College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 211816, China.
ACS Synth Biol. 2022 Jun 17;11(6):1992-2000. doi: 10.1021/acssynbio.1c00622. Epub 2022 May 31.
Rhamnolipids (RLs) are biosurfactants with great economic significance that have been used extensively in multiple industries. is a promising microorganism for sustainable RL production. However, current CTAB-MB based screening of RL-producing strains is time-consuming, labor-intensive, and unable to distinguish mono- and di-RL. In this study, we developed a novel transcription-associated fluorescence-activated droplet sorting (FADS) method to specifically target the di-RL hyperproducers. We first investigated critical factors associated with this method, including the specificity and sensitivity for discriminating di-RL overproducers from other communities. Validation of genotype-phenotype linkage between the GFP intensity, transcription, and di-RL production showed that transcription is closely correlated with di-RL production, and the GFP intensity is responsive to transcription, respectively. Using this platform, we screened out ten higher di-RL producing microorganisms, which produced 54-208% more di-RL than the model PAO1. In summary, the droplet-based microfluidic platform not only facilitates a more specific, reliable, and rapid screening of colonies with desired phenotypes, but also shows that intracellular transcription-associated GFP intensity can be used to measure the yield of di-RL between populations of droplets containing different environmental colonies. This method also can be integrated with transposon mutation libraries to target mutants.
鼠李糖脂（RLs）是一种具有重要经济意义的生物表面活性剂，已广泛应用于多个行业。是一种很有前途的可持续 RL 生产的微生物。然而，目前基于 CTAB-MB 的 RL 产生菌的筛选既耗时又费力，而且无法区分单 RL 和双 RL。在本研究中，我们开发了一种新的转录相关荧光激活液滴分选（FADS）方法，专门针对双 RL 高产菌。我们首先研究了与该方法相关的关键因素，包括区分双 RL 过生产菌与其他群落的特异性和敏感性。GFP 强度、转录与双 RL 生产之间的表型-基因型关联的验证表明，转录与双 RL 生产密切相关，而 GFP 强度对转录有反应。使用该平台，我们筛选出了 10 种更高产双 RL 的微生物，其双 RL 产量比模型 PAO1 高出 54-208%。总之，基于液滴的微流控平台不仅可以更具体、更可靠、更快速地筛选具有所需表型的 菌落，还表明含有不同环境菌落的液滴群体之间，细胞内转录相关 GFP 强度可用于测量双 RL 的产量。该方法还可以与转座子突变文库集成，以靶向 突变体。
