HIGH EFFICIENCY PRODUCTION OF CANNABIDIOLIC ACID
The present disclosure features compositions and methods for producing one or more cannabinoids, such as cannabidiolic acid (CBDa), in a host cell, such as a yeast cell, that is genetically modified to express the enzymes of a cannabinoid biosynthetic pathway. Using the compositions and methods of the present invention, the host cell may be genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway, such as an enzyme having CBDa synthase (CBDaS) activity.
1 . A genetically modified host cell capable of producing CBDa or CBD, wherein the genetically modified host cell comprises one or more heterologous nucleic acids that each, independently, encodes an enzyme having CBDaS activity.
2 . The genetically modified host cell of claim 1 , wherein the enzyme having CBDaS activity is a fusion protein.
3 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence of a CBDaS or a portion thereof.
4 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 1, 4, 7, 8, 9, 10, 11, 13, 15, 17, 19, 21, 4, 7, 8, 9, 10, 11, 13, 15, 17, 19, 21, 134, 135, 136, 137, 147, 148, 149, 150, or 151.
5 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence of a carrier protein or a portion thereof, an amino acid sequence of a signal sequence or a portion thereof, or an amino acid sequence of a linker or a portion thereof.
6 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 34, 36, 73, 77, 81, 87, 89, 90, 91, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112.
7 . (canceled)
8 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54.
9 . (canceled)
10 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 152, or 172.
11 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence of a protease recognition site.
12 . The genetically modified host cell of claim 11 , wherein the protease recognition site is selected from the group of amino acid sequences consisting of RR, KR, RRK, RRQ, RRW, RRE, LDKR, LDKREAEA, and KREAEA.
13 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence of a mating factor alpha (MFα) or a portion thereof.
14 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 153, 154, 155, 156, or 157.
15 . The genetically modified host cell of claim 2 , wherein the fusion protein comprises two or more of:
a) an amino acid sequence of a CBDaS or a portion thereof;
b) an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 1, 4, 7, 8, 9, 10, 11, 13, 15, 17, 19, 21, 4, 7, 8, 9, 10, 11, 13, 15, 17, 19, 21, 134, 135, 136, 137, 147, 148, 149, 150, or 151;
c) an amino acid sequence of a carrier protein or a portion thereof;
d) an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 34, 36, 73, 77, 81, 87, 89, 90, 91, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112;
e) an amino acid sequence of a signal sequence or a portion thereof;
f) an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54;
g) an amino acid sequence of a linker or a portion thereof;
h) an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NOS: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 152, or 172;
i) an amino acid sequence of a protease recognition site;
j) a protease recognition site selected from the group of amino acid sequences consisting of RR, KR, RRK, RRQ, RRW, RRE, LDKR, LDKREAEA, and KREAEA;
k) an amino acid sequence of a mating factor alpha (MF) or a portion thereof; or
l) an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NOS: 153, 154, 155, 156, or 157.
16 . The genetically modified host cell of claim 1 , wherein the enzyme having CBDaS activity or the amino acid sequence of a CBDaS or a portion thereof comprises one or more of the following mutations when aligned with and in reference to SEQ ID NO: 136: N45Q, N65Q, S168N, N296Q, N304Q, N328Q, N498Q, T47S, T67S, S170T, T298S, T306S, S330T, or T500S.
17 . The genetically modified host cell of claim 1 , wherein the enzyme having CBDaS activity or the amino acid sequence of a CBDaS or a portion thereof comprises one or more amino acid substitutions occurring at position(s) 29, 31, 43, 49, 53, 56, 57, 65, 71, 78, 79, 93, 95, 103, 117, 125, 129, 143, 147, 151, 161, 183, 213, 235, 241, 263, 264, 285, 286, 303, 314, 325, 336, 339, 396, 436, 518, or 540 when aligned with and in reference to SEQ ID NO: 137.
18 . (canceled)
19 . The genetically modified host cell of claim 1 , wherein the enzyme having CBDaS activity or the amino acid sequence of a CBDaS or a portion thereof comprises one or more amino acid substitutions selected from the group consisting of:
a) R53T, N78D, V147D, H235D, I263V, K325N, V540C;
b) R53T, P65D, L71D, N79D, L93D, V147D, W183N, H235D, S336C;
c) L71D, N78D, G117A, V147D, W183N, I263V, K325N, S336C, V540C;
d) R53T, P65D, L71D, N78D, N79D, L93D, V147D, W183N, H235D, K325N, S336C, V540C;
e) L71D, L93D, V147D, H235D, I263V;
f) R53T, V147D, 1151L, W183N, H235D, S336C, V540C;
g) R53T, N78D, N79D, G117A, V147D, S336C;
h) R53T, P65D, L71D, N79D, L93D, V147D, W183N, H235D, S336C;
i) R53T, L71D, N78D, G117A, V147D, H235D, S336C, V540C;
j) R53T, P65D, N78D, G117A, V147D, H235D, K325N, S336C, V540C;
k) R53T, P65D, N78D, L93D, V147D, W183N, H235D, V540C;
1, R53T, N78D, V147D, W183N, H235D, I263V, S336C;
m) R53T, N79D, V147D, W183N, H235D, I263V, K325N, S336C;
n) R53T, P65D, L71D, N78D, V147D, H235D, I263V, S336C, V540C;
0, R53T, L71D, G117A, V147D, H235D, I263V, V540C;
p) R53T, L71D, N78D, G117A, V147D, H235D, 1263V, K325N, S336C, V540C;
q) R53T, P65D, N78D, N79D, V147D, S336C, V540C;
r) R53T, N78D, N79D, V147D, W183N, H235D, I263V, K325N;
s) R53T, 1151L, H235D, K325N, S336C; and
t) R53T, P65D, L71D, N79D, L93D, V147D, W183N, H235D, S336C,
when aligned with and in reference to SEQ ID NO: 137.
20 . A genetically modified host cell comprising an enzyme having at least 80% sequence identity to the amino acid sequence of any of the enzymes having CBDaS activity or to the amino acid sequence of a CBDaS or a portion thereof in claim 19 .
21 - 22 . (canceled)
23 . A method for producing CBDa or CBD, comprising:
a) culturing the genetically modified host cell of claim 1 in a medium with a carbon source under conditions suitable for making CBDa or CBD; and
b) recovering CBDa or CBD from the genetically modified host cell or the medium.
24 . A fermentation composition comprising CBDa or CBD, comprising:
a) the genetically modified host cell of claim 1 ; and
b) CBDa or CBD produced by the genetically modified host cell.
25 . (canceled)
26 . A non-naturally occurring enzyme having CBDaS activity, comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 4, 7, 8, 9, 10, 11, 13, 15, 17, 19, 21, 134, 135, 136, 137, 147, 148, 149, 150, or 151.
27 - 51 . (canceled)