The document provides methods for biosynthesizing isobutene using one or more isolated enzymes such as one or more of a hydratase such as an enzyme classified under EC 4.2.1.- and a decarboxylating thioesterase, or using recombinant host cells expressing one or more such enzymes.

The present invention provides several non-naturally occurring sulfotransferase enzymes that have been engineered to react with aryl sulfate compounds as sulfo group donors, instead of the natural substrate 3-phosphoadenosine 5-phosphosulfate (PAPS), and with heparosan-based polysaccharides, particularly heparan sulfate, as sulfo group acceptors. Each of the engineered sulfotransferase enzymes have a biological activity characterized by the position within the heparosan-based polysaccharide that receives the sulfo group, including glucosaminyl N-sulfotransferase activity, hexuronyl 2-O sulfotransferase activity, glucosaminyl 6-O sulfotransferase activity, or glucosaminyl 3-O sulfotransferase activity. Methods of using the engineered sulfotransferases to produce sulfated heparosan-based polysaccharides, including polysaccharides having anticoagulant activity, are also provided.

The present invention provides methods and materials useful delivering liquids, including liquids comprising nucleic acid molecules into cells. In particular, the present invention provides methods for delivering saline solution, exogenous compositions, and isolated vectors to kidney cells, using the renal vein as a guide and under hydrodynamic pressure. The delivery methods and materials herein are useful to research, prognose, ameliorate symptoms of kidney injury, and treat kidney pathologies.

The present disclosure relates to synthesis of heparin, which may be bioequivalent to porcine USP Heparin Sodium. The synthesis may involve three intermediates starting from heparosan.

The invention features compositions and methods for site-specific modification of proteins by incorporation of an aldehyde tag. Enzymatic modification at a sulfatase motif of the aldehyde tag through action of a formylglycine generating enzyme (FGE) generates a formylglycine (FGly) residue. The aldehyde moiety of FGly residue can be exploited as a chemical handle for site-specific attachment of a moiety of interest to a polypeptide.

The present invention provides a 2-OST mutant exhibiting a high activity. Specifically, the present invention provides a 2-O-sulfation enzyme mutant, having a substitution of a leucine residue at position 321 with a basic amino acid residue in any one amino acid sequence of: (a) the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 2; (c) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2; (d) the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; (e) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; (f) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; and having a 2-O-sulfate transfer activity.

Methods and systems for synthesizing biological products within biomimetic condensates that enhance the concentration of enzymes, substrates, co-factors, and other molecules involved in the synthesis. The one or more enzymes involved in catalysis can be engineered to comprise low-complexity amino acid sequences or phase separation domains that can be controlled to drive reversible liquid-liquid phase separation, wherein the resulting biomimetic condensates comprise a traversable phase boundary between their dense internal portion and the less-crowded aqueous composition within which the condensates are maintained. Alternatively, biomimetic condensates can be first formed within the aqueous composition from affinity-tagged scaffold proteins that do not take part in catalysis, but are capable of recruiting into the condensate catalytic enzymes fused to an affinity tag partner. One or more enzymes can comprise or be recruited inside of the biomimetic condensate to generate a wide variety of desired chemical products.

The invention features compositions and methods for site-specific modification of proteins by incorporation of an aldehyde tag. Enzymatic modification at a sulfatase motif of the aldehyde tag through action of a formylglycine generating enzyme (FGE) generates a formylglycine (FGly) residue. The aldehyde moiety of FGly residue can be exploited as a chemical handle for site-specific attachment of a moiety of interest to a polypeptide.

The invention provides a method for producing sulfated polysaccharides by reacting a PAPS production/regeneration system utilizing the metabolic activity of a microorganism or a treated matter thereof with a microorganism expressing a sulfation enzyme or a treated matter or extract thereof upon mixing of inexpensive raw materials such as magnesium sulfate. The invention also provides a method for producing PAPS from inexpensive raw materials. The methods involve preparing a transformant (a) of a bacterium of the genus Corynebacterium, which contains a gene encoding an ATP sulfurylase and a gene encoding an APS kinase, which are expressible, and in which a cell plasma membrane of the transformant (a) is substance-permeable, or a treated matter of the transformant (a), and conducting a reaction for producing PAPS by using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant (a) or the treated matter thereof.

The invention provides a method for producing a sulfated polysaccharide by generating a sulfated polysaccharide by incorporating, in a reaction solution in the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparosan, a transformant (a) of a bacterium of the genus Corynebacterium, comprising at least a gene encoding an ATP sulfurylase and a gene encoding an APS kinase, and at least one selected from a transformant (b) of a microorganism belonging to prokaryotes, comprising at least a gene encoding a C5-epimerase, a transformant (c) of a microorganism belonging to prokaryotes, comprising at least a gene encoding a 2-O-sulfotransferase, a transformant (d) of a microorganism belonging to prokaryotes, comprising at least a gene encoding a 6-O-sulfotransferase, and a transformant (e) of a microorganism belonging to prokaryotes, comprising at least a gene encoding a 3-O-sulfotransferase.