Lim Hyun Gyu, Srinivasan Aparajitha, Menchavez Russel, Yunus Ian S, Noh Myung Hyun, White Megan, Chen Yan, Gin Jennifer W, Palsson Bernhard O, Lee Taek Soon, Petzold Christopher J, Eng Thomas, Mukhopadhyay Aindrila, Feist Adam M
Department of Bioengineering, University of California San Diego, 9500 Gilman Dr., La Jolla, CA, 92093, USA; Joint BioEnergy Institute, 5885 Hollis Street, 4th Floor, Emeryville, CA, 94608, USA; Department of Biological Sciences and Bioengineering, Inha University, Inha-ro 100, Michuhol-gu, Incheon, 22212, South Korea.
Joint BioEnergy Institute, 5885 Hollis Street, 4th Floor, Emeryville, CA, 94608, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, 94702, USA.
Metab Eng. 2025 Sep;91:322-335. doi: 10.1016/j.ymben.2025.05.007. Epub 2025 May 19.
Isoprenol (3-methyl-3-buten-1-ol) is a precursor to aviation fuels and other commodity chemicals and can be microbially synthesized from renewable carbon streams. Its production has been demonstrated in Pseudomonas putida KT2440 but its titers, rates, and yields have yet to reach commercially viable levels, potentially due to toxicity to the bacterial chassis. We hypothesized that utilization of Tolerization Adaptive Laboratory Evolution (TALE) would generate P. putida hosts more tolerant to isoprenol and suitable for enhanced production phenotypes. Here, we performed a comprehensive TALE campaign using three strains, the wild-type and two strains lacking subsets of known isoprenol catabolism and transport functions in quadruplicate independently evolved lineages. Several evolved clones from each starting strain displayed robust growth (up to 0.2 h) at 8 g/L of isoprenol, where starting strains could not grow. Whole genome resequencing of the 12 independent strain lineages identified convergent mutations. Reverse engineering each of the four commonly mutated regions individually (gnuR, ttgB-PP_1394, PP_3024-PP_5558, PP_1695) resulted in a partial recovery of the tolerance phenotypes observed in the evolved strains. Additionally, a proteomics-guided deletion of the master motility regulator, fleQ, in an evolved clone alleviated the tolerance vs. production trade-off, restoring isoprenol titers and consumption to levels observed in the starting strains. Collectively, this work demonstrated that an integrated strategy of laboratory evolution and rational engineering was effective to develop robust biofuel production hosts with minimized product toxicity.
异戊醇（3-甲基-3-丁烯-1-醇）是航空燃料和其他商品化学品的前体，可通过微生物从可再生碳流中合成。其生产已在恶臭假单胞菌KT2440中得到证实，但其滴度、速率和产量尚未达到商业可行水平，这可能是由于对细菌底盘有毒性。我们假设利用耐受性适应性实验室进化（TALE）将产生对异戊醇更具耐受性且适合增强生产表型的恶臭假单胞菌宿主。在这里，我们使用三种菌株进行了全面的TALE实验，这三种菌株分别是野生型以及两种在四个独立进化谱系中缺乏已知异戊醇分解代谢和转运功能子集的菌株。每个起始菌株的几个进化克隆在8 g/L异戊醇下表现出强劲的生长（高达0.2 h），而起始菌株在此浓度下无法生长。对12个独立菌株谱系进行全基因组重测序确定了趋同突变。分别对四个常见突变区域（gnuR、ttgB - PP_1394、PP_3024 - PP_5558、PP_1695）进行逆向工程，部分恢复了进化菌株中观察到的耐受性表型。此外，在一个进化克隆中通过蛋白质组学指导删除主要运动调节因子fleQ，缓解了耐受性与生产之间的权衡，使异戊醇滴度和消耗量恢复到起始菌株中观察到的水平。总的来说，这项工作表明实验室进化和理性工程的综合策略对于开发具有最小化产品毒性的强大生物燃料生产宿主是有效的。
