Engineering bacterial biomanufacturing: characterization and manipulation of Sphingomonas sp. LM7 extracellular polymers.

Biologically produced materials are an attractive alternative to traditional materials such as metals and plastics and offer improved functionalities such as better biodegradability and biocompatibility. Polysaccharides are an example of biologically produced materials that can have a range of chemical and physical properties including high stiffness to weight ratios and thermal stability. Polysaccharides synthesized by bacteria can come with many advantages such as being non-toxic and are mechanically robust relative to proteins and lipids, which are also secreted by bacteria to generate a biofilm. Biomanufacturing offers benefits compared to traditional manufacturing including low resource investment and equipment requirements, providing an alternative to sourcing fossil fuel byproducts, and relatively low temperatures needed for production. However, many biologically produced materials require complex and lengthy purification processes before use. This paper (1) identifies the material properties of a novel polysaccharide, dubbed promonan, isolated from the extracellular polymeric substances of Sphingomonas sp. LM7; (2) demonstrates that these properties can be manipulated to suit specific applications; and (3) presents two alternative methods of processing to shorten purification time by more than 50% while maintaining comparable material properties.

An exopolysaccharide pathway from a freshwater Sphingomonas isolate.

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties. IMPORTANCE: Bacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced by Sphingomonas sp. LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering.

Engineering Bacterial Biomanufacturing: Characterization and Manipulation of Sphingomonas sp. LM7 Extracellular Polymers.

Biologically produced materials are an attractive alternative to traditional materials such as metals and plastics and offer improved functionalities such as better biodegradability and biocompatibility. Polysaccharides are an example of a biologically produced materials that can have a range of chemical and physical properties including high stiffness to weight ratios and thermal stability. Biomanufactured bacterial polysaccharides can come with many advantages such as being non-toxic and are mechanically robust relative to proteins and lipids, which are also secreted by bacteria to generate a biofilm. One major goal in biomanufacturing is to produce quality material quickly and cost-effectively. Biomanufacturing offers additional benefits compared to traditional manufacturing including low resource investment and equipment requirements, providing an alternative to sourcing fossil fuel byproducts, and relatively low temperatures needed for production. However, many biologically produced materials require complex and lengthy purification processes before use. This paper 1) identifies the material properties of a novel polysaccharide, dubbed promonan, isolated from the extracellular polymeric substances of Sphingomonas sp. LM7; 2) demonstrates that these properties can be manipulated to suit specific applications; and 3) presents two alternative methods of processing to shorten purification time by more than 50% while maintaining comparable material.

A novel exopolysaccharide pathway from a freshwater Sphingomonas isolate.

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. To better understand the breadth of polysaccharide production in nature we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming that LM7 assembles a novel wzy/wzx-dependent polysaccharide. We extracted EPS from LM7 cultures and showed that it contains a linear chain of 3- and 4- linked glucose, galactose, and glucuronic acid residues. Finally, we found that the EPS pathway we identified diverges from those of adhesive polysaccharides such as the holdfast that are conserved in higher Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering of polysaccharides with valuable properties.