Provided is an SgRNA combination, comprising an SgRNA specifically targeting the GGTA1 gene, an SgRNA specifically targeting the CMAH gene and an SgRNA specifically targeting the β4GalNT2 gene. Also provided is a CRISPR/Cas9 vector combination, comprising a GGTA1-CRISPR/Cas9 vector, a CMAH-CRISPR/Cas9 vector and a β4GalNT2-CRISPR/Cas9 vector. Also provided is an applicaton of the CRISPR/Cas9 vector combination in knocking out the GGTA1 gene, the CMAH gene and the β4GalNT2 gene. The knockout rates of the three genes with the specifically targeted SgRNA sequences are respectively 56%, 63%, and 41%. A three genes knockoutpig can be obtained, wherein the three genes related to immune rejectionare knocked out, and heart valves of said pig can be acquired.

Described herein are methods and genetically engineered fungal cells useful for producing target molecules containing mammalian-like complex N-glycans or containing intermediates in a mammalian glycosylation pathway.

The current inventive technology includes novel systems, methods and compositions for the in vivo production, modification and isolation of terpene compounds from Cannabis or other plants. In particular, the invention provides systems and methods for high level in vivo biosynthesis of water-soluble terpene compounds. In one preferred embodiment, the present invention generally relates to the conversion of Cannabis sativa or hemp derived terpenes into water-soluble terpene compounds, such as terpene glycosides, glycosylated-xylosylated terpenes, and an acetylated glycoside terpenes. In one preferred aspect, the invention may include system for producing water-soluble terpene compounds in yeast and tobacco cell suspension cultures, as well as an in planta system using transgenic Cannabis or hemp plants.

In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes capable of catalyzing the transfer of a monosaccharide moiety from a NDP-sugar to the 3′ carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a “1-3 UGT.” In other aspects, the invention provides polynucleotides encoding the 1-3 UGT, and host cells comprising the same. In still other aspects, the invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzyme and host cells of this disclosure.

The present invention concerns genetically modified Mycoplasma bacteria. Also intended are methods of generating attenuated Mycoplasma bacteria and their use to produce heterologous gene products. Further intended are pharmaceutical compositions comprising the attenuated Mycoplasma bacteria described herein.

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a method for the production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end by a cell as well as the purification of the di- or oligosaccharide from the cultivation. Furthermore, the disclosure provides a cell metabolically engineered for production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end.

The present disclosure relates a polypeptide having UGT activity, which polypeptide comprises an amino acid sequence which, when aligned with a polypeptide having UGT activity comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid corresponding to any of amino acids at positions 35, 189, 280, 284, 285, 334 or 373,
said positions being defined with reference to SEQ ID NO: 2 and wherein the polypeptide has one or more modified properties as compared with a reference polypeptide having UGT activity. A polypeptide according to the disclosure may be used in a recombinant cell for the production of steviol or a steviol glycoside.

This disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. This disclosure provides a method for the production of a mixture of at least two different oligosaccharides by a cell as well as the purification of at least one of the oligosaccharides from the cultivation. In addition, this disclosure provides a method for the production of a mixture of at least two different oligosaccharides by a metabolically engineered cell as well as the purification of at least one of the oligosaccharides from the cultivation.

Methods for recombinant production of steviol glycoside and compositions containing steviol glycosides are provided by this invention.

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a cell metabolically engineered for production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R). Furthermore, the disclosure provides a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R by a cell as well as the purification of the alpha-1,3 glycosylated form Fuc-a1,2-Gal-R from the cultivation.