Unveiling the role of novel carbohydrate-binding modules in laminarin interaction of multimodular proteins from marine Bacteroidota during phytoplankton blooms.

Laminarin, a ß(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803T, thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.

Metabolic engineering enables Bacillus licheniformis to grow on the marine polysaccharide ulvan.

BACKGROUND: Marine algae are responsible for half of the global primary production, converting carbon dioxide into organic compounds like carbohydrates. Particularly in eutrophic waters, they can grow into massive algal blooms. This polysaccharide rich biomass represents a cheap and abundant renewable carbon source. In nature, the diverse group of polysaccharides is decomposed by highly specialized microbial catabolic systems. We elucidated the complete degradation pathway of the green algae-specific polysaccharide ulvan in previous studies using a toolbox of enzymes discovered in the marine flavobacterium Formosa agariphila and recombinantly expressed in Escherichia coli. RESULTS: In this study we show that ulvan from algal biomass can be used as feedstock for a biotechnological production strain using recombinantly expressed carbohydrate-active enzymes. We demonstrate that Bacillus licheniformis is able to grow on ulvan-derived xylose-containing oligosaccharides. Comparative growth experiments with different ulvan hydrolysates and physiological proteogenomic analyses indicated that analogues of the F. agariphila ulvan lyase and an unsaturated ß-glucuronylhydrolase are missing in B. licheniformis. We reveal that the heterologous expression of these two marine enzymes in B. licheniformis enables an efficient conversion of the algal polysaccharide ulvan as carbon and energy source. CONCLUSION: Our data demonstrate the physiological capability of the industrially relevant bacterium B. licheniformis to grow on ulvan. We present a metabolic engineering strategy to enable ulvan-based biorefinery processes using this bacterial cell factory. With this study, we provide a stepping stone for the development of future bioprocesses with Bacillus using the abundant marine renewable carbon source ulvan.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Reaching out in anticipation: bacterial membrane extensions represent a permanent investment in polysaccharide sensing and utilization.

Outer membrane extensions are common in many marine bacteria. However, the function of these surface enlargements or extracellular compartments is poorly understood. Using a combined approach of microscopy and subproteome analyses, we therefore examined Pseudoalteromonas distincta ANT/505, an Antarctic polysaccharide degrading gamma-proteobacterium. P. distincta produced outer membrane vesicles (MV) and vesicle chains (VC) on polysaccharide and non-polysaccharide carbon sources during the exponential and stationary growth phase. Surface structures of carbohydrate-grown cells were equipped with increased levels of highly substrate-specific proteins. At the same time, proteins encoded in all other polysaccharide degradation-related genomic regions were also detected in MV and VC samples under all growth conditions, indicating a basal expression. In addition, two alkaline phosphatases were highly abundant under non-limiting phosphate conditions. Surface structures may thus allow rapid sensing and fast responses in nutritionally deprived environments. It may also facilitate efficient carbohydrate processing and reduce loss of substrates and enzymes by diffusion as important adaptions to the aquatic ecosystem.

Biphasic cellular adaptations and ecological implications of Alteromonas macleodii degrading a mixture of algal polysaccharides.

Algal polysaccharides are an important bacterial nutrient source and central component of marine food webs. However, cellular and ecological aspects concerning the bacterial degradation of polysaccharide mixtures, as presumably abundant in natural habitats, are poorly understood. Here, we contextualize marine polysaccharide mixtures and their bacterial utilization in several ways using the model bacterium Alteromonas macleodii 83-1, which can degrade multiple algal polysaccharides and contributes to polysaccharide degradation in the oceans. Transcriptomic, proteomic and exometabolomic profiling revealed cellular adaptations of A. macleodii 83-1 when degrading a mix of laminarin, alginate and pectin. Strain 83-1 exhibited substrate prioritization driven by catabolite repression, with initial laminarin utilization followed by simultaneous alginate/pectin utilization. This biphasic phenotype coincided with pronounced shifts in gene expression, protein abundance and metabolite secretion, mainly involving CAZymes/polysaccharide utilization loci but also other functional traits. Distinct temporal changes in exometabolome composition, including the alginate/pectin-specific secretion of pyrroloquinoline quinone, suggest that substrate-dependent adaptations influence chemical interactions within the community. The ecological relevance of cellular adaptations was underlined by molecular evidence that common marine macroalgae, in particular Saccharina and Fucus, release mixtures of alginate and pectin-like rhamnogalacturonan. Moreover, CAZyme microdiversity and the genomic predisposition towards polysaccharide mixtures among Alteromonas spp. suggest polysaccharide-related traits as an ecophysiological factor, potentially relating to distinct 'carbohydrate utilization types' with different ecological strategies. Considering the substantial primary productivity of algae on global scales, these insights contribute to the understanding of bacteria-algae interactions and the remineralization of chemically diverse polysaccharide pools, a key step in marine carbon cycling.