Isoprene synthase variants for improved production of isoprene

Patent metadata

Field	Value
Publication number	US-9273298-B2
Application number	US-201113283564-A
Country	US
Kind code	B2
Filing date	Oct 27, 2011
Priority date	Oct 27, 2010
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

Abstract

The present invention provides methods and compositions comprising at least one isoprene synthase enzyme with improved specific productivity. In particular, the present invention provides variant plant isoprene synthases for increased isoprene production in host cells.

First claim

We claim:

1. A recombinant microbial host cell comprising a nucleic acid encoding a polypeptide having isoprene synthase activity and providing improved growth activity when the polypeptide is expressed in a host cell, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises a substitution at a residue corresponding to K134, K138, L143, I156, E159, F163, S166, H167, E170, K414, or Q421 of SEQ ID NO:1, wherein the substitution is selected from the group consisting of K134P, K138C, L143F, L143V, I156G, E159G, E159Q, F163C, F163E, F163Q, F163V, F163Y, S166C, S166D, S166G, S166P, S166V, H167M, E170G, E170H, E170K, E170N, E170R, E170S, E170W, K414F, K414G, K414N, K414P, and Q421R, and wherein a host cell expressing the polypeptide has at least 50% more growth compared to the growth of a host cell expressing a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 under similar growth conditions.

2. A recombinant microbial host cell comprising a nucleic acid encoding a polypeptide having isoprene synthase activity and providing improved growth activity when the polypeptide is expressed in a host cell, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises a substitution at a residue corresponding to E29, N47, S86, K94, E131, K134, I156, V162, K169, K178, E179, S231, R242, F369, K414, or Q421 of SEQ ID NO:1, wherein the substitution is selected from the group consisting of E29N, N47V, S86C, K94A, E131F, K134E, K134P, I156G, V162P, K169C, K178E, E179T, S231D, S231K, S231R, S231T, S231V, R242N, R242I, F369C, K414C, K414F, K414G, K414N, and Q421D, and wherein a host cell expressing the polypeptide has at least 50% more growth compared to the growth of a host cell expressing a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 under similar growth conditions.

3. A recombinant microbial host cell comprising a nucleic acid encoding a polypeptide having isoprene synthase activity and providing improved growth activity when the polypeptide is expressed in a host cell, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises a substitution at a residue corresponding to K50, D81, K134, I137, L143, I156, E159, S166, H167, K169, E170, or K414 of SEQ ID NO:1, wherein the substitution is selected from the group consisting of K50S, D81F, K134E, K134P, I137N, L143V, I156G, E159D, E159G, E159Q, S166C, S166W, H167M, H167N, K169C, E170H, E170K, E170W, K414C, K414F, K414G, K414N, and K414P, and wherein a host cell expressing the polypeptide has at least 30% more growth compared to the growth of a host cell expressing a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 under similar growth conditions.

4. A recombinant microbial host cell comprising a nucleic acid encoding a polypeptide having isoprene synthase activity and providing improved growth activity when the polypeptide is expressed in a host cell, wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises a substitution at a residue corresponding to V30, V84, K134, I140, L143, F163, S166, K169, E170 or S172 of SEQ ID NO:1, wherein the substitution is selected from the group consisting of V30K, V84T, K134C, K134D, K134E, I140S, I140T, L143F, L143I, L143M, L143V, F163I, F163M, S166P, S166V, K169Q, E170H, E170K, and S172V, and wherein the polypeptide has at least 20% increase in specific activity of isoprene synthase compared to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO: 1 and at least 20% more growth in a host cell expressing the polypeptide compared to the growth of a host cell expressing a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO: 1 under similar growth conditions.

5. A recombinant microbial host cell comprising a nucleic acid encoding a polypeptide with isoprene synthase properties wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises a substitution at a residue corresponding to N47, G87, I156, V162, E170, S231, R242, V409, or K414 of SEQ ID NO:1, wherein the substitution is selected from the group consisting of N47V, G87R, I156G, V162P, E170H, S231T, R242N, V409T, and K414F, and wherein the polypeptide has at least 30% increase in specific activity of isoprene synthase as compared to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 and at least 30% more growth when the polypeptide is expressed in a host cell, as compared to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 under similar growth conditions.

6. A recombinant microbial host cell comprising a nucleic acid encoding a polypeptide with isoprene synthase properties wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:1 and comprises a substitution at a residue corresponding to E2, Y18, L19, S21, T24, E26, S27, E29, K37, V42, N47, N48, E49, L56, D81, R82, V84, T93, K94, T95, S120, K123, N126, E131, N132, K134, I137, A139, L143, L151, N155, S166, H167, K169, E170, L171, K175, E179, L180, Q197, I229, S231, T240, R242, R245, R246, V247, T251, A271, S282, L306, D317, N319, F369, Q371, L376, K379, S380, G389, W392, K393, V408, V409, Q421, K422, Y423, R429, C437, S444, I447, S455, C458, R461, G464, S466, A470, S473, V500, T502, L506, T513, E525 or V531 of SEQ ID NO:1, wherein the substitution is selected from the group consisting of E2A or K or P, Y18D or E or K or S, L19Y, S21W, T24L or V, E26C, S27D or N, E29N, K37C or D or Por Q or S, V42M, N47D or S, N48D or G or T, E49L or V, L56E or F or G or I or K or T or V or Y, D81Q, R82N or T or V or Y, V84M, T93C or F or R or S, K94G or P, T95D or F or G or I or N or W, S120C or G or M or Q, K123V, N126E, E131H or K or L or M or T or W or Y, N132I or P, K134A, I137T, A139C or Q, L143C or D or E or H or K or M or Q or T or V or Y, L151A or F, N155A or C or G or H or Q or R or S or W, S166N, H167F or I or N or Q or V, K169A or C or H or N or Q or V, E170L or S or W or Y, L171A or N or Q or T or V or Y, K175C or F or I or Q or R, E179D, L180A or I, Q197C or D or N, I229C, S231A, T240C, R242G, R245C or K or M or Q or T or V, R246N, V247L or M, T251D or E or N or P or Q or S, A271T, S282Y, L306C, D317N, N319M, F369C or D or E or G or S, Q371F, L376I or M, K379G or Q, S380E, G389A or D or E or K or N or Q or S or V, W392Y, K393C or I or T or V, V408T, V409T, Q421H, K422D, Y423N or S, R429E or F or Q, C437M, S444D or E, I447T, S455A, C458T, R461A, G464C or M or N or Q or S, S466D, A470I or L, S473I, V500A or C, T502M, L506M, T513C or G or K or N, E525F or R, and V531E or H or K or Q or R or S, and wherein the polypeptide has (i) minimum performance indices (PI) relative to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO: 1 for specific activity and expression are greater than or equal to a PI of 0.9 and where at least one PI relative to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 for specific activity or growth is greater than or equal to a PI of 1.0; or (ii) minimum performance indices (PI) relative to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 for specific activity and expression are greater than or equal to a PI of 0.8 and where at least one PI relative to a parent polypeptide which does not comprise said substitution at a residue corresponding to SEQ ID NO:1 for specific activity or growth is greater than or equal to a PI of 1.2; or (iii) minimum performance indices (PI) relative to

Assignees

• Beck Zachary Q
• Estell David A
• Miller Jeffrey V
• Rife Christopher L
• Wells Derek H
• Danisco Us Inc
• Goodyear Tire & Rubber

Inventors

• Beck Zachary Q
• Estell David A
• Miller Jeffrey V
• Rife Christopher L
• Wells Derek H

Classifications

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12Y402/03027

  Isoprene synthase (4.2.3.27) · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N15/79

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title