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

A technology of ferulic acid and vanillin, applied in the field of identification of natural vanilla products, can solve problems such as product errors and wrong evaluations

Active Publication Date: 2016-11-09
BGN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is clear that in specific cases, without knowledge of the production conditions or influencing factors, the interpretation of analytical results can lead to incorrect assessments and misidentification of a product as synthetic, when in fact it may have been produced from Plant and animal materials or obtained from microbial enzymatic biotransformation / biocatalysis and should therefore be considered natural products

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0303] Example 1. Generation of ferulic acid from eugenol in a 25ml (0.025L) shake flask

[0304] Short-chain alcohol dehydrogenase (MtSAD1) and aldehyde dehydrogenase (AtADH) were cloned from Medicago truncatula and Arabidopsis thaliana, respectively. MtSAD1 and AtADH were cloned from Medicago truncatula (ecotype A17) and Arabidopsis thaliana (ecotype Columbia-0). Total plant RNA was extracted using Trizol Plus RNA Purification Kit (Invitrogen Inc.). Using Im Prom-II from Promega Inc. TM The reverse transcription system was used for cDNA synthesis according to the manufacturer's manual. Using the primers listed in Table 2, the gene was amplified from the synthesized cDNA using New England Biolab's Phusion PCR kit.

[0305] After digesting the PCR product with corresponding restriction enzymes, MtSAD1 was inserted into the Nde I and Xho I cloning sites of the pRSFDuet-1 vector. Insert AtADH into the Bgl II and Xho I cloning sites of pCDFDuet-1. After sequence confirmation...

Embodiment 2A

[0342] Example 2A. Ferulic acid production using eugenol in a 30 L volume in a 50 L fermenter

[0343] A 1 ml glycerol stock solution of E. coli cells containing CgVaoA-pETDuet and MtSADrbsAtADH-pRSFDuet was inoculated into 1 L of LB medium containing 100 mg / L ampicillin and 30 mg / L kanamycin and seeded at 37 Cultivate overnight at °C, and then transfer to 30 L LB medium containing 100 mg / L ampicillin and 30 mg / L kanamycin in a 50 L fermenter. The initial temperature was set at 37 °C, stirring at 300 rpm, the dissolved oxygen (DO) was kept above 30%, the air was 0.6 vvm, and the pH was not controlled at the beginning. After 1-1.5 hours, the temperature was lowered to 30 °C and 2 L of 22.5 g / L lactose was added to bring the final concentration to 15 g / L to start the induction. At this point the volume is about 33-34 L. Keep the air at 0.6vvm, increase the agitation to 350rpm, and keep the DO above 15%. After 14-16 hours of fermentation, the substrate eugenol was added, the t...

Embodiment 2B

[0351] Example 2B Biotransformation of Ferulic Acid to Vanillin

[0352] Purified ferulic acid (FA) converted from eugenol using our VaoA-MtSAD1-AtADH system was used as substrate for vanillin production. FA was converted to natural vanillin (NV) using Amycobacterium sp. strain (Zhp06) and the method described in US 2013 / 0115667A1 and CN102321563B1. Applicant's results showed that a yield of 13.4 g / L vanillin was obtained from 21.6 g / LFA, corresponding to a molar yield of vanillin of 79.2% ( Figure 8 ). During fermentation, the accumulation of vanillin was roughly linear. However, Applicants observed a rapid decrease in FA in the first 24 hours, suggesting that S. chlorosporium converts FA to a different intermediate before FA is converted to vanillin.

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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 Amycolalopsis sp. strain (Zhp06) and / or a recombinant E.coli strain.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C § 119(e) to U.S. Provisional Application 61 / 899,456, filed November 4, 2013, the contents of which are incorporated herein by reference in their entirety. technical field [0003] The present disclosure generally relates to methods and / or materials for catalyzing the bioconversion of eugenol to ferulic acid in bacteria, yeast, or other cellular systems using plant dehydrogenases. The present disclosure also relates to the production of vanillin, the production of vanillin using the methods and materials, and the identification of the vanilla product as a natural vanilla product. Background technique [0004] Vanilla flavor is one of some of the most commonly used spices in the world. They are used to flavor a variety of foods such as ice cream, dairy products, desserts, confectionary, baked goods and alcohol. They are also used in perfumes, pharmaceuticals and personal...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12P7/24C12P7/40C12P7/42C07C47/58
CPCC12P7/22C12P7/24C12P7/40C12P7/42C12N15/70
Inventor R·周X·余
Owner BGN TECH
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