Switching metabolic flux by engineering tryptophan operon-assisted CRISPR interference system in Klebsiella pneumoniae
| Autor: | Pingfang Tian, Qingyang Li, Peng Zhao, Tianwei Tan |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Glycerol
0106 biological sciences Bioengineering 3-Hydroxypropionic acid 01 natural sciences Applied Microbiology and Biotechnology trp operon 03 medical and health sciences chemistry.chemical_compound Biosynthesis 010608 biotechnology Operon Clustered Regularly Interspaced Short Palindromic Repeats 030304 developmental biology 0303 health sciences CRISPR interference Strain (chemistry) Chemistry Tryptophan Bioproduction Klebsiella pneumoniae Metabolic Engineering Biochemistry Flux (metabolism) Biotechnology |
| Zdroj: | Metabolic Engineering. 65:30-41 |
| ISSN: | 1096-7176 |
| DOI: | 10.1016/j.ymben.2021.03.001 |
| Popis: | One grand challenge for bioproduction of desired metabolites is how to coordinate cell growth and product synthesis. Here we report that a tryptophan operon-assisted CRISPR interference (CRISPRi) system can switch glycerol oxidation and reduction pathways in Klebsiella pneumoniae, whereby the oxidation pathway provides energy to sustain growth, and the reduction pathway generates 1,3-propanediol and 3-hydroxypropionic acid (3-HP), two economically important chemicals. Reverse transcription and quantitative PCR (RT-qPCR) showed that this CRISPRi-dependent switch affected the expression of glycerol metabolism-related genes and in turn improved 3-HP production. In shake-flask cultivation, the strain coexpressing dCas9-sgRNA and PuuC (an aldehyde dehydrogenase native to K. pneumoniae for 3-HP biosynthesis) produced 3.6 g/L 3-HP, which was 1.62 times that of the strain only overexpressing PuuC. In a 5 L bioreactor, this CRISPRi strain produced 58.9 g/L 3-HP. When circulation feeding was implemented to alleviate metabolic stress, biomass was substantially improved and 88.8 g/L 3-HP was produced. These results indicated that this CRISPRi-dependent switch can efficiently reconcile biomass formation and 3-HP biosynthesis. Furthermore, this is the first report of coupling CRISPRi system with trp operon, and this architecture holds huge potential in regulating gene expression and allocating metabolic flux. |
| Databáze: | OpenAIRE |
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