期刊
PLANT BIOTECHNOLOGY JOURNAL
卷 20, 期 10, 页码 1968-1982出版社
WILEY
DOI: 10.1111/pbi.13879
关键词
amine oxidase; Chlamydomonas reinhardtii; CRISPR; Cas9; microalga; ornithine decarboxylase; polyamines
资金
- European Regional Development Fund (ERDF)
- Ministry of Economic Affairs, Innovation, Digitization and Energy of the State of North Rhine-Westphalia by grant 'Cluster Industrial Biotechnology (CLIB) Kompetenzzentrum Biotechnologie (CKB)' [34.EFRE-0300095/1703FI04]
- Projekt DEAL
This study systematically investigated the native putrescine metabolism in the green microalga Chlamydomonas reinhardtii and successfully achieved CO2-based bio-production of putrescine. The study provides important insights into the putrescine biosynthesis pathway and offers a promising approach for sustainable biotechnology.
The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO2-based bio-production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9-based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5-fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co-production of cadaverine and 4-aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10-fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.
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