Identification of the Key Enzymes in WL Gum Biosynthesis and Critical Composition in Viscosity Control.

As an important microbial exopolysaccharide, the sphingan WL gum could be widely used in petroleum, food, and many other fields. However, its lower production is still limiting its wider application. Therefore, to gain insights into the bottlenecks of WL gum production by identifying the key enzymes in the WL gum biosynthesis pathway, more than 20 genes were over-expressed in Sphingomonas sp. WG and their effects on WL gum production and structure were investigated. Compared to the control strain, the WL gum production of welB over-expression strain was increased by 19.0 and 21.0% at 36 and 84 h, respectively. The WL gum production of both atrB and atrD over-expression strains reached 47 g/L, which was approximately 34.5% higher than that of the control strain at 36 h. Therefore, WelB, AtrB, and AtrD may be the key enzymes in WL production. Interestingly, the broth viscosity of most over-expression strains decreased, especially the welJ over-expression strain whose viscosity decreased by 99.3% at 84 h. Polysaccharides' structural features were investigated to find the critical components in viscosity control. The uronic acid content and total sugar content was affected by only a few genes, therefore, uronic acid and total sugar content may be not the key composition. In comparison, the acetyl degrees were enhanced by over-expression of most genes, which meant that acetyl content may be the critical factor and negatively correlated with the apparent viscosity of WL gum. This work provides useful information on the understanding of the bottlenecks of WL gum biosynthesis and will be helpful for the construction of high WL gum-yielding strains and rheological property controlling in different industries.

[Development of an LB cloning system and its application in expression of fusion genes in Sphingomonas sp. WG].

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.

Hybrid Histidine Kinase WelA of Sphingomonas sp. WG Contributes to WL Gum Biosynthesis and Motility.

Sphingomonas sp. WG produced WL gum with commercial utility potential in many industries. A hybrid sensor histidine kinase/response regulator WelA was identified to regulate the WL gum biosynthesis, and its function was evaluated by gene deletion strategy. The WL gum production and broth viscosity of mutant ΔwelA was only 44% and 0.6% of wild type strain at 72 h. The transcriptomic analysis of differentially expressed genes showed that WelA was mapped to CckA; ChpT, and CtrA in the CckA-ChpT-CtrA pathway was up-regulated. One phosphodiesterase was up-regulated by CtrA, and the intracellular c-di-GMP was decreased. Most genes involved in WL gum biosynthesis pathway was not significantly changed in ΔwelA except the up-regulated atrB and atrD and the down-regulated pmm. Furthermore, the up-regulated regulators ctrA, flaEY, flbD, and flaF may participate in the regulation of flagellar biogenesis and influenced motility. These results suggested that CckA-ChpT-CtrA pathway and c-di-GMP were involved in WL gum biosynthesis regulation. This work provides useful information on the understanding of molecular mechanisms underlying WL gum biosynthesis regulation.

Towards a more labor-saving way in microbial ammonium oxidation: A review on complete ammonia oxidization (comammox).

In the Anthropocene, nitrogen pollution is becoming an increasing challenge for both mankind and the Earth system. Microbial nitrogen cycling begins with aerobic nitrification, which is also the key rate-limiting step. For over a century, it has been accepted that nitrification occurs sequentially involving ammonia oxidation, which produces nitrite followed by nitrite oxidation, generating nitrate. This perception was changed by the discovery of comammox Nitrospira bacteria and their metabolic pathway. In addition, this also provided us with new knowledge concerning the complex nitrogen cycle network. In the comammox process, ammonia can be completely oxidized to nitrate in one cell via the subsequent activity of the enzyme complexes, ammonia monooxygenase, hydroxylamine dehydrogenase, and nitrite oxidodreductase. Over the past five years, research on comammox made great progress. However, there still exist a lot of questions, including how much does comammox contribute to nitrification? How large is the diversity and are there new strains to be discovered? Do comammox bacteria produce the greenhouse gas N2O, and how or to which extent may they contribute to global climate change? The above four aspects are of great significance on the farmland nitrogen management, aquatic environment restoration, and mitigation of global climate change. As large number of comammox bacteria and pathways have been detected in various terrestrial and aquatic ecosystems, indicating that the comammox process may exert an important role in the global nitrogen cycle.

The Function of ß-1,4-Glucuronosyltransferase WelK in the Sphingan WL Gum Biosynthesis Process in Marine Sphingomonas sp. WG.

The marine-derived polysaccharide WL gum produced by Sphingomonas sp. WG showed commercial utility potential in ink, food, and oil industries. A ß-1,4-glucuronosyltransferase WelK was predicted to catalyze the transfer of glucuronic acid from UDP-glucuronic acid to glucosyl-α-pyrophosphorylpolyprenol intermediate in the WL gum biosynthesis process. Its function was evaluated by bioinformatical analysis, gene knocking out, and overexpressing strategies. Compared to the wild strain, the WL gum production and broth viscosity of the mutant ∆welK were decreased by 71.5% and 99.2% when cultured for 48 h. The gene disruption led to the failure of product preparation. Homologous expression of welK in the native organism can effectively improve WL gum production. When glucose concentration was 6.7%, the WL gum production by the welK-overexpressing strain cultured for 60 h and 84 h reached 32.65 and 43.13 g/L, 134.1%, and 114% of the wild strain. The polysaccharide composition and qRT-PCR analysis showed that the glucuronic acid content was closely related to the expression level of welK. Thus, WelK was proved to play a critical role in the WL gum synthesis and will be an attractive target for metabolic engineering. Our experiment provided a genetic manipulation method for the functional characterization of genes in Sphingomonas sp. WG.