This disclosure is in the technical field of synthetic biology and metabolic engineering. The disclosure provides engineered viable bacteria having a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans and Glucosylglycerol (O), glycan, and trebalose. The disclosure further provides methods for the production of bioproduct by the viable bacteria and uses thereof. Furthermore, the disclosure is in the technical field of fermentation of metabolically engineered microorganisms producing bioproduct.

The disclosure is in the technical field of synthetic biology and metabolic engineering. The disclosure provides engineered viable bacteria. In particular, the disclosure provides viable bacteria with mutated outer membrane biosynthetic pathway leading to disruption of the pathway, preferably substantially lacking lipopolysaccharide (LPS, endotoxin) within the outer membrane. The disclosure further provides methods of generating viable bacteria and uses thereof. The disclosure also provides compositions and methods for inducing immune responses and for researching and developing therapeutic agents. Furthermore, the disclosure is in the technical field of fermentation of metabolically engineered microorganisms producing bioproduct or metabolite.

The present disclosure relates to novel modular methods for generating a diversity of N-glycans of high mannose, hybrid and complex types. The present disclosure also relates to exemplary arrays of the synthesized N-glycans spotted onto aluminium oxide coated slides. These arrays can be used to detect and analyze binding interactions between the synthesized N-glycans and glycan binding molecules, such as HIV-1 neutralizing antibodies. The present disclosure also relates to methods for identifying agents that bind to various types of molecules on the arrays and to defining the structural elements of the molecules on the arrays that bind to those agents. The arrays and methods provided herein may be used for general epitope identification, drug discovery and as analytical tools. The present disclosure also provides useful glycans and epitope determinants that are useful in detecting, diagnosing, recurrence monitoring and preventing pathological diseases such as HIV.

This disclosure relates to the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure relates to the technical field of fermentation of metabolically engineered microorganisms. This disclosure describes engineered micro-organisms that produce oligosaccharides that are free of KDO-lactose impurities and/or KDO-oligosaccharide impurities.

This disclosure relates to the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure relates to the technical field of fermentation of metabolically engineered microorganisms. This disclosure describes engineered micro-organisms that produce oligosaccharides that are free of KDO-lactose impurities and/or KDO-oligosaccharide impurities.

The present invention describes the use of thio-phosphate in the metabolic engineering of E. coli. Thio-phosphate can be used to increase the metabolic flux in important synthetic pathways to enhance the production of bioproducts. The pathways impacted include the following: fatty acid synthesis, isoprenoid syntheses, Vit K2 synthesis, ribonucleotide synthesis, and the synthesis of phosphoribosyl pyrophosphate (PRPP) derivatives like 5-aminoimidazole-4-carboxamide (AICA riboside), histidine, and tryptophan. Thus, thio-phosphate can be used to assist in the production of these molecules and/or their derivatives. Enhanced production of AICA in Bacillus megaterium is also demonstrated.

The present invention describes the use of thio-phosphate in the metabolic engineering of E. coli. Thio-phosphate can be used to increase the metabolic flux in important synthetic pathways to enhance the production of bioproducts. The pathways impacted include the following: fatty acid synthesis, isoprenoid syntheses, Vit K2 synthesis, ribonucleotide synthesis, and the synthesis of phosphoribosyl pyrophosphate (PRPP) derivatives like 5-aminoimidazole-4-carboxamide (AICA riboside), histidine, and tryptophan. Thus, thio-phosphate can be used to assist in the production of these molecules and/or their derivatives. Enhanced production of AICA in Bacillus megaterium is also demonstrated.

Provided herein are host cells capable of producing hybrid oligosaccharides and polysaccharides, wherein said hybrid oligosaccharides and polysaccharides do not comprise a hexose at the reducing end of their first repeat unit. Also provided herein are hybrid oligosaccharides or polysaccharides and bioconjugates which can be produced by the host cells described herein, wherein said bioconjugates comprise a carrier protein linked to a hybrid oligosaccharide or polysaccharide that does not comprise a hexose at the reducing end of its first repeat unit.

Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry (“MS”), or Ultra Violet (“UV”) detection and/or a combination thereof.

Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry (“MS”), or Ultra Violet (“UV”) detection and/or a combination thereof.