Function Enhancement of a Metabolic Module via Endogenous Promoter Replacement for Pseudomonas sp. JY-Q to Degrade Nicotine in Tobacco Waste Treatment.

Nicotine-degrading Pseudomonas sp. JY-Q is a preferred strain utilized in reconstituted tobacco process for tobacco waste treatment. However, its efficiency of nicotine metabolism still requires to be improved via genomic technology such as promoter engineering based on genomic information. Concerning upstream module of nicotine metabolic pathway, we found that two homologous genes of nicotine dehydrogenase (nicA2 and nox) coexisted in strain JY-Q. However, the transcriptional amount of nox was 20-fold higher than that of nicA2. Thus, the nicA2 expression required improvement. Combinatorial displacement was accomplished for two predicted endogenous promoters, named as PnicA2 and Pnox for nicA2 and nox, respectively. The mutant with Pnox as the promoters for both nicA2 and nox exhibited the best nicotine metabolic capacity which increased by 66% compared to the wild type. These results suggested that endogenous promoter replacement is also feasible for function improvement of metabolic modules and strain enhancement of biodegradation capacity to meet real environment demand.

Gaseous Formaldehyde Degrading by Methylobacterium sp. XJLW.

Formaldehyde is harmful to human beings. It is widely used in chemical industry, medicine, and agriculture and is frequently discharged into the sewage. Microbial metabolism of formaldehyde has attracted increasing attention for its potential application in formaldehyde removal, especially for indoor gaseous formaldehyde degradation. Methylobacterium sp. XJLW capable of degrading formaldehyde was isolated and exhibited a strong activity for liquid formaldehyde degradation. In the present study, the survival rate of XJLW was evaluated under drought, 30 °C, 4 °C, 15 °C, 35 °C, and 40 °C. After 4 days, the average survival rate under 30°C is the greatest (83.97%) among the five temperatures. Whether the temperature was above or below 30°C, the average survival rate decreased significantly. However, the resistance of XJLW to reduced temperatures seemed better than that to increased temperatures. The average survival rate under 15°C and 4°C was 71.1% and 58.67%, while that under 35 °C and 40 °C was 49.47% and 0.1%. Two batches of gaseous formaldehyde treatments were carried out in an analog device with super absorbent polymer (SAP) as the carrier materials of XJLW. The results showed that XJLW could effectively degrade gaseous formaldehyde in the analog device for a long period.

Co-occurrence of functional modules derived from nicotine-degrading gene clusters confers additive effects in Pseudomonas sp. JY-Q.

Pseudomonas sp. JY-Q was isolated from nicotine-rich environment and could degrade and tolerate high-content nicotine. Its specific genetic architecture comprised duplicated homologous nicotine-degrading clusters for different functional modules on the whole pathway. Its adaptive and genomic properties caused our concern whether the duplicated homologous gene clusters confer additive effects on nicotine degradation and result in strain JY-Q strong capability. After deletion of representative genes from duplicated homologous gene clusters of upstream module Nic1, midstream module Spm, and downstream module Nic2, the nicotine degradation efficiency of the wild type and mutant strains were examined. As the first genes of clusters Nic1-1 and Nic1-2, nicA2 and nox are both involved in nicotine degradation, but nox exhibited more contribution to nicotine metabolism due to the higher transcriptional amount of nox than that of nicA2. Likewise, the sub-clusters spm1 and spm2 showed additive effect on nicotine metabolism. As two hpo-like genes of clusters Nic2-1 and Nic2-2, hpo1, and hpo2 also showed additive effect on the nicotine degrading, but hpo1 provided more contribution than hpo2. The third hpo-like gene in cluster NA (nicotinic acid degrading), nicX is not necessary for 2,5-dihydroxypyridine transformation when hpo1 and hpo2 exist. A variety of transposases and integrases observed around Nic1 and Nic2 cluster genes suggests that the duplicated genes could evolve from horizontal gene transfer (HGT)-related dissemination. This study provide an insight into a novel adaptability mechanism of strains in extreme environment such as high nicotine concentration, and potential novel targets to enhance strain synthesis/degradation ability for future applications.

Comparative genomics and transcriptomics insights into the C1 metabolic model of a formaldehyde-degrading strain Methylobacterium sp. XJLW.

A formaldehyde-degrading strain Methylobacterium sp. XJLW was isolated and exhibited a special phenotype for formaldehyde utilization. The accumulation of formic acid in large quantities and lower cell growth was detected when XJLW utilized formaldehyde as the sole carbon source, suggesting XJLW has a potentially novel pathway to transfer formaldehyde to methanol and then enter the serine cycle for C1 metabolism. This mechanism requires exploration via molecular omics. Thus, the complete genome of XJLW was sequenced, and the transcriptome difference was also analyzed based on the RNA-seq data of strain XJLW cultivated with methanol and glucose, respectively. XJLW has a chromosome DNA and a mega-plasmid DNA. Ten percent of genes on chromosome DNA are strain-specific in genus Methylobacterium. Transcriptome analysis results showed that 623 genes were significantly up-regulated and that 207 genes were significantly down-regulated for growth in methanol. Among the up-regulated genes, 90 genes belong to strain-specific regions and are densely distributed in three areas. A specific gene (A3862_27225) annotated as methyltransferase was found ranking in the top 4 of up-regulated genes. This methyltransferase may play a role in the specific C1 metabolism of XJLW. Methylobacterium sp. XJLW should contain a potential methyl transport pathway via the novel methyltransferase, which is different from known pathways. These findings provide the basis for additional possibilities, which improve the formaldehyde-degrading ability of Methylobacterium sp. XJLW.