Refine
Year of publication
- 2023 (2) (remove)
Document Type
- Article (2)
Language
- English (2)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Keywords
- 3-Hydroxybutyric acid (1)
- Alphaproteobacteria (1)
- Background expression (1)
- Cumate (1)
- Dicarboxylic acids (1)
- Enzyme engineering (1)
- Inducible Promoter (1)
- Methylorubrum extorquens (1)
- Methylorubrum extorquens AM1 (1)
- Thioesterase (1)
Institute
Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.
Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
(2023)
Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches.