Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production
Abstract Paenibacillus polymyxa is a non‐pathogenic, Gram‐positive bacterium endowed with a rich and versatile metabolism. However interesting, this bacterium has been seldom used for bioproduction thus far. In this study, we engineered P. polymyxa for isobutanol production, a relevant bulk chemical...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2024-03-01
|
| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.14438 |
| _version_ | 1827304457463922688 |
|---|---|
| author | Meliawati Meliawati Daniel C. Volke Pablo I. Nikel Jochen Schmid |
| author_facet | Meliawati Meliawati Daniel C. Volke Pablo I. Nikel Jochen Schmid |
| author_sort | Meliawati Meliawati |
| collection | DOAJ |
| description | Abstract Paenibacillus polymyxa is a non‐pathogenic, Gram‐positive bacterium endowed with a rich and versatile metabolism. However interesting, this bacterium has been seldom used for bioproduction thus far. In this study, we engineered P. polymyxa for isobutanol production, a relevant bulk chemical and next‐generation biofuel. A CRISPR‐Cas9‐based genome editing tool facilitated the chromosomal integration of a synthetic operon to establish isobutanol production. The 2,3‐butanediol biosynthesis pathway, leading to the main fermentation product of P. polymyxa, was eliminated. A mutant strain harbouring the synthetic isobutanol operon (kdcA from Lactococcus lactis, and the native ilvC, ilvD and adh genes) produced 1 g L−1 isobutanol under microaerobic conditions. Improving NADPH regeneration by overexpression of the malic enzyme subsequently increased the product titre by 50%. Network‐wide proteomics provided insights into responses of P. polymyxa to isobutanol and revealed a significant metabolic shift caused by alcohol production. Glucose‐6‐phosphate 1‐dehydrogenase, the key enzyme in the pentose phosphate pathway, was identified as a bottleneck that hindered efficient NADPH regeneration through this pathway. Furthermore, we conducted culture optimization towards cultivating P. polymyxa in a synthetic minimal medium. We identified biotin (B7), pantothenate (B5) and folate (B9) to be mutual essential vitamins for P. polymyxa. Our rational metabolic engineering of P. polymyxa for the production of a heterologous chemical sheds light on the metabolism of this bacterium towards further biotechnological exploitation. |
| first_indexed | 2024-04-24T17:35:55Z |
| format | Article |
| id | doaj.art-462df2868a654271967fcdfc67e60dd1 |
| institution | Directory Open Access Journal |
| issn | 1751-7915 |
| language | English |
| last_indexed | 2024-04-24T17:35:55Z |
| publishDate | 2024-03-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj.art-462df2868a654271967fcdfc67e60dd12024-03-28T04:50:35ZengWileyMicrobial Biotechnology1751-79152024-03-01173n/an/a10.1111/1751-7915.14438Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol productionMeliawati Meliawati0Daniel C. Volke1Pablo I. Nikel2Jochen Schmid3Institute of Molecular Microbiology and Biotechnology University of Münster Münster GermanyThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark Kgs. Lyngby DenmarkThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark Kgs. Lyngby DenmarkInstitute of Molecular Microbiology and Biotechnology University of Münster Münster GermanyAbstract Paenibacillus polymyxa is a non‐pathogenic, Gram‐positive bacterium endowed with a rich and versatile metabolism. However interesting, this bacterium has been seldom used for bioproduction thus far. In this study, we engineered P. polymyxa for isobutanol production, a relevant bulk chemical and next‐generation biofuel. A CRISPR‐Cas9‐based genome editing tool facilitated the chromosomal integration of a synthetic operon to establish isobutanol production. The 2,3‐butanediol biosynthesis pathway, leading to the main fermentation product of P. polymyxa, was eliminated. A mutant strain harbouring the synthetic isobutanol operon (kdcA from Lactococcus lactis, and the native ilvC, ilvD and adh genes) produced 1 g L−1 isobutanol under microaerobic conditions. Improving NADPH regeneration by overexpression of the malic enzyme subsequently increased the product titre by 50%. Network‐wide proteomics provided insights into responses of P. polymyxa to isobutanol and revealed a significant metabolic shift caused by alcohol production. Glucose‐6‐phosphate 1‐dehydrogenase, the key enzyme in the pentose phosphate pathway, was identified as a bottleneck that hindered efficient NADPH regeneration through this pathway. Furthermore, we conducted culture optimization towards cultivating P. polymyxa in a synthetic minimal medium. We identified biotin (B7), pantothenate (B5) and folate (B9) to be mutual essential vitamins for P. polymyxa. Our rational metabolic engineering of P. polymyxa for the production of a heterologous chemical sheds light on the metabolism of this bacterium towards further biotechnological exploitation.https://doi.org/10.1111/1751-7915.14438 |
| spellingShingle | Meliawati Meliawati Daniel C. Volke Pablo I. Nikel Jochen Schmid Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production Microbial Biotechnology |
| title | Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production |
| title_full | Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production |
| title_fullStr | Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production |
| title_full_unstemmed | Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production |
| title_short | Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production |
| title_sort | engineering the carbon and redox metabolism of paenibacillus polymyxa for efficient isobutanol production |
| url | https://doi.org/10.1111/1751-7915.14438 |
| work_keys_str_mv | AT meliawatimeliawati engineeringthecarbonandredoxmetabolismofpaenibacilluspolymyxaforefficientisobutanolproduction AT danielcvolke engineeringthecarbonandredoxmetabolismofpaenibacilluspolymyxaforefficientisobutanolproduction AT pabloinikel engineeringthecarbonandredoxmetabolismofpaenibacilluspolymyxaforefficientisobutanolproduction AT jochenschmid engineeringthecarbonandredoxmetabolismofpaenibacilluspolymyxaforefficientisobutanolproduction |