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Biotechnology For Biofuels Jan 2021Isobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms....
BACKGROUND
Isobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms. Shimwellia blattae (p424IbPSO) is a robust strain resistant to high concentration of isobutanol that can achieve a high production rate of this alcohol. Nevertheless, this strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. Isobutanol production under anaerobic conditions leads to a depletion of NADPH, which is necessary for two enzymes in the metabolic pathway. In this work, two independent approaches have been studied to mitigate the co-substrates imbalance: (i) using a NADH-dependent alcohol dehydrogenase to reduce the NADPH dependence of the pathway and (ii) using a transhydrogenase to increase NADPH level.
RESULTS
The addition of the NADH-dependent alcohol dehydrogenase from Lactococcus lactis (AdhA) to S. blattae (p424IbPSO) resulted in a 19.3% higher isobutanol production. The recombinant strain S. blattae (p424IbPSO, pIZpntAB) harboring the PntAB transhydrogenase produced 39.0% more isobutanol than the original strain, reaching 5.98 g L of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol.
CONCLUSIONS
The isobutanol biosynthesis pathway in S. blattae (p424IbPSO) uses the endogenous NADPH-dependent alcohol dehydrogenase YqhD to complete the pathway. The addition of NADH-dependent AdhA leads to a reduction in the consumption of NADPH that is a bottleneck of the pathway. The higher consumption of NADH by AdhA reduces the availability of NADH required for the transformation of pyruvate into lactic acid and ethanol. On the other hand, the expression of PntAB from E. coli increases the availability of NADPH for IlvC and YqhD and at the same time reduces the availability of NADH and thus, the production of lactic acid and ethanol. In this work it is shown how the expression of AdhA and PntAB enzymes in Shimwellia blattae increases yield from 11.9% to 14.4% and 16.4%, respectively.
PubMed: 33407735
DOI: 10.1186/s13068-020-01862-1 -
Protein Expression and Purification Jan 2021The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the...
The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.
Topics: Bacillus megaterium; Bacterial Proteins; Binding Sites; Biodegradation, Environmental; Cloning, Molecular; Enterobacteriaceae; Escherichia coli; Gene Expression; Genetic Vectors; Halogens; Iron-Sulfur Proteins; Kinetics; Models, Molecular; Nucleoside Q; Oxidoreductases; Phyllobacteriaceae; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Streptococcus thermophilus; Vibrio; Vitamin B 12
PubMed: 32871253
DOI: 10.1016/j.pep.2020.105743 -
Enzyme and Microbial Technology Mar 2024Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise...
Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl, NaS and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H-oxidizing C. necator H16, a novel host for the expression of RDases.
Topics: Escherichia coli; Bacteria; Methylene Chloride; Ascorbic Acid; Biodegradation, Environmental
PubMed: 38147780
DOI: 10.1016/j.enzmictec.2023.110390