Difference between revisions of "Part:BBa K5246043"

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Caulobacter crescentus CB2/CB2A HfsE-HfsJ-HfsG-HfsH-HfsK-HfsL Polysaccharide tetrad assembly

Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. baltica - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.

This is the complete holdfast tetrad assembly system. Parts of this composite can be found:BBa_K5246044 and BBa_K5246045.

This part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

Biology and Usage

Biology

Caulobacter crescentus is a common freshwater gram-negative oligotrophic bacterium of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, C. crescentus daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure called a holdfast, allowing them to adhere to surfaces and perform cell division. [1][2]

Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface[3][4]. The C. crescentus holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in Escherichia coli. The holdfast synthesis (hfs) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway. [4][5][6] The synthesis of holdfast polysaccharides (Fig.1) occurs through a mechanism analogous to the Wzx/Wzy-dependent group I capsular polysaccharide biosynthesis pathway observed in Escherichia coli. The process is initiated in the cytoplasm by the glycosyltransferase (1) HfsE, which transfers an activated glucose-phosphate from UDP to an undecaprenyl-phosphate (Und-P) lipid carrier (1) [7]. Subsequent monosaccharide residues are added to the lipid carrier to form a repeating unit by the action of three glycosyltransferases: (2) HfsJ (adding N-mannosamine uronic acid or D-xylose), (3) hfsG (adds N-acetylglucosamine) and (4) HfsL (most likely adding another N-acetylglucosamine) [8]. Then some of the N-acetyl-D-glucosamine within these repeat units undergoes enzymatic modification through the activity of the deacetylases (5) HfsH and HfsK, which “incorporates” into the tetrad of another saccharide - D-glucosamine [9]. The completed repeat of four monomers is then flipped over the inner membrane to the periplasm by flippase HfsF (6) [8]. In the periplasm, the repeat unit is transferred to copolymerases HfsC and HfsI (7), which assemble holdfast into a mature polysaccharide [10]. Subsequently, following the polymerization, holdfast saccharides are exported through a multi-protein export channel made of HfsB, HfsA, and HfsD (8-10) [11]. After excretion, holdfast polymer is relocated to the anchoring Hfa group of proteins (11), where they function by holding the mature polysaccharide on the cell's surface of, e.g. C. crescentus or H. baltica , and securing it to the surface [8].

Usage

Genes from this composite part are responsible for tetrasaccharide assembly in the holdfast biosynthesis pathway. HfsE (BBa_K5246005 ) transfers an activated glucose-phosphate from UDP to an undecaprenyl-phosphate (Und-P) lipid carrier, HfsJ (BBa_K5246010 ) adds N-mannosamine uronic acid. then HfsG (BBa_K5246007 ) and HfsL (BBa_K5246012 ) joins two N-acetyl-D-glucosamines (HfsG transfers to mannosaminuronic acid, HfsL to another N-acetyl-D-glucosamine) then last N-acetyl-D-glucosamine is deacetylated by HfsH and HfsK (BBa_K5246008 ) (BBa_K5246011 )

In the end, we have a glycolipid - a lipid carrier undecaprenyl phosphate (UndP) with an oligosaccharide (glucose--mannosaminuronic acid--N-acetyl-D-glucosamine--D-glucosamine) attached to it.

Sequence and Features

Experimental characterization