Bracalente Fernando, Tripaldi Matías, Galván Virginia, Tsai Yi-Ting, Takano Eriko, Altabe Silvia, Gramajo Hugo, Arabolaza Ana
Microbiology Division, IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Ocampo y Esmeralda, 2000, Rosario, Argentina.
Manchester Institute of Biotechnology, Department of Chemistry, School of Natural Sciences, Faculty of Science and Engineering, Manchester Institute of Biotechnology (MIB), University of Manchester, United Kingdom.
Metab Eng. 2025 Jul;90:78-91. doi: 10.1016/j.ymben.2025.03.005. Epub 2025 Mar 7.
Microbial fatty acids (FAs) hold significant potential as alternatives for the oleochemical industry. However, expanding the functional and structural diversity of microbial FA-derived products is essential to fully leverage this potential. Methyl-branched-chain FAs (MBFAs) are of particular interest as high-performance industrial compounds. This study examines the ability of the Escherichia coli FA biosynthesis pathway to produce a diverse mixture of random MBFAs (R-MBFAs) by utilizing both the natural malonyl-ACP substrate and the branched-chain methylmalonyl-ACP (mm-ACP) as an unnatural elongation unit. First, E. coli was engineered to accumulate methylmalonyl-CoA (mm-CoA) through a methylmalonate or a propionate-dependent pathway, and the capacity of E. coli FASII enzymes to synthesize mm-ACP and utilize it as a substrate was confirmed by the production of R-MBFAs. However, low R-MBFA accumulation and propionate-induced growth inhibition was observed. To improve R-MBFA yields, various malonyl-/mm-CoA acyltransferase (AT) enzymes were expressed, and their efficacy in generating mm-ACP was indirectly assessed through R-MBFA production levels. When expressing selected ATs, including native malonyl CoA-acyl carrier protein transacylase FabD, propionate-induced growth inhibition was alleviated and R-MBFA titers ranged from 5.9% to 7.7% of total FAs. Further strain optimization, analyzing two thioesterase (TE) activities and overexpression of the E. coli transciptional regulator FadR, significantly boosted R-MBFA titers. While an engineered strain carrying the Mus musculus TE domain (TE) produced 55.2 mg/L of R-MBFAs, representing an 11.8% of total FAs, another strain combining the overexpression of the cytosolic version of the TE TesA from E. coli ('TesA) and FadR produced approximately 1.1 g/L of total FAs, with an R-MBFA fraction of 6.7% (70.5 mg/L), marking the highest yield recorded in shake-flask cultures. Finally, these two recombinant E. coli strains were grown in laboratory-scale fed-batch fermentations, and produced approximately 10 g/L of total FAs and over 1-1.2 g/L of R-MBFAs, underscoring the potential for large-scale production of these valuable FA-derived compounds.
微生物脂肪酸（FAs）作为油脂化学工业的替代品具有巨大潜力。然而，扩大微生物脂肪酸衍生产品的功能和结构多样性对于充分利用这一潜力至关重要。甲基支链脂肪酸（MBFAs）作为高性能工业化合物尤其受到关注。本研究考察了大肠杆菌脂肪酸生物合成途径利用天然丙二酰-ACP底物和支链甲基丙二酰-ACP（mm-ACP）作为非天然延伸单元来生产多种随机甲基支链脂肪酸（R-MBFAs）混合物的能力。首先，通过丙二酸单酯或丙酸依赖途径对大肠杆菌进行工程改造以积累甲基丙二酰辅酶A（mm-CoA），并通过R-MBFAs的产生证实了大肠杆菌FASII酶合成mm-ACP并将其用作底物的能力。然而，观察到R-MBFA积累量低且丙酸诱导生长抑制。为了提高R-MBFA产量，表达了各种丙二酰/甲基丙二酰辅酶A酰基转移酶（AT），并通过R-MBFA产量水平间接评估它们产生mm-ACP的功效。当表达选定的AT时，包括天然丙二酰辅酶A-酰基载体蛋白转酰基酶FabD，丙酸诱导的生长抑制得到缓解，R-MBFA滴度占总脂肪酸的5.9%至7.7%。进一步的菌株优化、分析两种硫酯酶（TE）活性以及大肠杆菌转录调节因子FadR的过表达显著提高了R-MBFA滴度。携带小家鼠硫酯酶结构域（TE）的工程菌株产生了55.2mg/L的R-MBFAs，占总脂肪酸的11.8%，而另一种结合了大肠杆菌胞质型硫酯酶TesA（'TesA）和FadR过表达的菌株产生了约1.1g/L的总脂肪酸，R-MBFA比例为6.7%（70.5mg/L），这是摇瓶培养中记录的最高产量。最后，这两种重组大肠杆菌菌株在实验室规模的补料分批发酵中生长，产生了约10g/L的总脂肪酸和超过1-1.2g/L的R-MBFA，突出了大规模生产这些有价值的脂肪酸衍生化合物的潜力。
