The critical roles of propanethiol oxidoreductase and sulfide-quinone oxidoreductase in the propanethiol catabolism pathway in Pseudomonas putida S-1.

Propanethiol (PT) is a hazardous pollutant that poses risks to both the environment and human well-being. Pseudomonas putida S-1 has been identified as a microorganism capable of utilizing PT as its sole carbon source. However, the metabolic pathway responsible for PT degradation in P. putida S-1 has remained poorly understood, impeding its optimization and practical application. In this study, we investigated the catabolic network involved in PT desulfurization with P. putida S-1 and identified key gene modules crucial to this process. Notably, propanethiol oxidoreductase (PTO) catalyzes the initial degradation of PT, a pivotal step for P. putida S-1's survival on PT. PTO facilitates the oxidation of PT, resulting H2S, H2O2, and propionaldehyde (PA). Catalase-peroxidase catalyzes the conversion of H2O2 to oxygen and water, while PA undergoes gradual conversion to Succinyl-CoA, which is subsequently utilized in the tricarboxylic acid cycle. H2S is digested in a comprehensive desulfurization network where sulfide-quinone oxidoreductase (SQOR) predominantly converts it to sulfane sulfur. The transcriptome analysis suggests that sulfur can be finally converted to sulfite or sulfate and exported out of the cell. The PT degradation capacity of P. putida S-1 was enhanced by increasing the transcription level of PTO and SQOR genes in vivo.IMPORTANCEThis work investigated the PT catabolism pathway in Pseudomonas putida S-1, a microorganism capable of utilizing PT as the sole carbon source. Critical genes that control the initiation of PT degradation were identified and characterized, such as pto and sqor. By increasing the transcription level of pto and sqor genes in vivo, we have successfully enhanced the PT degradation efficiency and growth rate of P. putida S-1. This work does not only reveal a unique PT degradation pathway but also highlights the potential of enhancing the microbial desulfurization process in the bioremediation of thiol-contaminated environment.

Synthetic bacterial consortia enhanced the degradation of mixed priority phthalate ester pollutants.

Dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP) are hazardous chemicals listed as priority pollutants that disrupt endocrine systems. According to available reports, these six priority phthalate esters (PAEs) are considered the most polluting; however, no studies have been conducted on the efficient remediation of these PAEs. We therefore designed and constructed a synthetic bacterial consortium capable of the simultaneous and efficient degradation of six priority PAEs in minimal inorganic salt medium (MSM) and soil. The consortium comprised Glutamicibacter sp. ZJUTW, which demonstrates priority for degrading short-chain PAEs; Cupriavidus sp. LH1, which degrades phthalic acid (PA) and protocatechuic acid (PCA), intermediates of the PAE biodegradation process; and Gordonia sp. GZ-YC7, which efficiently degrades long-chain priority PAEs, including DEHP and DOP. In MSM containing the six mixed PAEs (250 mg/L each), the ZJUTW + YC + LH1 consortium completely degraded the four short-chain PAEs within 48 h, and DEHP (100%) and DOP (62.5%) within 72 h. In soil containing the six mixed PAEs (DMP, DEP, BBP, and DOP, 400 mg/kg each; DBP and DEHP, 500 mg/kg, each), the ZJUTW + YC + LH1 consortium completely degraded DMP, DEP, BBP, and DBP within 6 days, and 70.84% of DEHP and 66.24% of DOP within 2 weeks. The consortium efficiently degraded the six mixed PAEs in both MSM and soil. We thus believe that this synthetic microbial consortium is a strong candidate for the bioremediation of environments contaminated with mixed PAE pollutants.

Rational engineering of BaLal_16 from a novel Bacillus amyloliquefaciens strain to improve catalytic performance.

L-amino acid ligases (Lals) are promising biocatalysts for the synthesis of dipeptides with special biological properties. However, their poor (or broad) substrate specificity limits their industrial applications. To address this problem, a molecular engineering method for Lals was developed to enhance their catalytic performance. Based on substrate channeling, entrances to the active site for different substrates were identified, and the "gate" located around the active site pocket, which plays an essential role in substrate recognition, was then engineered to facilitate acceptance of L-Gln. Two mutants (L110Y and N108F/L110Y) were discovered to display significantly increased catalytic activity toward L-Ala and L-Gln in the biosynthesis of Ala-Gln. The catalytic efficiency (kcat/ Km) of the L110Y and N108F/L110Y mutants was improved by 2.64-fold and 4.06-fold, respectively, compared with that of the wild type. N108F/L110Y was then further applied for batch production of Ala-Gln, which showed that the released Pi yield was 694.47 µM, which was an increase of approximately 21.4 %, and the yield of Ala-Gln was approximately 2.59 mM-1 L-1 mg-1. Collectively, these findings suggest the potential practical application of this method in the rational design of Lals for increased catalytic performance.

l-amino acid ligase: A promising alternative for the biosynthesis of l-dipeptides.

Given their special action mechanisms and structural simplicity, L-amino acid ligases (Lals) are considered to be desirable tools for the catalytic biosynthesis of dipeptides. Ywf E (BacD) was the first Lal identified and was shown to be involved in the biosynthesis of a potent antibacterial, bacilysin, since then, various novel Lals have been discovered. Each Lal has different substrate spectra and is capable of synthesizing different dipeptides. Owning to their great potentials for producing bioactive dipeptides of industrial importance, in this review, recent developments of Lals are discussed, including their structures, action mechanisms, applications and the advantages and disadvantages of different Lals. In addition, protein engineering of Lals to improve their substrate specificity and catalytic performance is also discussed.