Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis

Hermann, Maria; Teleki, Attila; Weitz, Sandra; Nieß, Alexander; Freund, Andreas; Bengelsdorf, Frank R.; Dürre, Peter

doi:10.3389/fbioe.2021.647853

Cited by 12 publications

(11 citation statements)

References 57 publications

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“…Since the metabolic engineering of n -butanol in Clostridia has been extensively studied, ,− future work could focus on the fine-tuning of the isobutanol pathway. This could be achieved by reconstructing or activating the isobutanol synthesis pathway, − using different promoter combinations to adjust the expression level of key enzymes according to the determination results of intermediate metabolites, ,, promoting NADPH supply by engineering transhydrogenase (PntAB) and the pentose phosphate pathway (PPP), or modifying the key enzymes (and their cofactor preference) in the isobutanol pathway. , At the same time, the pathways of byproducts, like ethanol, acetone, lactate, acetate, and butyrate, should be downregulated or inactivated to guide the carbon flow to the butanol pathways. ,, …”

Section: Resultsmentioning

confidence: 99%

Genetic Manipulation of a Twin Clostridia Consortium for Co-production of n-Butanol and Isobutanol by Consolidated Bioprocessing

Wen

makishah

et al. 2022

ACS Sustainable Chem. Eng.

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It is attractive to co-produce n-butanol and isobutanol from lignocellulosic biomass in engineered strains or consortia, because they can be coseparated and combined for use as biofuel. However, very few attempts have been made to date. To address that, a heterologous isobutanol pathway was first introduced to enable Clostridium beijerinckii to coproduce n-butanol and isobutanol. Following medium optimization, the recombinant C. beijerinckii produced 194 mg/L isobutanol and 7.16 g/L n-butanol from glucose. C. cellulovorans was then genetically engineered by overexpressing adhE1, kivD, and yqhD to synthesize n-butanol and isobutanol with alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. It produced 156 mg/L isobutanol and 1.81 g/L n-butanol from 39.5 g/L AECC within 120 h. Finally, the above two recombinant strains were mix-cultured and optimized. The optimal consortium degraded 55.1 g/L AECC and produced 1.05 g/L isobutanol and 6.22 g/L n-butanol, which were 6.73-and 3.44-fold higher than those obtained from single culture of recombinant C. cellulovorans. Besides, the titer and yield of total butanol are 15.6% and 11.9%, respectively, higher than those of recombinant consortia that only produce n-butanol. Synthetic biology empowers artificial consortia with expanded functions, as well as improved performance, showing great potential in consolidated bioprocessing.

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Section: Resultsmentioning

confidence: 99%

Genetic Manipulation of a Twin Clostridia Consortium for Co-production of n-Butanol and Isobutanol by Consolidated Bioprocessing

Wen

makishah

et al. 2022

ACS Sustainable Chem. Eng.

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“…Finally, growing the strains in gassed and stirred bioreactors will certainly improve the fermentation outcome. In fact, a C. ljungdahlii:ilvE strain carrying pKAIA showed under such conditions an increased isobutanol production by factor 6.5 (Hermann et al, 2021).…”

Section: Discussionmentioning

confidence: 98%

Isobutanol Production by Autotrophic Acetogenic Bacteria

Weitz

Hermann

Linder

et al. 2021

Front. Bioeng. Biotechnol.

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Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.

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“…0.02 µmol/gDCW. 28 Interestingly, methanol metabolism appears to result in upregulation of the pyruvate-consuming PD (elim_c2580, 2581) (log 2 fold change of 1.9 and 2.3 respectively for adding methanol/CO 2 to formate/0.4D growth).…”

Section: Formate Triggers a Beneficial Intracellular State During Met...mentioning

confidence: 99%

Molecular understanding ofEubacterium limosumchemostat methanol metabolism

Wood

Gonzalez-Garcia

Daygon

et al. 2022

Preprint

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Methanol is a promising renewable energy carrier that can be used as a favourable substrate for biotechnology, due to its high energy efficiency conversion and ease of integration within existing infrastructure. Some acetogenic bacteria have the native ability to utilise methanol, along with other C1substrates such as CO2and formate, to produce valuable chemicals. Continuous cultures favour economically viable bioprocesses, however, the performance of acetogens has not been investigated at the molecular level when grown on methanol. Here we present steady-state chemostat quantification of the metabolism ofEubacterium limosum, finding maximum methanol uptake rates up to 640±22 mmol/gDCW/d, with significant fluxes to butyrate. To better understand metabolism of acetogens under methanol growth conditions, we sampled chemostats for proteomics and metabolomics. Changes in protein expression and intracellular metabolomics highlighted key aspects of methanol metabolism, and highlighted bottleneck conditions preventing formation of the more valuable product, butanol. Interestingly, a small amount of formate in methylotrophic metabolism triggered a cellular state known in other acetogens to correlate with solventogenesis. Unfortunately, this was prevented by post-translation effects including an oxidised NAD pool. There remains uncertainty around ferredoxin balance at the methylene-tetrahydrofolate reductase (MTHFR) and at the Rnf level.

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Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis

Cited by 12 publications

References 57 publications

Genetic Manipulation of a Twin Clostridia Consortium for Co-production of n-Butanol and Isobutanol by Consolidated Bioprocessing

Genetic Manipulation of a Twin Clostridia Consortium for Co-production of n-Butanol and Isobutanol by Consolidated Bioprocessing

Isobutanol Production by Autotrophic Acetogenic Bacteria

Molecular understanding ofEubacterium limosumchemostat methanol metabolism

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