Characterization of a λ-Carrageenase Mutant with the Generation of Long-Chain λ-Neocarrageenan Oligosaccharides.

λ-carrageenan oligosaccharides can be widely applied in the food, pharmaceutical, medicine and cosmetic industries due to their abundant bioactivities, and they are important products for the high-value utilization of λ-carrageenan. However, oligosaccharides with different degrees of polymerization have different properties, and the final products of λ-carrageenase reported so far are mainly λ-neocarrabiose, λ-neocarratetraose and λ-neocarrahexaose without longer-chain oligosaccharides. Further research is consequently required. Herein, a mutant λ-carrageenase was constructed by deleting the pyrroloquinoline quinone-like domain of OUC-CglA derived from Maribacter vaceletii. Interestingly, it was discovered that the majority of final products of the mutant OUC-CglA-DPQQ were long-chain oligosaccharides with a polymerization degree of 10-20, which underwent significant changes compared to that of OUC-CglA. Additionally, without the pyrroloquinoline quinone-like domain, fewer inclusion bodies were produced throughout the expression process, and the yield of the λ-carrageenase increased about five-fold. However, compared to its parental enzyme, significant changes were made to its enzymatic properties. Its optimal temperature and pH were 15 °C and pH 7.0, and its specific activity was 51.59 U/mg. The stability of the enzyme decreased. Thus, it was found that the deleting domain was related to the formation of inclusion bodies, the stability of the enzyme, the activity of the enzyme and the composition of the products.

Metabolic engineering of oleaginous yeast in the lipogenic phase enhances production of nervonic acid.

Insufficient biosynthesis efficiency during the lipogenic phase can be a major obstacle to engineering oleaginous yeasts to overproduce very long-chain fatty acids (VLCFAs). Taking nervonic acid (NA, C24:1) as an example, we overcame the bottleneck to overproduce NA in an engineered Rhodosporidium toruloides by improving the biosynthesis of VLCFAs during the lipogenic phase. First, evaluating the catalytic preferences of three plant-derived ketoacyl-CoA synthases (KCSs) rationally guided reconstructing an efficient NA biosynthetic pathway in R. toruloides. More importantly, a genome-wide transcriptional analysis endowed clues to strengthen the fatty acid elongation (FAE) module and identify/use lipogenic phase-activated promoter, collectively addressing the stagnation of NA accumulation during the lipogenic phase. The best-designed strain exhibited a high NA content (as the major component in total fatty acid [TFA], 46.3%) and produced a titer of 44.2 g/L in a 5 L bioreactor. The strategy developed here provides an engineering framework to establish the microbial process of producing valuable VLCFAs in oleaginous yeasts.

Biochemical Characterization of a Cold-Adapted λ-Carrageenase OUC-CglA from Maribacter vaceletii: An Efficient Tool for λ-Carrageenan Degradation.

λ-Carrageenase with high activity is an effective and environmentally friendly tool enzyme for the preparation of λ-carrageenan oligosaccharides with various biological activities. Herein, a novel GH150 (glycoside hydrolases family 150) λ-carrageenase OUC-CglA from Maribacter vaceletii was heterologously expressed, purified, and characterized. The recombinant OUC-CglA performs strict selectivity toward λ-carrageenan with a specific activity of 418.7 U/mg under its optimal reaction conditions of 20 °C and pH 7.0. Additionally, OUC-CglA is a typical cold-adapted λ-carrageenase because it unfolds 90% and 63% of its maximum activity at 15 and 10 °C, respectively. The hydrolysis process suggests that OUC-CglA is an endotype λ-carrageenase with the final products consisting of λ-neocarrabiose, λ-neocarratetraose, λ-neocarrahexaose, and other long-chain λ-neocarrageenan oligosaccharides. As a result, high activity, cold-adaptation, and principal products of OUC-CglA make it a potential biocatalyst for the effective preparation of λ-carrageenan oligosaccharides.

Enzymatic Verification and Comparative Analysis of Carrageenan Metabolism Pathways in Marine Bacterium Flavobacterium algicola.

Marine bacteria usually contain polysaccharide utilization loci (PUL) for metabolizing red algae polysaccharides. They are of great significance in the carbon cycle of the marine ecosystem, as well as in supporting marine heterotrophic bacterial growth. Here, we described the whole κ-carrageenan (KC), ι-carrageenan (IC), and partial λ-carrageenan (LC) catabolic pathways in a marine Gram-negative bacterium, Flavobacterium algicola, which is involved carrageenan polysaccharide hydrolases, oligosaccharide sulfatases, oligosaccharide glycosidases, and the 3,6-anhydro-d-galactose (d-AHG) utilization-related enzymes harbored in the carrageenan-specific PUL. In the pathways, the KC and IC were hydrolyzed into 4-sugar-unit oligomers by specific glycoside hydrolases. Then, the multifunctional G4S sulfatases would remove their nonreducing ends' G4S sulfate groups, while the ι-neocarratetrose (Nι4) product would further lose the nonreducing end of its DA2S group. Furthermore, the neocarrageenan oligosaccharides (NCOSs) with no G4S and DA2S groups in their nonreducing ends would completely be decomposed into d-Gal and d-AHG. Finally, the released d-AHG would enter the cytoplasmic four-step enzymatic process, and an l-rhamnose-H+ transporter (RhaT) was preliminarily verified for the function for transportation of d-AHG. Moreover, comparative analysis with the reported carrageenan metabolism pathways further implied the diversity of microbial systems for utilizing the red algae carrageenan. IMPORTANCE Carrageenan is the main polysaccharide of red macroalgae and is composed of d-AHG and d-Gal. The carrageenan PUL (CarPUL)-encoded enzymes exist in many marine bacteria for decomposing carrageenan to provide self-growth. Here, the related enzymes in Flavobacterium algicola for metabolizing carrageenan were characterized for describing the catabolic pathways, notably, although the specific polysaccharide hydrolases existed that were like previous studies. A multifunctional G4S sulfatase also existed, which was devoted to the removal of G4S or G2S sulfate groups from three kinds of NCOSs. Additionally, the transformation of three types of carrageenans into two monomers, d-Gal and d-AHG, occurred outside the cell with no periplasmic reactions that existed in previously reported pathways. These results help to clarify the diversity of marine bacteria using macroalgae polysaccharides.