Journal
BIOTECHNOLOGY JOURNAL
Volume 16, Issue 6, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.202000413
Keywords
2‐ keto acid pathway; isobutanol; metabolomics; methanol; Methylorubrum extorquens
Funding
- National Key R&D Program of China [2018YFA0901500]
- National Natural Science Foundation of China [21776149, 22078169, 32000003]
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The study investigated the potential of using pyruvate to produce isobutanol in engineered Methylorubrum extorquens AM1. By optimizing the metabolic pathways and utilizing an isobutanol-tolerant strain, the production of isobutanol was significantly increased, showcasing the feasibility of converting methanol to valuable chemicals using pyruvate as a precursor.
Background Methylorubrum extorquens AM1 can be engineered to convert methanol to value-added chemicals. Most of these chemicals derive from acetyl-CoA involved in the serine cycle. However, recent studies on methylotrophic metabolism have suggested that C3 pyruvate is a good potential precursor for broadening the types of synthesized products. Methods and Results In the present study, we found that isobutanol was a model chemical that could be generated from pyruvate through a 2-keto acid pathway. Initially, the engineered M. extorquens AM1 could only produce a trace amount of isobutanol at 0.62 mgL(-1) after introducing the heterologous 2-ketoisovalerate decarboxylase and alcohol dehydrogenase. Furthermore, the metabolomic analysis revealed that insufficient carbon fluxes through 2-ketoisovalerate and pyruvate were the key limitation steps for efficient biosynthesis of isobutanol. Based on this analysis, the titer of isobutanol was improved by over 20-fold after overexpressing alsS gene encoding acetolactate synthase and deleting ldhA gene for lactate dehydrogenase. Moreover, substituting the cell chassis with the isobutanol-tolerant strain isolated from adaptive evolution of M. extorquens AM1 further increased the production of isobutanol by 1.7-fold, resulting in the final titer of 19 mgL(-1) in flask cultivation. Conclusion Our current findings provided promising insights into engineering methylotrophic cell factories capable of converting methanol to isobutanol or value-added chemicals using pyruvate as the precursor.
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