Yarrowia lipolytica has recently emerged as a prominent microbial host for production of terpenoids. Its robust metabolism and growth in wide range of substrates offer several advantages at industrial scale. In the present study, we investigate the metabolic potential of Y. lipolytica to produce isoprene. Sustainable production of isoprene has been attempted through engineering several microbial hosts; however, the engineering studies performed so far are challenged with low titers. Engineering of Y. lipolytica, which have inherent high acetyl-CoA flux could fuel precursors into the biosynthesis of isoprene and thus is an approach that would offer sustainable production opportunities. The present work, therefore, explores this opportunity wherein a codon-optimized IspS gene (single copy) of Pueraria montana was integrated into the Y. lipolytica genome. With no detectable isoprene level during the growth or stationary phase of modified strain, attempts were made to overexpress enzymes from MVA pathway. GC-FID analyses of gas collected during stationary phase revealed that engineered strains were able to produce detectable isoprene only after overexpressing HMGR (or tHMGR). The significant role of HMGR (tHMGR) in diverting the pathway flux toward DMAPP is thus highlighted in our study. Nevertheless, the final recombinant strains overexpressing HMGR (tHMGR) along with Erg13 and IDI showed isoprene titers of ~500 μg/L and yields of ~80 μg/g. Further characterization of the recombinant strains revealed high lipid and squalene content compared to the unmodified strain. Overall, the preliminary results of our laboratory-scale studies represent Y. lipolytica as a promising host for fermentative production of isoprene.

中文翻译：

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解脂耶氏酵母的代谢工程生产异戊二烯

解脂耶氏酵母最近已成为生产萜类化合物的重要微生物宿主。它在各种底物中的强劲代谢和生长在工业规模上提供了几个优势。在本研究中，我们研究了解脂耶氏酵母产生异戊二烯的代谢潜力。异戊二烯的可持续生产已尝试通过改造几种微生物宿主；然而，迄今为止进行的工程研究面临着低滴度的挑战。解脂耶氏酵母的工程，其具有固有的高乙酰辅酶A通量，可以将前体燃料用于异戊二烯的生物合成，因此是一种提供可持续生产机会的方法。因此，目前的工作探索了这个机会，其中密码子优化Pueraria montana的IspS基因（单拷贝）被整合到解脂耶氏酵母基因组中。由于在修饰菌株的生长或静止阶段没有可检测到的异戊二烯水平，因此尝试过表达来自 MVA 途径的酶。对静止期收集的气体的 GC-FID 分析表明，工程菌株只有在过表达HMGR（或tHMGR）后才能产生可​​检测的异戊二烯。因此，我们的研究强调了HMGR ( tHMGR ) 在将通路通量转向 DMAPP方面的重要作用。然而，最终过表达HMGR ( tHMGR ) 的重组菌株与Erg13和IDI的异戊二烯滴度约为 500 μg/L，产量约为 80 μg/g。与未修饰的菌株相比，重组菌株的进一步表征揭示了高脂质和角鲨烯含量。总体而言，我们实验室规模研究的初步结果表明解脂耶氏酵母是发酵生产异戊二烯的有希望的宿主。