Compositions and methods for making (R)-reticuline and precursors thereof
Inventors
FACCHINI, Peter James • Farrow, Scott Cameron • Beaudoin, Guillaume Arthur Welch
Assignees
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Publication Number
US-11613770-B2
Publication Date
2023-03-28
Expiration Date
Abstract
Methods that may be used for the manufacture of the chemical compound (R)-Reticuline and synthesis precursors thereof. Compositions useful for the synthesis (R)-Reticuline and synthesis precursors are also provided.
Core Innovation
The invention relates to methods for preparing (R)-Reticuline or an (R)-Reticuline precursor using enzyme-encoded conversions in a non-plant host cell. A chimeric nucleic acid sequence is introduced into the host cell, wherein the chimeric nucleic acid sequence encodes a polypeptide having an amino acid sequence that corresponds to at least 85% sequence identity to the polypeptide encoded by SEQ ID NO: 322, and comprises operably linked components encoding CYP450 and AKR with one or more nucleic acid sequence capable of controlling expression in the host cell.
The host cell is grown to produce CYP450, AKR, and (R)-Reticuline or the (R)-Reticuline precursor. The described system enables epimerization of benzylisoquinoline derivatives to (R)-reticuline/(R)-reticuline precursors, and includes oxidized benzylisoquinoline derivative intermediates and corresponding reticuline precursor intermediates.
In certain embodiments, the CYP450 and AKR components are provided as a CYP450-AKR fusion polypeptide or through chimeric nucleic acid constructs. The non-plant host cell is a yeast cell or a bacteria cell, and the resulting system is intended to enable living cells to produce (R)-Reticuline or selected (R)-reticuline precursors.
Claims Coverage
The provided material identifies one core method claim directed to preparing (R)-Reticuline or an (R)-Reticuline precursor in a non-plant cell using a chimeric nucleic acid sequence encoding CYP450 and AKR with expression control elements, followed by growing the host cell. Dependent claims further specify host-cell type, sequence-identity thresholds, stereospecific product output, and cellular growth medium components.
Chimeric expression system in a non-plant cell
Introducing into a non-plant cell a chimeric nucleic acid sequence encoding a polypeptide having at least 85% sequence identity to the polypeptide encoded by SEQ ID NO: 322, the chimeric sequence comprising operably linked components including a first nucleic acid sequence encoding CYP450, a second nucleic acid sequence encoding AKR, and one or more nucleic acid sequence capable of controlling expression in a host cell.
Host-cell production by growing the transformed cell
Growing the host cell to produce CYP450, AKR, and (R)-Reticuline or the (R)-Reticuline precursor.
Host cell as yeast
Using a yeast cell as the non-plant cell in the method.
Host cell as bacteria
Using a bacteria cell as the non-plant cell in the method.
CYP450/AKR polypeptide identity threshold
Using a chimeric nucleic acid sequence encoding a polypeptide whose amino acid sequence has at least 90% sequence identity to the polypeptide encoded by SEQ ID NO: 322.
Production of (R)-Reticuline
Carrying out the method such that the host cell produces (R)-Reticuline.
Cellular growth medium with specified (S)-benzyltetrahydroisoquinoline-related compounds
Growing the host cell in a cellular growth medium containing at least one specified (S)-benzyltetrahydroisoquinoline-related compound selected from the listed group.
Overall, the claim set focuses on an engineered chimeric nucleic acid expression system in a non-plant host cell encoding CYP450 and AKR with expression control, followed by growing the host cell to produce (R)-Reticuline or an (R)-Reticuline precursor. Dependent claims narrow to yeast or bacteria hosts, tighter sequence identity relative to SEQ ID NO: 322, a specific (R)-product outcome, and culturing with selected (S)-benzyltetrahydroisoquinoline-related compounds in the growth medium.
Stated Advantages
Improved, efficient, commercial-scale manufacture not relying on chemical synthesis.
Enabling living cells not naturally capable to produce (R)-reticuline.
Downstream utility for morphine and codeine manufacturing.
Documented Applications
Use of AKR/CYP450 nucleic acids in detection/genotyping and modulation of expression in plants.
Manufacturing (R)-reticuline used as a downstream feedstock for morphine and codeine manufacturing.
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