Construction of acid-producing Clostridium saccharoperbutylacetonicum strains by deletion, overexpression, and interfering with regulation of genes

Autor: Baur, Saskia Tabea
Přispěvatelé: Dürre, Peter, Eikmanns, Bernhard
Jazyk: angličtina
Rok vydání: 2022
Předmět:
DOI: 10.18725/oparu-42087
Popis: Clostridium saccharoperbutylacetonicum N1-4(HMT) was isolated in 1960 as hyper-butanol producer and widely used for butanol and hydrogen production by fermentation. In this work, the strain was genetically modified to create strains for either acetate or butyrate production. BLASTp and transcriptome analyses were performed to identify target genes for deletion and overexpression. Thereby, the genes encoding enzymes mainly responsible for acid and solvent production were identified. Acetate production was achieved by elimination of the by-products butyrate and butanol via deletion of crt (Cspa_c04330) encoding the only crotonase. This was the first time that this gene was deleted in C. saccharoperbutylacetonicum. It resulted in drastic changes in the redox balance, leading to extremely increased lactate and ethanol concentrations. Further increase in acetate production was achieved by homologous overexpression of pta and ack encoding phosphotransacetylase and acetate kinase (Cspa_c13010 and Cspa_c13020) in the strain C. saccharoperbutylacetonicum ��crt [pMTL83151_PA_PbgaL] resulting in an increase of up to 116 % and 138 % of acetate levels produced by C. saccharoperbutylacetonicum N1-4(HMT) and C. saccharoperbutylacetonicum ��crt, respectively. Butyrate was established as a second major product besides butanol in the strain C. saccharoperbutylacetonicum ��bld��pta [pMTL83151_BCS_PbgaL]. The strain lacks the genes bld and pta (Cspa_c56880 and Cspa_c13010) encoding butyraldehyde dehydrogenase and phosphotransacetylase and harbors a plasmid containing the bcs operon consisting of crt, bcd with etfB and etfA, and hbd (Cspa_c04330 to Cspa_c04370) encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits �� and ��, and 3-hydroxybutyryl-CoA dehydrogenase. The molar ratio of butyrate to the other products shifted to 1:1.6 compared to 1:55.4 for C. saccharoperbutylacetonicum N1-4(HMT). Additional overexpression of ptb and buk (Cspa_c02520 and Cspa_c02530) encoding phosphotransbutyrylase and butyrate kinase did not lead to further increase of butyrate production. The regulation of the sol mRNA and the asRNA Assolrna complementary to the whole sol operon was investigated. The expression of Assolrna during different growth phases was evaluated using transcriptomic data showing a parallel increase and decrease of transcription levels of sol mRNA and Assolrna. Growth experiments with deletion, complementation, and overexpression strains of Assolrna combined with qRT-PCR analyses were performed to analyze 103 changes in solvent formation and transcription of sol mRNA and Assolrna. The results show that Assolrna is crucial for transcription of sol mRNA since no transcription of the sol operon was detected in the deletion strain lacking Assolrna transcription (C. saccharoperbutylacetonicum ��Pasr::Pasr**). Additionally, it was shown that Assolrna stabilizes or enables transcription of the sol mRNA when the sol mRNA:Assolrna ratio is larger than 2:1. When the ratio reaches 1:1 or if Assolrna is more abundant than the sol mRNA, the stabilizing effect is converted into a destabilizing effect regarding the sol mRNA. Finally, a model for the regulatory mechanism of the regulation of the sol operon by Assolrna was proposed. Using the transcriptomic data, other asRNAs were identified exposing the same parallel transcription pattern as sol mRNA and Assolrna. Therefore, it is possible that they show a similar regulatory mechanism. This allows construction of various production strains by interference with regulatory mechanisms leading to better product yields or stricter elimination of by-products as it is the case for the strong decrease of solvent formation in C. saccharoperbutylacetonicum ��Pasr::Pasr**.
Databáze: OpenAIRE
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