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.

Enzymatic degradation, antioxidant and rheological properties of a sphingan WL gum from Sphingomonas sp. WG.

A molecular weight (Mw) controllable degradation strategy using the lyase WelR as the efficient tool was established, and the relationship between the Mw and the rheological properties and antioxidant activity of WL gum was systematically investigated. Four different WL samples WL1-WL4 with a gradient Mw change (from 4.70 × 106 to 1.45 × 106 Da) were obtained by controlling the enzymatic reaction conditions. As the Mw decreased, its apparent viscosity, intrinsic viscosity, viscous modulus (G″) and elastic modulus (G') decreased. More interestingly, in contrast to the native WL, the G″ of the degraded WL became higher than G'. Besides, the biodegraded WL samples possessed much higher hydroxyl radicals scavenging activity than the original WL. WL4 with the lowest Mw showed the highest HO radical scavenging activity, about 94.65% at 1 mg/mL. This work provided a useful method to obtain a series of WL samples with controllable Mw and properties, which will broaden the application of sphingans.

[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.