Enhanced In Vitro Cascade Catalysis of Glycerol into Pyruvate and Acetoin by Integration with Dihydroxy Acid Dehydratase from Paralcaligenes ureilyticus

Recently, an in vitro enzymatic cascade was constructed to transform glycerol into the high-value platform chemical pyruvate. However, the low activity of dihydroxy acid dehydratase from Sulfolobus solfataricus (SsDHAD) limited the efficiency. In this study, the enzymatic reduction of pyruvate catalyzed by d-lactate dehydrogenase from Pseudomonas aeruginosa PAO1 was used to assay the activities of dihydroxy acid dehydratases. Dihydroxy acid dehydratase from Paralcaligenes ureilyticus (PuDHT) was identified as the most efficient candidate for glycerate dehydration. After the optimization of the catalytic temperature for the enzymatic cascade, comprising alditol oxidase from Streptomyces coelicolor A3, PuDHT, and catalase from Aspergillus niger, 20.50 ± 0.27 mM of glycerol was consumed in 4 h to produce 18.95 ± 0.97 mM of pyruvate with a productivity 12.15-fold higher than the previous report using SsDHAD. The enzymatic cascade was further coupled with the pyruvate decarboxylase from Zymomonas mobile for the production of another platform compound, acetoin. Acetoin at a concentration of 8.52 ± 0.12 mM was produced from 21.62 ± 0.19 mM of glycerol with a productivity of 1.42 ± 0.02 mM h−1.

Download Full-text

Engineering of a thermophilic dihydroxy-acid dehydratase to enhance its dehydration ability on glycerate to pyruvate and its application in in vitro synthetic enzymatic biosystems

10.22541/au.162601310.07045038/v1 ◽

2021 ◽

Author(s):

Juan Wang ◽

Ge Qu ◽

Leipeng Xie ◽

Chao Gao ◽

Yingying Jiang ◽

...

Keyword(s):

Protein Engineering ◽

Binding Pocket ◽

Saturation Mutagenesis ◽

Triple Mutant ◽

Dihydroxy Acid ◽

Acid Dehydratase ◽

High Throughput Method ◽

Low Activity ◽

Formed Hydrogen

The low activity of dihydroxy-acid dehydratase (DHAD) on dehydration of glycerate to pyruvate hampers its applications in the biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydratation activity. A novel high-throughput method was established. A triple-mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunk substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-times of the reaction rate than that of wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes.

Download Full-text

ParB of Pseudomonas aeruginosa: Interactions with Its Partner ParA and Its Target parS and Specific Effects on Bacterial Growth

Journal of Bacteriology ◽

10.1128/jb.186.20.6983-6998.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6983-6998 ◽

Cited By ~ 68

Author(s):

Aneta A. Bartosik ◽

Krzysztof Lasocki ◽

Jolanta Mierzejewska ◽

Christopher M. Thomas ◽

Grazyna Jagura-Burdzy

Keyword(s):

Pseudomonas Aeruginosa ◽

Streptomyces Coelicolor ◽

Consensus Sequence ◽

Terminal Part ◽

Dimerization Domain ◽

Interaction Domain ◽

C Terminus ◽

Cell Components

ABSTRACT The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

Download Full-text

Sulfane Sulfur Regulates LasR-Mediated Quorum Sensing and Virulence in Pseudomonas aeruginosa PAO1

Antioxidants ◽

10.3390/antiox10091498 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1498

Author(s):

Guanhua Xuan ◽

Chuanjuan Lü ◽

Huangwei Xu ◽

Kai Li ◽

Huaiwei Liu ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Hydrogen Sulfide ◽

Quorum Sensing ◽

Sulfide Oxidation ◽

Pseudomonas Aeruginosa Pao1 ◽

Sulfane Sulfur ◽

Target Dna ◽

Production Of Hydrogen

Sulfane sulfur, such as inorganic and organic polysulfide (HSn− and RSn−, n > 2), is a common cellular component, produced either from hydrogen sulfide oxidation or cysteine metabolism. In Pseudomonas aeruginosa PAO1, LasR is a quorum sensing master regulator. After binding its autoinducer, LasR binds to its target DNA to activate the transcription of a suite of genes, including virulence factors. Herein, we report that the production of hydrogen sulfide and sulfane sulfur were positively correlated in P. aeruginosa PAO1, and sulfane sulfur was able to modify LasR, which generated Cys188 persulfide and trisulfide and produced a pentasulfur link between Cys201 and Cys203. The modifications did not affect LasR binding to its target DNA site, but made it several-fold more effective than unmodified LasR in activating transcription in both in vitro and in vivo assays. On the contrary, H2O2 inactivates LasR via producing a disulfide bond between Cys201 and Cys203. P. aeruginosa PAO1 had a high cellular sulfane sulfur and high LasR activity in the mid log phase and early stationary phase, but a low sulfane sulfur and low LasR activity in the declination phase. Thus, sulfane sulfur is a new signaling factor in the bacterium, adding another level of control over LasR-mediated quorum sensing and turning down the activity in old cells.

Download Full-text