic recombination in this strain and produce high amounts of DCAs from methyl laurate using engineered .
In the current study, we first developed efficient genetic engineering tools for the industrial application of . To increase homologous recombination (HR) efficiency during transformation, the cell cycle of the yeast was synchronized to the S/G2 phase using hydroxyurea. The HR efficiency at and loci increased from 56.3% and 41.7%, respectively, to 97.9% in both cases. The original HR efficiency at and loci was nearly 0% during the early stationary and logarithmic phases of growth, and increased to 4.8% and 25.6%, respectively. We used the developed tools to construct UHP4, in which β-oxidation was completely blocked. The strain produced 92.5 g/l of dodecanedioic acid (DDDA) from methyl laurate over 126 h in 5-l fed-batch fermentation, with a productivity of 0.83 g/l/h.
UHP4 produced more DDDA methyl laurate than . Hence, we demonstrated that is a powerful microbial platform for vegetable oil-based DCA production. In addition, by using the developed genetic engineering tools, this emerging yeast could be used for the production of a variety of fatty acid derivatives, such as fatty alcohols, fatty aldehydes, and ω-hydroxy fatty acids.
作为一种可持续的工业生产过程，利用植物油生产二羧酸（DCA）作为聚酰胺、聚酯、香料、增塑剂、润滑剂和粘合剂的前体，一直备受关注。尽管酵母已被用作生产DCA的宿主，但仍在不断研究其他菌株以满足生产力阈值和工业需求。在这方面，已筛选出潜在的候选菌株酵母。然而，这种不常见菌株缺乏遗传和生理信息是一个值得进一步研究的障碍。为克服这一限制，我们试图开发一种方法来促进该菌株的基因重组，并利用工程改造的酵母从月桂酸甲酯中大量生产DCA。
在本研究中，我们首先为酵母的工业应用开发了高效的基因工程工具。为提高转化过程中的同源重组（HR）效率，使用羟基脲将酵母细胞周期同步到S/G2期。在两个位点的HR效率分别从56.3%和41.7%提高到97.9%。在生长的早期稳定期和对数期，两个位点的原始HR效率几乎为0%，分别提高到4.8%和25.6%。我们使用开发的工具构建了UHP4，其中β-氧化被完全阻断。该菌株在5升补料分批发酵中，126小时内从月桂酸甲酯生产了92.5克/升的十二烷二酸（DDDA），生产力为0.83克/升/小时。
UHP4比酵母生产更多的DDDA甲酯。因此，我们证明酵母是基于植物油生产DCA的强大微生物平台。此外，通过使用开发的基因工程工具，这种新兴酵母可用于生产各种脂肪酸衍生物，如脂肪醇、脂肪醛和ω-羟基脂肪酸。
