Harahap Andre Fahriz Perdana, Treinen Chantal, Zyl Leonardo Joaquim Van, Williams Wesley Trevor, Conrad Jürgen, Pfannstiel Jens, Klaiber Iris, Grether Jakob, Hiller Eric, Vahidinasab Maliheh, Perino Elvio Henrique Benatto, Lilge Lars, Burger Anita, Trindade Marla, Hausmann Rudolf
Department of Bioprocess Engineering (150k), Institute of Food Science and Biotechnology, University of Hohenheim, Fruwirthstr. 12, 70599 Stuttgart, Germany.
Cellular Agriculture, TUM School of Life Sciences, Technical University of Munich, Gregor-Mendel-Str. 4,85354 Freising, Germany.
Microorganisms. 2025 Jul 15;13(7):1664. doi: 10.3390/microorganisms13071664.
DSM 100043 had been previously proven to produce a novel glucoselipid biosurfactant which has a very low critical micelle concentration (CMC) as well as very good stability against a wide range of pH, temperature, and salinity. In this study, we performed a function-based library screening from a DSM 100043 genome library to identify responsible genes for biosynthesis of this glucoselipid. The identified open reading frames (ORFs) were cloned into several constructs in for gene permutation analysis and the individual products were analyzed using high-performance thin-layer chromatography (HPTLC). Products of interest from positive expression strains were purified and analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) for further structure elucidation. Function-based screening of 5400 clones led to the identification of an operon containing three ORFs encoding acetyltransferase GlcA (ORF1), acyltransferase GlcB (ORF2), and phosphatase/HAD GlcC (ORF3). pCAT2, with all three ORFs, resulted in the production of identical DSM 100043 glucosedilipid with Glu-C-C as the main congener. ORF2-deletion strain pAFP1 primarily produced glucosemonolipids, with Glu-C and Glu-C as the major congeners, predominantly esterified at the C-2 position of the glucose moiety. Furthermore, fed-batch bioreactor cultivation of pCAT2 using glucose as the carbon source yielded a maximum glucosedilipid titer of 2.34 g/L after 25 h of fermentation, which is 55-fold higher than that produced by batch cultivation of DSM 100043 in the previous study.
DSM 100043先前已被证明能产生一种新型糖脂生物表面活性剂，其临界胶束浓度（CMC）非常低，并且在很宽的pH、温度和盐度范围内具有非常好的稳定性。在本研究中，我们从DSM 100043基因组文库中进行了基于功能的文库筛选，以鉴定负责该糖脂生物合成的基因。将鉴定出的开放阅读框（ORF）克隆到几种构建体中进行基因置换分析，并使用高效薄层色谱（HPTLC）分析各个产物。对阳性表达菌株中感兴趣的产物进行纯化，并通过液相色谱/电喷雾电离串联质谱（LC-ESI-MS/MS）和核磁共振（NMR）进行分析，以进一步阐明结构。对5400个克隆进行基于功能的筛选，鉴定出一个操纵子，其中包含三个编码乙酰转移酶GlcA（ORF1）、酰基转移酶GlcB（ORF2）和磷酸酶/HAD GlcC（ORF3）的ORF。含有所有三个ORF的pCAT2产生了与DSM 100043相同的葡萄糖二脂，其中Glu-C-C为主要同系物。ORF2缺失菌株pAFP1主要产生葡萄糖单脂，以Glu-C和Glu-C为主要同系物，主要在葡萄糖部分的C-2位酯化。此外，以葡萄糖为碳源对pCAT2进行补料分批生物反应器培养，发酵25小时后葡萄糖二脂的最高产量为2.34 g/L，这比先前研究中DSM 100043分批培养产生的产量高55倍。
