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Title:
STABILIZATION OF AMINES WITH SULFUR COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/2020/232027
Kind Code:
A1
Abstract:
Presented herein are color stabilized diamine compositions comprising a diamine and a sulfur compound. The diamine can have an aromatic moiety. The sulfur compound includes methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, 2-propenethiol, 1-butanethiol, 2-butanethiol, t-butyl mercaptan, pentyl mercaptan, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythriol, 3-mercaptopropane-1,2-diol, 3-mercapto-1-propanesulfonic acid, or any combination thereof.

Inventors:
MACLEAN MICHAEL (US)
EDGAR STEVEN M (US)
Application Number:
PCT/US2020/032529
Publication Date:
November 19, 2020
Filing Date:
May 12, 2020
Export Citation:
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Assignee:
ZYMERGEN INC (US)
International Classes:
C07C209/46; C07C209/78; C07C211/51; C08G73/02; C12N15/77; C12N15/81
Domestic Patent References:
WO2018203947A22018-11-08
Foreign References:
CN101735116A2010-06-16
US20070066578A12007-03-22
Other References:
NCBI, GenBank Accession No. WP_021474081.1, `MULTISPECIES: aminodeoxychorism ate synthase component I [Arthrobacter]`, 27 February 2018 the whole document
NCBI, GenBank Accession No. WP_060542866.1, `chorismate mutase [Pseudomonas sp. Leaf48]`, 27 January 2016 the whole document
DATABASE Protein 7 November 2018 (2018-11-07), ANONYMOUS: "prephenate dehydrogenase [Paenibacillus wynnii]", XP055754086, retrieved from NCBI
NCBI, GenBank Accession No. WP_100598879.1, `aminodeoxychorismate synthase c omponent I [Streptomyces sp. CB01635]`, 27 February 2018 the whole document
DATABASE Protein 12 October 2019 (2019-10-12), ANONYMOUS: "chorismate mutase family protein [Streptomyces pristinaespiralis] - Protein - NCBI", XP055754103, retrieved from NCBI
NCBI, GenBank Accession No. WP_099586770.1, `MULTISPECIES: prephenate dehydr ogenase [Pseudomonas]`, 13 July 2018 the whole document
Attorney, Agent or Firm:
BACA, Helen S. et al. (US)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1 . A method for stabilizing a diamine comprising:

preparing a diamine having a purity of at least 95 mol%, at least 96 mol%, at least 97 mol%, at least 98 mol%, at least 99 mol%, or at least 99.5 mol%; and

combining the diamine with a sulfur compound, wherein the sulfur compound is added to a concentration of not greater than 10 wt%, not greater than 8 wt%, not greater than 6 wt%, not greater than 5 wt%, not greater than 4 wt%, not greater than 3 wt%, not greater than 2 wt%, not greater than 1 .5 wt%, not greater than 1 wt%, not greater than 0.8 wt%, not greater than 0.6 wt%, not greater than 0.4 wt%, not greater than 0.3 wt%, not greater than 0.2 wt%, or not greater than 0.01 wt% based on the combined weight of the diamine and the sulfur compound.

2. The method according to any one of the preceding claims, wherein the diamine comprises an aromatic moiety.

3. The method according to any one of the preceding claims, wherein the diamine is

any combination thereof.

4. The method according to any one of the preceding claims, wherein the sulfur compound is selected from a thiol, a thioester, a thioketone, a thioaldehyde, a thiocarboxylic acid, a thioamide, a thio amino acid, or any combination thereof.

5. The method according to any one of the preceding claims, wherein the sulfur compound is selected from methanethiol, ethanethiol, 1 -propanethiol, 2-propanethiol, 2- propenethiol, 1 -butanethiol, 2-butanethiol, t-butyl mercaptan, pentyl mercaptan, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 3-mercaptopropane-1 ,2-diol, 3- mercapto-1 -propanesulfonic acid, or any combination thereof.

6. The method according to any one of the preceding claims, wherein the diamine is prepared by fermentation from a microbe.

7. The method according to any one of the preceding claims, wherein the diamine is prepared by chemical modification of a fermented compound.

8. The method according to claim 7, wherein the fermented compound is selected from the group consisting of:

9. A copolymer comprising:

a diamine moiety, and

a sulfur moiety;

wherein the copolymer further has at least two properties selected from:

(i) a thickness of not greater than 100 microns, not greater than 90 microns, not greater than 80 microns, not greater than 70 microns, not greater than 60 microns, not greater than 50 microns, not greater than 40 microns, not greater than 35 microns, not greater than 30 microns, or not greater than 25 microns;

(ii) a tensile modulus according to ASTM D882 of at least 5 GPa, at least 5.2

GPa, at least 5.4 GPa, at least 5.6 GPa, at least 5.8 GPa, at least 6 GPa, at least 6.2 GPa, at least 6.4 GPa, at least 6.6 GPa, at least 6.8 GPa, at least 7 GPa, at least 7.2

GPa, at least 7.4 GPa, at least 7.6 GPa, at least 7.8 GPa, at least 8 GPa, at least 8.2

GPa, at least 8.5 GPa, at least 9 GPa, or at least 10 GPa; (iii) a first optical transparency according to ASTM D1746-15 at 380 nm of less than 50%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, or less than 1 %, and a second optical transparency according to ASTM

D1746-15 at 400 nm of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 96%;

(iv) a Yellowing Index according to ASTM E313-15e1 of not greater than 2.5, not greater than 2.4, not greater than 2.3, not greater than 2.2, not greater than 2.1 , not greater than 2.0, not greater than 1 .9, not greater than 1 .8, not greater than 1 .7, not greater than 1 .6, not greater than 1 .5, not greater than 1 .4, or not greater than 1 .3;

(v) a haze as determined according to ASTM D1003-13 of not greater than

1 .5%, not greater than 1 .3%, not greater than 1 .1 %, not greater than 1 .0%, not greater than 0.8%, not greater than 0.6%, not greater than 0.5%, not greater than 0.4%, or not greater than 0.3%;

(vi) a pencil hardness of greater than 1 H, greater than 2H, greater than 3H, greater than 4H, greater than 5H, or greater than 6H;

(vii) a coefficient of moisture expansion (‘CME’) as determined according to ASTM D5229/D5229M-14 of not greater than 50 ppm, not greater than 45 ppm, not greater than 40 ppm, not greater than 35 ppm, not greater than 30 ppm, not greater than 25 ppm, not greater than 20 ppm, or not greater than 15 ppm;

(viii) an elongation at break as determined according to ASTM D5034-09 (2017) of at least 10%, at least 15%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 35%, or at least 40%; or

(ix) a folding endurance as determined according to ASTM D2176-16 at a radius of 1 mm of at least 10,000 folds, at least 20,000 folds, at least 50,000 folds, at least 80,000 folds, at least 100,000 folds, at least 150,000 folds, at least 180,000 folds, at least 200,000 folds, at least 250,000 folds, at least 300,000 folds, at least 500,000 folds, or at least 1 ,000,000 folds.

10. The copolymer according to claim 9, comprising at least three properties selected from (i) through (ix), at least four properties selected from (i) through (ix), at least five properties selected from (i) through (ix), at least six properties selected from (i) through (ix), at least seven properties selected from (i) through (ix), or all properties selected from (i) through (ix).

1 1 . The copolymer according to any one of claims 9 and 10, wherein the diamine moiety is selected from the group consisting of: wherein X is selected from Z,

Y is selected independently for each occurrence from CH2, CH2CH2, or ; and Z is selected independently for each occurrence from an NH or an oxygen.

12. The copolymer according to any one of claims 9 through 1 1 , wherein the diamine moiety is selected from

13. The copolymer according to any one of claims 9 through 12, wherein the copolymer has a sulfur content of at least 0.01 wt%, at least 0.02 wt%, at least 0.05 wt%, at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, or at least 0.5 wt% based on elemental analysis.

14. A color stabilized diamine composition comprising

a compound selected from the group consisting of: , wherein X is selected from Z,

Y is selected independently for each occurrence from CH2, CH2CH2, , or ; and Z is NH2; and

a sulfur compound selected from methanethiol, ethanethiol, 1 -propanethiol, 2- propanethiol, 2-propenethiol, 1 -butanethiol, 2-butanethiol, t-butyl mercaptan, pentyl mercaptan, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 3- mercaptopropane-1 ,2-diol, 3-mercapto-1 -propanesulfonic acid, or any combination thereof.

15. The color stabilized diamine composition according to claim 14 wherein the sulfur compound is present at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.5 wt%, at least 0.8 wt%, at least 1 wt%, at least 1.2 wt%, at least 1.5 wt%, or at least 2 wt%, based on the weight of the color stabilized diamine composition.

16. The color stabilized diamine composition according to any one of claims 14 and

15, wherein the sulfur compound is present at not greater than 10 wt%, not greater than 9 wt%, not greater than 8 wt%, not greater than 7 wt%, not greater than 6 wt%, not greater than 5 wt%, not greater than 4 wt%, not greater than 3 wt%, not greater than 2.5 wt%, not greater than 2.2 wt%, not greater than 2 wt%, not greater than 1 .8 wt%, not greater than 1.5 wt%, or not greater than 1.2 wt%.

17. The color stabilized diamine composition according to any one of claims 14 through 16, wherein the sulfur compound consists essentially of glutathione, cysteine, 2- mercaptoethanol, dithiothreitol, dithioerythriol, 3-mercaptopropane-1 ,2-diol, or any combination thereof.

18. The color stabilized diamine composition according to any one of claims 14 through 17, wherein the diamine consists essentially of

or any combination thereof.

19. An engineered microbial cell that expresses:

a heterologous chorismite aminotransferase;

a heterologous 4-amino-4-deoxychorismate mutase; and

a heterologous 4-amino-4-deoxyphrenate dehydrogenase.

20. The engineered microbial cell of claim 19, wherein the engineered microbial cell is a bacterial cell.

21 . The engineered microbial cell of claim 20, wherein the bacterial cell is a Corynebacteria glutamicum cell.

22. The engineered microbial cell of any one of claims 19-21 , wherein :

the heterologous chorismite aminotransferase has at least 70% amino acid sequence identity with an Anthrobacter sp. ATCC 21022 chorismite

aminotransferase comprising SEQ ID NO:3;

the heterologous 4-amino-4-deoxychorismate mutase has at least 70% amino acid sequence identity with a Pseudomonas sp. Leaf 48 4-amino-4- deoxychorismate mutase comprising SEQ ID NO:17; and

the heterologous 4-amino-4-deoxyphrenate dehydrogenase has at least 70% amino acid sequence identity with a Paenibacillus wynnii 4-ami no-4- deoxyphrenate dehydrogenase comprising SEQ ID NO:28.

23. The engineered microbial cell of claim 19, wherein the engineered microbial cell is a yeast cell.

24. The engineered microbial cell of claim 23, wherein the yeast cell is a

Saccharomyces cerevisiae cell.

25. The engineered microbial cell of any one of claims 19, 23, or 24, wherein :

the heterologous chorismite aminotransferase has at least 70% amino acid sequence identity with a Streptomyces sp. CB01635 chorismite

aminotransferase comprising SEQ ID NO:13;

the heterologous 4-amino-4-deoxychorismate mutase has at least 70% amino acid sequence identity with a Streptomyces pristinaespiralis 4-amino-4- deoxychorismate mutase comprising SEQ ID NO:18; and

the heterologous 4-amino-4-deoxyphrenate dehydrogenase has at least 70% amino acid sequence identity with a Pseudomonas sp. 2822-17 4-amino-4- deoxyphrenate dehydrogenase comprising SEQ ID NO:38.

Description:
STABILIZATION OF AMINES WITH SULFUR COMPOUNDS

CROSS-REFERENCE TO RELATED APPLICATIONS [0001 ] This application claims the benefit of U.S. provisional application no. 62/847,225, filed May 13, 2019, which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] This application includes a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on May 7, 2020, is named ZMGNP032WO_seq_list_ST25.txt and is 141 ,336 bytes in size.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to stabilization of aromatic amines. More specifically, the present disclosure relates to stabilization of aromatic diamines with sulfur-containing compounds, especially aromatic diamines for polymers, such as polyamides, polyimides, polyamide-imides, polyureas, or any combination thereof.

BACKGROUND

[0004] Aromatic amines and polyamines find abundantly utility in all kinds of technologies ranging from electronics, plastics, and fabrics to cosmetics and pharmaceuticals. Many goods contain aromatic amines, aromatic polyamines, polyamines or an aromatic polyamine derivative. One disadvantage of aromatic amines and polyamines is the discoloration that occurs when the aromatic amines and polyamines are not stored properly. The discoloration is mostly due to an initial oxidation of the amine group and subsequent reactions. Aromatic moieties in the polyamine discolor faster than aliphatic amines. The discoloration is an undesired effect, especially when polyamines are incorporated in optical products such as optical films or surface compositions.

[0005] Most common antioxidants are ineffective in preventing the color formation. Or if they are effective, they interfere with further processing of the polyamine, e.g., polymerization.

SUMMARY

[0006] In a first aspect, a method for stabilizing a diamine includes preparing a diamine having a purity of at least 95 mol%. The diamine can further have a purity of at least 96 mol%, at least 97 mol%, at least 98 mol%, at least 99 mol%, or at least 99.5 mol%. The method can further include combining the diamine with a sulfur compound. The sulfur compound can be added to a concentration of not greater than 10 wt%, not greater than 8 wt%, not greater than 6 wt%, not greater than 5 wt%, not greater than 4 wt%, not greater than 3 wt%, not greater than 2 wt%, not greater than 1 .5 wt%, not greater than 1 wt%, not greater than 0.8 wt%, not greater than 0.6 wt, not greater than 0.4 wt%, not greater than 0.3 wt%, not greater than 0.2 wt%, not greater than 0.1 wt%, not greater than 0.05 wt%, not greater than 0.02 wt%, or not greater than 0.01 wt%, based on the combined weight of diamine and sulfur compound.

[0007] In a second aspect, a copolymer comprises a diamine moiety. The copolymer can further include a sulfur moiety. Moreover, the copolymer can further have at least two properties selected from the following:

(i) a thickness of not greater than 100 microns, not greater than 90 microns, not greater than 80 microns, not greater than 70 microns, not greater than 60 microns, not greater than 50 microns, not greater than 40 microns, not greater than 35 microns, not greater than 30 microns, or not greater than 25 microns;

(ii) a tensile modulus according to ASTM D882 of at least 5 GPa, at least 5.2 GPa, at least 5.4 GPa, at least 5.6 GPa, at least 5.8 GPa, at least 6 GPa, at least 6.2 GPa, at least 6.4 GPa, at least 6.6 GPa, at least 6.8 GPa, at least 7 GPa, at least 7.2 GPa, at least 7.4 GPa, at least 7.6 GPa, at least 7.8 GPa, at least 8 GPa, at least 8.2 GPa, at least 8.5 GPa, at least 9 GPa, or at least 10 GPa;

(iii) a first optical transparency according to ASTM D1746-15 at 380 nm of less than 50%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, or less than 1 %, and a second optical transparency according to ASTM

D1746-15 at 400 nm of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 96%;

(iv) a Yellowing Index according to ASTM E313-15e1 of not greater than 2.5, not greater than 2.4, not greater than 2.3, not greater than 2.2, not greater than 2.1 , not greater than 2.0, not greater than 1 .9, not greater than 1 .8, not greater than 1 .7, not greater than 1 .6, not greater than 1 .5, not greater than 1 .4, or not greater than 1 .3;

(v) a haze as determined according to ASTM D1003-13 of not greater than 1 .5%, not greater than 1 .3%, not greater than 1 .1 %, not greater than 1 .0%, not greater than 0.8%, not greater than 0.6%, not greater than 0.5%, not greater than 0.4%, or not greater than 0.3%;

(vi) a pencil hardness of greater than 1 H, greater than 2H, greater than 3H, greater than 4H, greater than 5H, or greater than 6H;

(vii) a coefficient of moisture expansion (‘CME’) as determined according to ASTM D5229/D5229M-14 of not greater than 50 ppm, not greater than 45 ppm, not greater than 40 ppm, not greater than 35 ppm, not greater than 30 ppm, not greater than 25 ppm, not greater than 20 ppm, or not greater than 15 ppm;

(viii) an elongation at break as determined according to ASTM D5034-09 (2017) of at least 10%, at least 15%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 35%, or at least 40%; or (ix) a folding endurance as determined according to ASTM D2176-16 at a radius of 1 mm of at least 10,000 folds, at least 20,000 folds, at least 50,000 folds, at least 80,000 folds, at least 100,000 folds, at least 150,000 folds, at least 180,000 folds, at least 200,000 folds, at least 250,000 folds, at least 300,000 folds, at least 500,000 folds, or at least 1 ,000,000 folds.

[0008] In a third aspect, a color stabilized diamine composition includes a compound selected from the group consisting of

In formula I, X can be selected from

Y can be selected independently for each occurrence from

CH 2 , CH 2 CH 2 , , or ; and Z is for each occurrence NH 2 . The sulfur

compound can be selected from methanethiol, ethanethiol, 1 -propanethiol, 2- propanethiol, 2-propenethiol, 1 -butanethiol, 2-butanethiol, t-butyl mercaptan, pentyl mercaptan, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 3- mercaptopropane-1 ,2-diol, 3-mercapto-1 -propanesulfonic acid, or any combination thereof. [0009] Other aspects of the disclosure relate to cells engineered to produce diamines or diamine precursors, including, but not limited to the following:

[0010] Engineered Cell Embodiment 1 : An engineered microbial cell that expresses: a heterologous chorismite aminotransferase; a heterologous 4-amino-4-deoxychorismate mutase; and a heterologous 4-amino-4-deoxyphrenate dehydrogenase.

[001 1 ] Engineered Cell Embodiment 2: The engineered microbial cell of embodiment 1 , wherein the engineered microbial cell is a bacterial cell.

[0012] Engineered Cell Embodiment 3: The engineered microbial cell of embodiment 2, wherein the bacterial cell is a Corynebacteria glutamicum cell.

[0013] Engineered Cell Embodiment 4: The engineered microbial cell of any one of embodiments 1 -3, wherein: the heterologous chorismite aminotransferase has at least 70% amino acid sequence identity with an Anthrobacter sp. ATCC 21022 chorismite aminotransferase including SEQ ID NO:3; the heterologous 4-amino-4- deoxychorismate mutase has at least 70% amino acid sequence identity with a

Pseudomonas sp. Leaf 48 4-amino-4-deoxychorismate mutase including SEQ ID NO: 17; and the heterologous 4-amino-4-deoxyphrenate dehydrogenase has at least 70% amino acid sequence identity with a Paenibacillus wynnii 4-amino-4-deoxyphrenate dehydrogenase including SEQ ID NO:28.

[0014] Engineered Cell Embodiment 5: The engineered microbial cell of embodiment 1 , wherein the engineered microbial cell is a yeast cell.

[0015] Engineered Cell Embodiment 6: The engineered microbial cell of embodiment 5, wherein the yeast cell is a Saccharomyces cerevisiae cell.

[0016] Engineered Cell Embodiment 7: The engineered microbial cell of any one of embodiments 1 , 5, or 6 wherein: the heterologous chorismite aminotransferase has at least 70% amino acid sequence identity with a Streptomyces sp. CB01635 chorismite aminotransferase including SEQ ID NO:13; the heterologous 4-amino-4- deoxychorismate mutase has at least 70% amino acid sequence identity with a

Streptomyces pristinaespiralis 4-amino-4-deoxychorismate mutase including SEQ ID NO: 18; and the heterologous 4-amino-4-deoxyphrenate dehydrogenase has at least 70% amino acid sequence identity with a Pseudomonas sp. 2822-17 4-amino-4- deoxyphrenate dehydrogenase including SEQ ID NO:38.

BRIEF DESCRIPTION OF THE DRAWINGS [0017] Figure 1 : Absorption Spectra of 4-(aminomethyl)aniline [‘AMA’} comprising various concentrations of DTT.

[0018] Figure 2: Absorption Spectrum of 4-(aminomethyl)aniline [‘AMA’] comprising various concentrations of cysteine.

[0019] Figure 3A-3C: The expression of three heterologous enzymes (chorismate aminotransferase, 4-amino-4-deoxychorismate mutase, and 4-amino-4-deoxyphrenate dehydrogenase) in Saccharomyces cerevisiae and Corynebacterium glutamicum enables production of precursors to 4-aminophenyl-ethylamine (‘4APEA,’ also described herein as 4-(2-aminoethyl)aniline [‘AEA’]); the pathway is shown in 3A. 3B

demonstrates the production of 40 mg/L of a non-native byproduct (4- aminophenylethanol [‘4APE’]) in S. cerevisiae, which indicates activity of the three heterologous enzymes. 3C demonstrates production of a precursor (4- aminophenylalanine [‘4APHE’]) one step away from 4APEA in C. glutamicum.

[0020] Figure 4: Integration of Promoter-Gene-Terminator into Corynebacteria glutamicum.

[0021 ] Figure 5: Integration of Promoter-Gene-Terminator into Saccharomyces cerevisiae.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Diamines

[0022] As stated in the Summary, in a first aspect, a method for stabilizing a diamine includes preparing a diamine having a purity of at least 95 mol%. The diamine can further have a purity of at least 96 mol%, at least 97 mol%, at least 98 mol%, at least 99 mol%, or at least 99.5 mol%. The method can further include combining the diamine with a sulfur compound. The sulfur compound can be added to a concentration of not greater than 10 wt%, not greater than 8 wt%, not greater than 6 wt%, not greater than 5 wt%, not greater than 4 wt%, not greater than 3 wt%, not greater than 2 wt%, not greater than 1 .5 wt%, not greater than 1 wt%, not greater than 0.8 wt%, not greater than 0.6 wt%, not greater than 0.4 wt%, not greater than 0.3 wt%, not greater than 0.2 wt%, or not greater than 0.1 wt%, based on the combined weight of diamine and sulfur compound.

[0023] In one embodiment, the diamine can comprise an aromatic moiety. The aromatic moiety can be a phenylene, a naphthylene, a bi-phenylene, or a heteroarylene. In yet one further embodiment, the diamine can be selected from:

or any combination thereof.

[0024] In yet one further embodiment, the sulfur compound can be selected from a thiol, a thioester, a thioketone, a thioaldehyde, a thiocarboxylic acid, a thioamide, a thio amino acid, or any combination thereof. More specifically, the sulfur compound can be selected from methanethiol, ethanethiol, 1 -propanethiol, 2-propanethiol, 2-propenethiol,

1 -butanethiol, 2-butanethiol, t-butyl mercaptan, pentyl mercaptan, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, cysteine, a cysteine derivative, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 3-mercaptopropane-1 ,2-diol, 3-mercapto-1 -propanesulfonic acid, or any combination thereof.

[0025] In yet another embodiment, the diamine is prepared by fermentation from a microbe. In another embodiment, the sulfur compound is prepared by fermentation from a microbe.

[0026] In another embodiment, the diamine can be prepared by chemical modification of a fermentation product. In addressing diamines made from a fermented compound, such fermented compound can be selected from the group:

[0027] As stated in the Summary, in a second aspect, a copolymer comprises a diamine moiety. The copolymer can further include a sulfur moiety. Moreover, the copolymer can further have at least two properties selected from the following properties (i) through (ix) :

(i) A thickness of not greater than 100 microns, not greater than 90 microns, not greater than 80 microns, not greater than 70 microns, not greater than 60 microns, not greater than 50 microns, not greater than 40 microns, not greater than 35 microns, not greater than 30 microns, or not greater than 25 microns. In embodiments, the thickness can be at least 50 nm, at least 100 nm, at least 0.5 micron, at least 1 micron, at least 2 microns, at least 5 microns. In yet another embodiment, the thickness can range between 50 nm and 100 microns, between 100 nm and 80 microns, or between 1 micron and 60 microns.

(ii) A tensile modulus according to ASTM D882 of at least 5 GPa, at least 5.2

GPa, at least 5.4 GPa, at least 5.6 GPa, at least 5.8 GPa, at least 6 GPa, at least 6.2

GPa, at least 6.4 GPa, at least 6.6 GPa, at least 6.8 GPa, at least 7 GPa, at least 7.2

GPa, at least 7.4 GPa, at least 7.6 GPa, at least 7.8 GPa, at least 8 GPa, at least 8.2

GPa, at least 8.5 GPa, at least 9 GPa, or at least 10 GPa. In one embodiment, the tensile modulus is not greater than 2000 GPa, not greater than 1000 GPa, not greater than 800 GPa, or not greater than 500 GPa. In some embodiments, the tensile modulus ranges between 5 GPa and 2000 GPa, between 6 GPa and 1000 GPa, or between 7 GPa and 800 GPa.

(iii) A first optical transparency according to ASTM D1746-15 at 380 nm of less than 50%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, or less than 1 %. In one embodiment, the first optical transparency can be at least 0.5%, at least 1 %, at least 2%, or at least 5%. In one embodiment, the first optical transparency can range between 0.5% and 30%, between 0.5% and 10%, or between 1 % and 8%. The property also includes a second optical transparency according to ASTM D1746-15 at 400 nm of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 82%, greater than 84%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, or greater than 96%. In one embodiment, the second optical transparency is less than 99.9%, less than 99.5%, less than 99%, less than 98%, or less than 95%. In one embodiment, the second optical transparency ranges between 50% and 99.9%.

(iv) A Yellowing Index according to ASTM E313-15e1 of not greater than 2.5, not greater than 2.4, not greater than 2.3, not greater than 2.2, not greater than 2.1 , not greater than 2.0, not greater than 1 .9, not greater than 1 .8, not greater than 1 .7, not greater than 1 .6, not greater than 1 .5, not greater than 1 .4, or not greater than 1 .3.

(v) A haze as determined according to ASTM D1003-13 of not greater than 1 .5%, not greater than 1 .3%, not greater than 1 .1 %, not greater than 1 .0%, not greater than 0.8%, not greater than 0.6%, not greater than 0.5%, not greater than 0.4%, or not greater than 0.3%.

(vi) A pencil hardness of greater than 1 H, greater than 2H, greater than 3H, greater than 4H, greater than 5H, or greater than 6H.

(vii) A coefficient of moisture expansion (‘CME’) as determined according to ASTM D5229/D5229M-14 of not greater than 50 ppm, not greater than 45 ppm, not greater than 40 ppm, not greater than 35 ppm, not greater than 30 ppm, not greater than 25 ppm, not greater than 20 ppm, or not greater than 15 ppm. (viii) An elongation at break as determined according to ASTM D5034-09 (2017) of at least 10%, at least 15%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 35%, or at least 40%.

(ix) A folding endurance as determined according to ASTM D2176-16 at a radius of 1 mm of at least 10,000 folds, at least 20,000 folds, at least 50,000 folds, at least 80,000 folds, at least 100,000 folds, at least 150,000 folds, at least 180,000 folds, at least 200,000 folds, at least 250,000 folds, at least 300,000 folds, at least 500,000 folds, or at least 1 ,000,000 folds.

[0028] In a third aspect, a color stabilized diamine composition includes a compound selected from the group consisting of

In formula I, X can be selected from Z,

Y can be selected independently for each occurrence from

CH 2 , CH 2 CH 2 , , or ; and Z is for each occurrence NH 2 . The sulfur

compound can be selected from methanethiol, ethanethiol, 1 -propanethiol, 2- propanethiol, 2-propenethiol, 1 -butanethiol, 2-butanethiol, t-butyl mercaptan, pentyl mercaptan, thiophenol, dimercaptosuccinic acid, thioacetic acid, coenzyme A, glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 3- mercaptopropane-1 ,2-diol, 3-mercapto-1 -propanesulfonic acid, or any combination thereof.

[0029] In one embodiment, the color stabilized diamine can include the sulfur compound to be present at least 0.01 wt%, such as at least 0.02 wt%, at least 0.03 wt%, at least 0.05 wt%, at least 0.1 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.5 wt%, at least 0.8 wt%, at least 1 wt%, at least 1 .2 wt%, at least 1.5 wt%, at least 2 wt%, based on the weight of the color stabilized diamine composition. In another

embodiment, the sulfur compound can be present at not greater than 10 wt%, not greater than 9 wt%, not greater than 8 wt%, not greater than 7 wt%, not greater than 6 wt%, not greater than 5 wt%, not greater than 4 wt%, not greater than 3 wt%, not greater than 2.5 wt%, not greater than 2.2 wt%, not greater than 2 wt%, not greater than 1 .8 wt%, not greater than 1 .5 wt%, not greater than 1 .2 wt%, not greater than 1 .0 wt%, or not greater than 0.8 wt%. In yet one further embodiment the sulfur compound can be present between 0.01 wt% and 5 wt%, such as between 0.02 wt% and 2 wt%, or between 0.03 wt% and 1 wt%.

[0030] In another embodiment, the sulfur compound can consist essentially of glutathione, cysteine, 2-mercaptoethanol, dithiothreitol, dithioerythriol, 3- mercaptopropane-1 ,2-diol, or any combination thereof. Moreover, in yet one further embodiment, the diamine can consist essentially of

or any combination thereof.

Cells Engineered to Produce Diamines

Definitions

[0031 ] The term“fermentation” is used herein to refer to a process whereby a microbial cell converts one or more substrate(s) into a desired product (such as 3-amino-4- hydroxybenzoic acid) by means of one or more biological conversion steps, without the need for any chemical conversion step.

[0032] The term“engineered” is used herein, with reference to a cell, to indicate that the cell contains at least one targeted genetic alteration introduced by man that distinguishes the engineered cell from the naturally occurring cell.

[0033] The term“native” is used herein to refer to a cellular component, such as a polynucleotide or polypeptide, that is naturally present in a

[0034] When used with reference to a polynucleotide or polypeptide, the term“non native” refers to a polynucleotide or polypeptide that is not naturally present in a particular cell.

[0035] When used with reference to the context in which a gene is expressed, the term “non-native” refers to a gene expressed in any context other than the genomic and cellular context in which it is naturally expressed. A gene expressed in a non-native manner may have the same nucleotide sequence as the corresponding gene in a host cell, but may be expressed from a vector or from an integration point in the genome that differs from the locus of the native gene.

[0036] The term“heterologous” is used herein to describe a polynucleotide or polypeptide introduced into a host cell. This term encompasses a polynucleotide or polypeptide, respectively, derived from a different organism, species, or strain than that of the host cell. In this case, the heterologous polynucleotide or polypeptide has a sequence that is different from any sequence(s) found in the same host cell. However, the term also encompasses a polynucleotide or polypeptide that has a sequence that is the same as a sequence found in the host cell, wherein the polynucleotide or polypeptide is present in a different context than the native sequence (e.g., a heterologous polynucleotide can be linked to a different promotor and inserted into a different genomic location than that of the native sequence). “Heterologous expression” thus encompasses expression of a sequence that is non-native to the host cell, as well as expression of a sequence that is native to the host cell in a non-native context.

[0037] As used with reference to polynucleotides or polypeptides, the term“wild-type” refers to any polynucleotide having a nucleotide sequence, or polypeptide having an amino acid, sequence present in a polynucleotide or polypeptide from a naturally occurring organism, regardless of the source of the molecule; i.e., the term“wild-type” refers to sequence characteristics, regardless of whether the molecule is purified from a natural source; expressed recombinantly, followed by purification; or synthesized. The term“wild-type” is also used to denote naturally occurring cells.

[0038] Enzymes are identified herein by the reactions they catalyze and, unless otherwise indicated, refer to any polypeptide capable of catalyzing the identified reaction. Unless otherwise indicated, enzymes may be derived from any organism and may have a native or mutated amino acid sequence. As is well known, enzymes may have multiple functions and/or multiple names, sometimes depending on the source organism from which they derive. The enzyme names used herein encompass orthologs, including enzymes that may have one or more additional functions or a different name.

[0039] The term“sequence identity,” in the context of two or more amino acid or nucleotide sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.

[0040] For sequence comparison to determine percent nucleotide or amino acid sequence identity, typically one sequence acts as a“reference sequence,” to which a “test” sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted using BLAST set to default parameters.

[0041 ] The term“titer,” as used herein, refers to the mass of a product (e.g. , 3-amino-4- hydroxybenzoic acid) produced by a culture of microbial cells divided by the culture volume.

Biosynthesis of 4-Aminophenyl Pyruvate

[0042] 4-aminophenyl pyruvate is a precursor to 4-aminophenyl-ethylamine (‘4APEA,’ also described herein as 4-(2-aminoethyl)aniline [‘AEA’], as shown in Fig. 3A. This compound can be produced from chorismate in three enzymatic steps, requiring the enzymes chorismate aminotransferase, 4-amino-4-deoxychohsmate mutase, and 4- amino-4-deoxyphrenate dehydrogenase. Accordingly, a microbial host that can produce chorismate can be engineered to produce 4-aminophenyl pyruvate by expressing forms of these three enzymes that are active in the microbial host.

Engineering for Microbial 4-Aminophenyl Pyruvate Production

[0043] Any chorismate aminotransferase, 4-amino-4-deoxychohsmate mutase, and 4- amino-4-deoxyphrenate dehydrogenase enzymes that are active in the microbial cell being engineered may be introduced into the cell, typically by introducing and expressing the gene(s) encoding the enzyme(s) using standard genetic engineering techniques. Suitable 2 chorismate aminotransferase, 4-amino-4-deoxychorismate mutase, and 4-amino-4-deoxyphrenate dehydrogenase enzymes may be derived from any source, including plant, archaeal, fungal, gram-positive bacterial, and gram-negative bacterial sources.

[0044] One or more copies of any of these genes can be introduced into a selected microbial host cell. If more than one copy of a gene is introduced, the copies can have the same or different nucleotide sequences. In some embodiments, one or both (or all) of the heterologous gene(s) is/ are expressed from a strong, constitutive promoter. In some embodiments, the heterologous gene(s) is/are expressed from an inducible promoter. The heterologous gene(s) can optionally be codon-optimized to enhance expression in the selected microbial host cell. Illustrative codon-optimization tables for various host cells are as follows: Bacillus subtilis Kazusa codon table:

Yarrowia lipolytica Kazusa codon table:

Corynebacteria glutamicum Kazusa codon table:

Saccharomyces cerevisiae Kazusa codon table:

Also useful is a modified, combined codon usage scheme for S. cereviae and C. glutamicum, which is reproduced below (Table 1 ).

Table 1 : Modified Codon Usage Table for Sc and Cg

Microbial Host Cells

[0045] Any microbe that can be used to express introduced genes can be engineered for fermentative production of 4-aminophenyl pyruvate as described above. In certain embodiments, the microbe is one that is naturally incapable of fermentative production of 4-aminophenyl pyruvate. In some embodiments, the microbe is one that is readily cultured, such as, for example, a microbe known to be useful as a host cell in fermentative production of compounds of interest. Bacteria cells, including gram positive or gram-negative bacteria can be engineered as described above. Examples include, in addition to C. glutamicum cells, Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B.

halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., P. alcaligenes, P. citrea, Lactobacillus spp. (such as L. lactis, L. plantarum), L. grayi, E. coli, E. faecium, E.

gallinarum, E. casseliflavus, and/or E. faecal is cells.

[0046] There are numerous types of anaerobic cells that can be used as microbial host cells in the methods described herein. In some embodiments, the microbial cells are obligate anaerobic cells. Obligate anaerobes typically do not grow well, if at all, in conditions where oxygen is present. It is to be understood that a small amount of oxygen may be present, that is, there is some level of tolerance level that obligate anaerobes have for a low level of oxygen. Obligate anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes.

[0047] Alternatively, the microbial host cells used in the methods described herein can be facultative anaerobic cells. Facultative anaerobes can generate cellular ATP by aerobic respiration (e.g., utilization of the TCA cycle) if oxygen is present. However, facultative anaerobes can also grow in the absence of oxygen. Facultative anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes, or can be alternatively grown in the presence of greater amounts of oxygen.

[0048] In some embodiments, the microbial host cells used in the methods described herein are filamentous fungal cells. (See, e.g., Berka & Barnett, Biotechnology

Advances, (1989), 7(2) :127-154). Examples include Trichoderma longibrachiatum, T. viride, T. koningii, T. harzianum, Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, Chrysosporium sp., C. lucknowense, Gliocladium sp., Aspergillus sp. (such as A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori ), Fusarium sp. (such as F. roseum, F. graminum, F. cerealis, F. oxysporuim, or F. venenatum ), Neurospora sp. (such as N. crassa or Hypocrea sp.), Mucor sp. (such as M. miehei ), Rhizopus sp., and Emericella sp. cells. In particular embodiments, the fungal cell engineered as described above is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A.

japonicus, T. reesei, T. viride, F. oxysporum, or F. solani. Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Patent Pub. No. 201 1/0045563.

[0049] Yeasts can also be used as the microbial host cell in the methods described herein. Examples include: Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Hansenula polymorpha, Pichia stipites, Kluyveromyces marxianus, Kluyveromyces spp., Yarrowia lipolytica and Candida sp. In some embodiments, the Saccharomyces sp. is S. cerevisiae (See, e.g., Romanos et al., Yeast, (1992), 8(6):423-488). Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Pat. No. 7,659,097 and U.S. Patent Pub. No. 201 1/0045563.

[0050] In some embodiments, the host cell can be an algal cell derived, e.g., from a green alga, red alga, a glaucophyte, a chlorarachniophyte, a euglenid, a chromista, or a dinoflagellate. (See, e.g., Saunders & Warmbrodt,“Gene Expression in Algae and Fungi, Including Yeast,” (1993), National Agricultural Library, Beltsville, Md.). Illustrative plasmids or plasmid components for use in algal cells include those described in U.S. Patent Pub. No. 201 1/0045563.

[0051 ] In other embodiments, the host cell is a cyanobacterium, such as

cyanobacterium classified into any of the following groups based on morphology:

Chlorococcales, Pleurocapsales, Oscillatoriales, Nostocales, Synechosystic or

Stigonematales (See, e.g., Lindberg et al., Metab. Eng., (2010) 12(1 ):70-79). Illustrative plasmids or plasmid components for use in cyanobacterial cells include those described in U.S. Patent Pub. Nos. 2010/0297749 and 2009/0282545 and in Inti. Pat. Pub. No.

WO 201 1/034863. Genetic Engineering Methods

[0052] Microbial cells can be engineered for fermentative 4-aminophenyl pyruvate production using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, see e.g.,“Molecular Cloning: A Laboratory Manual,” fourth edition (Sambrook et al., 2012); Oligonucleotide Synthesis” (M. J. Gait, ed., 1984);“Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications” (R. I. Freshney, ed., 6th Edition, 2010);“Methods in

Enzymology” (Academic Press, Inc.);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates);“PCR: The Polymerase Chain Reaction,” (Mullis et al., eds., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994).

[0053] Vectors are polynucleotide vehicles used to introduce genetic material into a cell. Vectors useful in the methods described herein can be linear or circular. Vectors can integrate into a target genome of a host cell or replicate independently in a host cell.

For many applications, integrating vectors that produced stable transformants are preferred. Vectors can include, for example, an origin of replication, a multiple cloning site (MCS), and/or a selectable marker. An expression vector typically includes an expression cassette containing regulatory elements that facilitate expression of a polynucleotide sequence (often a coding sequence) in a particular host cell. Vectors include, but are not limited to, integrating vectors, prokaryotic plasmids, episomes, viral vectors, cosmids, and artificial chromosomes.

[0054] Illustrative regulatory elements that may be used in expression cassettes include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0055] In some embodiments, vectors may be used to introduce systems that can carry out genome editing, such as CRISPR systems. See U.S. Patent Pub.

No. 2014/0068797, published 6 March 2014; see also Jinek M., et al.,“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science 337:816- 21 , 2012). In Type II CRISPR-Cas9 systems, Cas9 is a site-directed endonuclease, namely an enzyme that is, or can be, directed to cleave a polynucleotide at a particular target sequence using two distinct endonuclease domains (HNH and RuvC/RNase H- like domains). Cas9 can be engineered to cleave DNA at any desired site because Cas9 is directed to its cleavage site by RNA. Cas9 is therefore also described as an “RNA-guided nuclease.” More specifically, Cas9 becomes associated with one or more RNA molecules, which guide Cas9 to a specific polynucleotide target based on hybridization of at least a portion of the RNA molecule(s) to a specific sequence in the target polynucleotide. Ran, F.A., et at., (“In vivo genome editing using Staphylococcus aureus Cas9,” Nature 520(7546):186-91 , 2015, Apr 9], including all extended data) present the crRNA/tracrRNA sequences and secondary structures of eight Type II CRISPR-Cas9 systems. Cas9-like synthetic proteins are also known in the art (see U.S. Published Patent Application No. 2014-0315985, published 23 October 2014).

[0056] Example 2 describes illustrative integration approaches for introducing polynucleotides and other genetic alterations into the genomes of C. glutamicum and S. cerevisiae cells.

[0057] Vectors or other polynucleotides can be introduced into microbial cells by any of a variety of standard methods, such as transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE- Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. Transformants can be selected by any method known in the art. Suitable methods for selecting transformants are described in U.S. Patent Pub. Nos. 2009/0203102, 2010/0048964, and 2010/0003716, and International Publication Nos. WO 2009/076676, WO 2010/003007, and WO

2009/132220. Engineered Microbial Cells

[0058] The above-described methods can be used to produce engineered microbial cells that produce, and in certain embodiments, overproduce, 4-aminophenyl pyruvate. Engineered microbial cells can have at least 1 , 2, 3, 4, 5, 6 ,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more genetic alterations, such as 30-100 alterations, as compared to a native microbial cell, such as any of the microbial host cells described herein. Engineered microbial cells described in the Example below have one, two, or three genetic alterations, but those of skill in the art can, following the guidance set forth herein, design microbial cells with additional alterations. In some embodiments, the engineered microbial cells have not more than 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, or 4 genetic alterations, as compared to a native microbial cell. In various embodiments, microbial cells engineered for 4-aminophenyl pyruvate production can have a number of genetic alterations falling within the any of the following illustrative ranges: 1 -10, 1 -9, 1 - 8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, etc.

[0059] In some embodiments, an engineered microbial cell expresses at least three heterologous genes, e.g., a heterologous chorismate aminotransferase gene, a heterologous 4-amino-4-deoxychorismate mutase gene , and a heterologous 4-amino-4- deoxyphrenate dehydrogenase gene. In various embodiments, the microbial cell can include and express, for example: (1 ) a single copy of each of these genes, (2) two or more copies of one of these genes, which can be the same or different, or (3) two or more copies of both of these genes, wherein the copies of a given gene can be the same or different. The same is true for other heterologous genes that can be introduced into the engineered microbial cell.

[0060] The engineered microbial cells can contain introduced genes that have a native nucleotide sequence or that differ from native. For example, the native nucleotide sequence can be codon-optimized for expression in a particular host cell. Codon optimization for a particular host can, for example, be based on the codon usage tables found at www.kazusa.or.jp/codon/. The amino acid sequences encoded by any of these introduced genes can be native or can differ from native. In various embodiments, the amino acid sequences have at least 60 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a native amino acid sequence.

[0061 ] The approach described herein has been carried out in bacterial cells, namely C. glutamicum, and in yeast cells, namely S. cerevisiae. (See Example 2.)

Illustrative Engineered Bacterial Cells

[0062] In certain embodiments, the engineered bacterial (e.g. , C. glutamicum) cell expresses one or more heterologous chorismite aminotransferase(s) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an Anthrobacter sp. ATCC 21022 chorismite aminotransferase (e.g., SEQ ID NO:3); one or more 4-amino-4-deoxychorismate mutase(s) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a Pseudomonas sp. Leaf 48 4-amino-4-deoxychorismate mutase (e.g., SEQ ID NO:17); and/or one or more heterologous 4-amino-4-deoxyphrenate dehydrogenase(s) having at least 70 percent,

75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a Paenibacillus wynnii 4-amino-4-deoxyphrenate dehydrogenase (e.g., SEQ ID NO:28).

Illustrative Engineered Yeast Cells

[0063] In certain embodiments, the engineered yeast (e.g., S. cerevisiae) cell expresses heterologous chorismite aminotransferase(s) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a Streptomyces sp. CB01635 chorismite aminotransferase (e.g., SEQ ID NO:13); one or more 4-amino-4-deoxychorismate mutase(s) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a Streptomyces pristinaespiralis 4-amino-4-deoxychorismate mutase (e.g., SEQ ID NO:18) ; and/or one or more heterologous 4-amino-4- deoxyphrenate dehydrogenase(s) having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a Pseudomonas sp. 2822-17 4-amino-4-deoxyphrenate dehydrogenase (e.g., SEQ ID NO:38).

Culturing of Engineered Microbial Cells

[0064] Any of the microbial cells described herein can be cultured, e.g., for maintenance, growth, and/or 4-aminophenyl pyruvate production.

[0065] In some embodiments, the cultures are grown to an optical density at 600 nm of 10-500, such as an optical density of 50-150.

Culture Media

[0066] Microbial cells can be cultured in any suitable medium including, but not limited to, a minimal medium, i.e. , one containing the minimum nutrients possible for cell growth. Minimal medium typically contains: (1 ) a carbon source for microbial growth ;

(2) salts, which may depend on the particular microbial cell and growing conditions; and

(3) water. Suitable media can also include any combination of the following: a nitrogen source for growth and product formation, a sulfur source for growth, a phosphate source for growth, metal salts for growth, vitamins for growth, and other cofactors for growth.

[0067] Any suitable carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a microbial cell. In various embodiments, the carbon source is a carbohydrate (such as a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide), or an invert sugar (e.g., enzymatically treated sucrose syrup).

Illustrative monosaccharides include glucose (dextrose), fructose (levulose), and galactose; illustrative oligosaccharides include dextran or glucan, and illustrative polysaccharides include starch and cellulose. Suitable sugars include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). Other, less expensive carbon sources include sugar cane juice, beet juice, sorghum juice, and the like, any of which may, but need not be, fully or partially deionized.

[0068] The salts in a culture medium generally provide essential elements, such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize proteins and nucleic acids.

[0069] Minimal medium can be supplemented with one or more selective agents, such as antibiotics.

[0070] To produce 4-aminophenyl pyruvate, the culture medium can include, and/or is supplemented during culture with, glucose and/or a nitrogen source such as urea, an ammonium salt, ammonia, or any combination thereof.

Culture Conditions

[0071 ] Materials and methods suitable for the maintenance and growth of microbial cells are well known in the art. See, for example, U.S. Pub. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2004/033646, WO 2009/076676, WO 2009/132220, and WO 2010/003007, Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.

[0072] In general, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as about 20 S C to about 37 S C, about 6% to about 84% C0 2 , and a pH between about 5 to about 9). In some aspects, cells are grown at 35 S C. In certain embodiments, such as where thermophilic bacteria are used as the host cells, higher temperatures (e.g., 50 S C -75 S C) may be used. In some aspects, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Cells can be grown under aerobic, anoxic, or anaerobic conditions based on the requirements of the particular cell. [0073] Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in U.S. Publ. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2009/076676, WO 2009/132220, and WO 2010/003007. Batch and Fed-Batch fermentations are common and well known in the art, and examples can be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.

[0074] In some embodiments, the cells are cultured under limited sugar (e.g., glucose) conditions. In various embodiments, the amount of sugar that is added is less than or about 105% (such as about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of the amount of sugar that can be consumed by the cells. In particular embodiments, the amount of sugar that is added to the culture medium is approximately the same as the amount of sugar that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added sugar such that the cells grow at the rate that can be supported by the amount of sugar in the cell medium. In some embodiments, sugar does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited sugar conditions for times greater than or about 1 , 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours or even up to about 5-10 days. In various embodiments, the cells are cultured under limited sugar conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited sugar conditions can allow more favorable regulation of the cells.

[0075] In some aspects, the cells are grown in batch culture. The cells can also be grown in fed-batch culture or in continuous culture. Additionally, the cells can be cultured in minimal medium, including, but not limited to, any of the minimal media described above. The minimal medium can be further supplemented with 1 .0% (w/v) glucose (or any other six-carbon sugar) or less. Specifically, the minimal medium can be supplemented with 1 % (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1 % (w/v) glucose. In some cultures, significantly higher levels of sugar (e.g., glucose) are used, e.g., at least 10% (w/v), 20% (w/v), 30% (w/v), 40 % (w/v), 50% (w/v), 60% (w/v), 70% (w/v), or up to the solubility limit for the sugar in the medium. In some embodiments, the sugar levels fall within a range of any two of the above values, e.g. : 0.1 -10% (w/v), 1 .0-20% (w/v), 10-70 %

(w/v), 20-60 % (w/v), or 30-50 % (w/v). Furthermore, different sugar levels can be used for different phases of culturing. For fed-batch culture (e.g., of S. cerevisiae or C. glutamicum), the sugar level can be about 100-200 g/L (10-20 % (w/v)) in the batch phase and then up to about 500-700 g/L (50-70 % in the feed).

[0076] Additionally, the minimal medium can be supplemented 0.1 % (w/v) or less yeast extract. Specifically, the minimal medium can be supplemented with 0.1 % (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01 % (w/v) yeast extract. Alternatively, the minimal medium can be supplemented with 1 % (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5%

(w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1 % (w/v) glucose and with 0.1 % (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), or 0.02% (w/v) yeast extract. In some cultures, significantly higher levels of yeast extract can be used, e.g., at least 1.5% (w/v), 2.0% (w/v), 2.5% (w/v), or 3 % (w/v). In some cultures (e.g., of S. cerevisiae or C. glutamicum), the yeast extract level falls within a range of any two of the above values, e.g. : 0.5-3.0% (w/v), 1 .0-2.5% (w/v), or 1 .5-2.0% (w/v).

EXAMPLES

Example 1 : Preparation of 4-aminomethylaniline (‘AMA’), 4-(2-aminoethyl)aniline

(‘AEA’), and 4-(3-aminopropyl)aniline (‘APA’)

[0077] AMA, AEA, and APA can be prepared biologically, chemically, or by a combination of both methodologies. The following scheme depicts the three asymmetric diamines.

Scheme 1 : Structures of AMA, AEA, and APA.

[0078] Several biological routes can be considered for a biological production via fermentation of AMA, AEA, or APA. As an example, Scheme 2 depicts the shikimate pathway also known as the shikimic acid pathway which can serve as an avenue to aromatic amines including AMA, AEA, or APA. The pathway starts from phospho-enol pyruvate (PEP) and Erythrose-4-phosphate to form chorismate in seven enzymatic steps. Chorismate then undergoes enzymatic transamination to form 4-amino-4- deoxychorismate which serves as a precursor molecule for (i) synthesis of AMA on a first terminal pathway or (ii) AEA and APA on a second and third terminal pathway, respectively.

Scheme 2: Shikimate pathway

Biological synthesis of AM A from 4-amino-4-deoxychorismate

[0079] Referring to Scheme 3, 4-amino-4-deoxychorismate can be aromatized via elimination of pyruvic acid to form p-amino benzoic acid (PABA). This acid can serve as a precursor either via partial reduction or full reduction to form p-amino benzaldehyde or p-amino benzylalcohol. Either compound, aldehyde or alcohol can serve as a substrate for amination. This step can be performed biologically or chemically. In the case of amination of p-amino benzaldehyde the intermediary Schiff base would need another reduction with an appropriate enzyme (biologically) or a reduction agent, such as hydrogen over catalyst, e.g. Raney Nickel, or a homogenous agent, e.g., LiBH 4 or

NaBH 4 . Biological synthesis of AEA from 4-amino-4-deoxychorismate

[0080] Referring to Scheme 4, 4-amino-4-deoxychorismate can undergo enzymatic rearrangement to form 1 -carboxy-4-amino-1 ,4-dihydrophenyl-1 -(3’-2’-oxo-propanoic acid), which via elimination of formic acid forms p-amino-phenylpyruvic acid. This pyruvic acid derivative can be further modified, either biologically or chemically, to form p-amino-phenylalanine. Biological modification includes enzymatic conversion using glutamate. Chemical modification can include, reaction with ammonia or ammonia solution to form the Schiff base followed by heterogenous (e.g. H 2 /cat.) or homogenous (e.g. LiBH 4 or NaBH 4 ) reduction. The resulting p-amino-phenylalanine is only one step from AEA via decarboxylation which can be performed biologically or chemically as well.

Scheme 4: Enzyme pathways to AEA. Biological synthesis of APA from p-amino-phenylalanine

[0081 ] p-Amino-phenylalanine can also serve as a precursor for APA. Here, the biological or chemical synthetic route is designed to preserve the carboxy-carbon as the primary amine carbon of APA. p-Amino-phenylalanine can be deaminated, chemically via Hofmann elimination or biologically via a deaminase enzyme, to form p-amino- cinnamic acid. Reduction of the carboxy group, again chemically or biologically, renders p-amino-cinnamaldehyde or p-amino-cinnamyl alcohol. As conceptually described above, the aldehyde can be rendered to an amine chemically via ammonia treatment and reduction or biologically via a transaminase. For the cinnamyl alcohol, substitution of the hydroxy group with an amino group yields cinnamyl amine. Reduction of the alkene unsaturation, again chemically or biologically, yields APA.

Example 2: Construction and Selection of Strains of Corynebacterium glutamicum and Saccharomyces Cerevisiae for Production of Diamines or

Diamine Precursors by Fermentation

Plasmid/DNA Design

[0082] All strains tested for this work were transformed with plasmid DNA designed using proprietary software. Plasmid designs were specific to each of the host organisms engineered in this work. The plasmid DNA was physically constructed by a standard DNA assembly method. This plasmid DNA was then used to integrate metabolic pathway inserts by one of two host-specific methods, each described below.

C. glutamicum Pathway Integration

[0083] A“loop-in, single-crossover” genomic integration strategy has been developed to engineer C. glutamicum strains. Fig. 4 illustrates genomic integration of loop-in only and loop-in/loop-out constructs and verification of correct integration via colony PCR. Loop- in only constructs (shown under the heading“Loop-in”) contained a single 2-kb homology arm (denoted as“integration locus”), a positive selection marker (denoted as “Marker”)), and gene(s) of interest (denoted as“promoter-gene-terminator”). A single crossover event integrated the plasmid into the C. glutamicum chromosome. Integration events are stably maintained in the genome by growth in the presence of antibiotic (25pg/ml kanamycin). Correct genomic integration in colonies derived from loop-in integration were confirmed by colony PCR with UF/IR and DR/IF PCR primers.

[0084] Loop-in, loop-out constructs (shown under the heading“Loop-in, loop-out) contained two 2-kb homology arms (5’ and 3’ arms), gene(s) of interest (arrows), a positive selection marker (denoted“Marker”), and a counter-selection marker. Similar to “loop-in” only constructs, a single crossover event integrated the plasmid into the chromosome. Note: only one of two possible integrations is shown here. Correct genomic integration was confirmed by colony PCR and counter-selection was applied so that the plasmid backbone and counter-selection marker could be excised. This results in one of two possibilities: reversion to wild-type (lower left box) or the desired pathway integration (lower right box). Again, correct genomic loop-out is confirmed by colony PCR. (Abbreviations: Primers: UF = upstream forward, DR = downstream reverse, IR = internal reverse, IF = internal forward.)

S. cerevisiae Pathway Integration

[0085] A“split-marker, double-crossover” genomic integration strategy has been developed to engineer S. cerevisiae strains. Fig. 5 illustrates genomic integration of complementary, split-marker plasmids and verification of correct genomic integration via colony PCR in S. cerevisiae. Two plasmids with complementary 5’ and 3’ homology arms and overlapping halves of a URA3 selectable marker (direct repeats shown by the hashed bars) were digested with meganucleases and transformed as linear fragments.

A triple-crossover event integrated the desired heterologous genes into the targeted locus and re-constituted the full URA3 gene. Colonies derived from this integration event were assayed using two 3-primer reactions to confirm both the 5’ and 3’ junctions (UF/IF/wt-R and DR/IF/wt-F). For strains in which further engineering is desired, the strains can be plated on 5-FOA plates to select for the removal of URA3, leaving behind a small single copy of the original direct repeat. This genomic integration strategy can be used for gene knock-out, gene knock-in, and promoter titration in the same workflow.

Cell Culture

[0086] The workflow established for S. cerevisiae involved a hit-picking step that consolidated successfully built strains using an automated workflow that randomized strains across the plate. For each strain that was successfully built, up to four replicates were tested from distinct colonies to test colony-to-colony variation and other process variation. If fewer than four colonies were obtained, the existing colonies were replicated so that at least four wells were tested from each desired genotype. [0087] The colonies were consolidated into 96-well plates with selective medium (SD- ura for S. cerevisiae ) and cultivated for two days until saturation and then frozen with 16.6% glycerol at -80 °C for storage. The frozen glycerol stocks were then used to inoculate a seed stage in minimal media with a low level of amino acids to help with growth and recovery from freezing. The seed plates were grown at 30°C for 1 -2 days. The seed plates were then used to inoculate a main cultivation plate with minimal medium and grown for 48-88 hours. Plates were removed at the desired time points and tested for cell density (OD600), viability and glucose, supernatant samples stored for LC-MS analysis for product of interest.

Cell Density

[0088] Cell density was measured using a spectrophotometric assay detecting absorbance of each well at 600nm. Robotics were used to transfer fixed amounts of culture from each cultivation plate into an assay plate, followed by mixing with 175mM sodium phosphate (pH 7.0) to generate a 10-fold dilution. The assay plates were measured using a Tecan M1000 spectrophotometer and assay data uploaded to a LIMS database. A non-inoculated control was used to subtract background absorbance. Cell growth was monitored by inoculating multiple plates at each stage, and then sacrificing an entire plate at each time point.

[0089] To minimize settling of cells while handling large number of plates (which could result in a non-representative sample during measurement) each plate was shaken for 10-15 seconds before each read. Wide variations in cell density within a plate may also lead to absorbance measurements outside of the linear range of detection, resulting in underestimate of higher OD cultures. In general, the tested strains so far have not varied significantly enough for this be a concern. Liquid-Solid Separation

[0090] To harvest extracellular samples for analysis by LC-MS, liquid and solid phases were separated via centrifugation. Cultivation plates were centrifuged at 2000 rpm for 4 minutes, and the supernatant was transferred to destination plates using robotics. 75mI_ of supernatant was transferred to each plate, with one stored at 4°C, and the second stored at 80 °C for long-term storage.

Genetic Engineering Results

[0091 ] C. glutamicum and S. cerevisiae were engineered for production of APA or a precursor thereto by expressing three enzymes in the cells: chorismite

aminotransferase, 4-amino-4-deoxychrismate mutase, and 4-amino-4-deoxyphrenate dehydrogenase (Enzymes 1 -3 in Scheme 5, above). The results for various different strain designs are shown in Table 2. Enzyme sequence information is found in Table 3.

Table 2: Genetic Engineering Results

4APE = 4-aminophenylethanol

4APHE = 4-aminophenylalanine

All genes were codon-optimized using Table 1 (the Modified Codon Usage Table for Sc and Cg), above.

Table 3: SEQ ID NO Cross-Reference Table

Example 3: Chemical Syntheses of Diamines

Chemical synthesis of AM A

2.3 24

[0092] In addition to the above mentioned routes, AMA can be synthesized by direct reduction of 4-aminobenzylnitrile derivatives (22), or 4-nitrobenzylamine (20) using heterogeneous or homogeneous routes. Moreover 4-nitrobenzonitrile (19) can be reduced in a similar fashion either directly, or via 22 or 20. 19 and 22 can be synthesized through aromatic substitution from nitrobenzene derivative (18) and aniline derivative (21 ), respectively. Alternatively transformation of 4-nitrobenzaldehyde (23) to AMA can be facilitated via the oxamine 24, followed by heterogeneous or homogeneous reduction.

Chemical Synthesis of AEA

[0093] In addition to the above mentioned routes, AEA can also be synthesized by nitro reduction of 4-nitrophenylethanamine and derivatives (3) using heterogeneous or homogeneous routes and nitrile reduction via heterogeneous (Pd/C, Ni, etc.) or homogeneous means from either 4-aminophenylacetonitrile derivatives (6) or 4- nitrophenylacetonitrile (2) the former available commercially, or reduced from the latter via heterogeneous or homogeneous means. 2 and 3 can also be prepared by nitration of phenylacetonitrile (1) and phenylethanamine and derivatives (7) respectively. 6 can also be prepared through the substitution of amino toluene derivatives functionalized with leaving groups in the benzyl position (5) which in turn can be synthesized from aminotoluene derivatives (4). Chemical Synthesis of APA

[0094] APA can be synthesized by reduction of 1 -(4-aminophenyl)-3-aminopropene derivatives (15) or 1 -(4-nitrophenyl)-3-aminopropene derivatives (17), formed through metal catalyzed cross coupling reactions from the corresponding functionalized 4- aminobenzene (14) and 4-nitrobenzene (16) respectively, using heterogeneous or homogeneous reductions in one or two steps. Alternatively it can be made from the nitrile reduction of 4-nitrophenylpropionitrile (12) via 4-aminophenylpropionitrile (13). 12 can be formed through a rearrangement of amide 11 which is formed by substitution of 4-nitrophenylpropionic acid (9) after the generation of acid chloride 10. 9 can be generated by addition of meldrum’s acid to aldehyde 8. Example 4: Production of Diamines by Fermentation and Chemical Synthesis

Combination of both methodologies for the Synthesis of AMA

[0095] 4-Aminobenzylnithle derivatives (22) can be prepared from PABA using ethyl carbamate and thionyl chloride (Journal of Chemical Technology and Biotechnology, 83(10), 1441 -1444; 2008): from fermentation

[0096] 4-aminobenzylnitrile (22) can be subsequently hydrogenated using Raney nickel to AMA.

Combination of both methodologies for the Synthesis of AEA

[0097] AEA can be synthesized through a combination of synthetic transformations from precursors produced through fermentation or from biological or synthetic sources. Reduction of carboxylic acid 25 to the aldehyde 26 followed by homologation with nitromethane gives nitro analogue 27 which can be reduced to AEA by either homogeneous or heterogeneous means. Alternatively 25 may be reduced fully to the alcohol, which can then be converted into a suitable leaving group (5) to give the nitrile (6) through substitution as above. This can then be reduced by heterogeneous or homogeneous means to AEA. 6 can also be prepared from 29 or 28 through variations on the schmidt reaction. Aminoalcohol 30 can also be converted to AEA, either directly through amination, or by substitution of analogue 31 prior to cleavage of any protecting groups used. Combination of both methodologies for the Synthesis of APA

[0098] APA can be synthesized through a combination of synthetic transformations from precursors produced through fermentation or from biological or synthetic sources. Reduction of aminophenylacetic acid 29 to alcohol 30 prior to the conversion of the alcohol into a suitable leaving group in compound 31 allows for substitution to the aminophenylpropionitrile 13 which can be reduced to give APA.

[0099] Alternatively, 13 may be prepared through a combination of reduction and functional group transformation from the carboxylic acid from 33 via either 34 or 35. Partial reduction of the aminophenylpropionic acid 35 to aldehyde 36 allows for imine formation to give compound 37 which may be reduced to APA by synthetic means. Conversely, if 35 is reduced fully to aminophenylpropanol 38 then the alcohol may be converted to a better leaving group in 39 and this may undergo substitution to give APA.

Example 5: Amine Stability Screening Experiments

Efficacy of the stabilizers at preventing oxidation and thereby color change were screened in a by UV/Vis measurement in a 96 well plate over time with exposure to heat and air. Stabilizer concentrations were explored from 0.0005 to 5% stabilizer loading and sulfur compounds fared superior as shown in the figure below.

[0100] Figure 1 depicts the results for DTT and Figure 2 depicts the results for cysteine. Both graphs depict the absorbance spectra for AMA stabilized with the sulfur compound at various wt%. The absorbance spectra were measured after leaving samples at room temperature for 14 days. As can be seen from both graphs, a stabilizer concentration of as little as 0.5 wt% shows a shift of the absorbance spectra to the extent that the absorbance at 380 nm decreases substantially. As a result, there is insignificant to no absorption in the visible region (~ 380 nm - 750 nm).

INFORMAL SEQUENCE LISTING

The amino acid sequences of the enzymes using in the strain designs of Table 2 are listed below (in single-letter code).

Chorismate Aminotransferase

< 1 ; Protein/1 ; Pseudomonas rhizosphaerae>