Methods of making vanillin via the microbial fermentation of ferulic acid from eugenol using a plant dehydrogenase

• US 9,932,610 B2
• Filed: 11/04/2014
• Issued: 04/03/2018
• Est. Priority Date: 11/04/2013
• Status: Active Grant

• Current Assignee: Bgn Tech LLC
• Original Assignee: Bgn Tech LLC
• Inventors: Zhou, Rui, Yu, Xiaodan
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US15/033,980
• Publication Number:
  US 20160312250A1
• Time in Patent Office: 1,246 Days
• Field of Search: 435189, 435190, 435243, 4352528, 435132, 435155, 435156, 536 231, 536 232, 530350
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12P 7/22 aromatic
• CPC Class Codes: C12P 7/24 containing a carbonyl group
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids

Abstract

A bioconversion method of making vanillin including expressing VaoA gene in a mixture, expressing MtSAD1 gene in the mixture, feeding eugenol to the mixture, and converting ferulic acid to vanillin by incubating with a microbial Amycolatopsis sp. strain (Zhp06) and/or a recombinant E. coli strain.

Claims

1. A bioconversion method of making vanillin comprising:

expressing a Vanillyl-alcohol oxidase (VaoA) gene product obtained from Penicillum species and

a Saponin-deficient 1 (mtSAD1) gene product obtained from Medicago species expressed in a Escherichia coli (E. coli) host cell;

feeding eugenol to the E. coli host cell;

wherein

the E. coli host cell converts ferulic acid to vanillin, thereby making vanillin.

2. The bioconversion method of making vanillin of claim 1, wherein the MtSAD1 gene product comprises an amino acid sequence selected from the group consisting of:

SEQ ID No. 1;

an amino acid sequence with at least 90% or at least 95% identity to SEQ ID No. 1; and

an amino acid sequence expressed from E. coli.

3. The bioconversion method of making vanillin of claim 1, wherein the VaoA gene product comprises an amino acid sequence selected from the group consisting of:

SEQ ID No. 5;

an amino acid sequence with at or at least 90% or at least 95% identity to SEQ ID No. 5; and

an amino acid sequence expressed from E. coli.

4. The bioconversion method of making vanillin of claim 1 further comprising expressing an Alcohol Dehydrogenase (ADH) gene product of SEQ ID No. 3.

5. The bioconversion method of making vanillin of claim 1, further comprising the step of purifying the VaoA, MtSAD1 and/or ADH gene product as a recombinant protein.

6. The bioconversion method of making vanillin of claim 1, wherein converting ferulic acid to vanillin further comprises incubating ferulic acid with a microorganism selected from the group consisting of Pseudomonas sp. HR 199, Amycolatopsis sp. ATCC 39116, Amycolatopsis sp. HR167, Sphingomonas paucimobilis SYK-6, Pseudomonas fluorescens AN103, Streptomyces setonii, Streptomyces sannanensis, Amycolatopsis sp. strain (Zhp06) and a combination thereof.

7. A composition comprising vanillin obtained by the method of claim 1, wherein the vanillin has a δ

¹³C value range of −

25 to −

32 that differs from the δ

¹³C value range of vanillin obtained in other plant sources selected from the group consisting of rice, maize, sugar beet, wheat and curcumin; and

wherein the value range differs from vanillin obtained from lignin and guaiacol.

8. A method of flavoring a consumable product, the method comprising:

the vanillian prepared by the method of claim 1, wherein the vanillin is admixed with the consumable product.

9. The method of claim 8, wherein the consumable product is selected from the group consisting of a food product, a pharmaceutical composition, a dietary supplement, a nutraceutical, and a cosmetic product.