Trikka Fotini A, Nikolaidis Alexandros, Athanasakoglou Anastasia, Andreadelli Aggeliki, Ignea Codruta, Kotta Konstantia, Argiriou Anagnostis, Kampranis Sotirios C, Makris Antonios M
Institute of Applied Biosciences/CERTH, P.O. Box 60361, Thermi, 57001, Thessaloniki, Greece.
Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion, 71003, Greece.
Microb Cell Fact. 2015 Apr 24;14:60. doi: 10.1186/s12934-015-0246-0.
Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system.
Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation.
Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.
萜类化合物（类异戊二烯）在香料、香水、药物和生物燃料等领域有众多应用。随着新的生物合成途径被阐明，微生物产生的萜类化合物数量不断增加。然而，提高酵母中萜类化合物产量的努力大多利用了固醇生物合成途径的现有知识，而许多其他因素可能会影响工程系统的产量。
为了开发一种能够支持高滴度生产香紫苏醇（一种对香水行业非常重要的二萜）的酵母菌株，我们试图鉴定能够提高类胡萝卜素产量从而可能提高香紫苏醇产量的基因缺失。通过类胡萝卜素生成筛选，从4700个突变菌株中选出了最佳的100个缺失突变体，创建了一个子集用于进一步分析。为了鉴定协同作用以进一步提高产量的缺失组合，应用了迭代类胡萝卜素生成筛选，并且每次根据每个特定基因已知的遗传和物理相互作用的数量对表现最佳的基因缺失进行进一步排名。删除每轮中选择的基因，并将所得菌株用于新一轮的筛选。这种方法导致了一种源自EG60的单倍体菌株的开发，该菌株结合了六个缺失（rox1、dos2、yer134c、vba5、ynr063w和ygr259c），类胡萝卜素产量增加了40倍，香紫苏醇滴度增加了12倍，在摇瓶培养中达到750mg/L香紫苏醇。
通过迭代方法，我们鉴定出了酵母基因缺失的新组合，这些组合在不影响菌株生长和活力的情况下提高了类胡萝卜素和香紫苏醇的产量。大多数鉴定出的缺失以前未涉及固醇途径控制。使用不同的起始点应用相同的方法可能会产生具有相似或更好结果的替代缺失组合。
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