# US 2020/0399666 A1 · Method for preparation of diester derivatives of malonic acid

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- Number: US 2020/0399666 A1
- Document ID: US20200399666A1
- Status: Published application
- Kind: A1
- Application number: 16/969,517
- Filed: 2019-02-12
- Published: 2020-12-24
- Earliest claimed: 2018-02-13
- Assignees: Lygos, Inc.
- Applicants: Lygos, Inc.
- Inventors: Jeffrey A. Dietrich; Eric Steen; Johan van Walsem
- Web page: https://patentgrep.com/patent/US20200399666A1

## CPC

- C12P 7/46: Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
- C07C 67/58: by liquid-liquid treatment
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS

## Abstract

Methods for the preparation of bio-based malonic acid and diester derivatives of malonic acid are provided. The disclosed methods produce diester derivatives of malonic acid with fewer impurities, which is useful for many industrial processes. The diester derivatives of malonic acid can be purified from existing sources of malonic acid, or from malonic acid made from a renewable carbon source.

## Claims

### Claim 1

A method for isolating diester derivatives of malonic acid from fermentation broth, comprising:
separating calcium malonate crystals of at least 10 microns in diameter from the fermentation broth by centrifugation;
dissolving malonic acid from the calcium malonate crystals in a gypsum reactive crystallizer;
recrystallizing the dissolved malonic acid in a malonic acid crystallizer;
refluxing the crystalline malonic acid with a dialkyl carbonate and sulfuric acid to generate diester derivatives of the malonic acid; and
isolating the diester derivatives of malonic acid via distillation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the centrifugation is done using a hydrocyclone apparatus.

### Claim 3 (depends on 1)

The method of claim 1, wherein the centrifugation is done using a decanter centrifuge.

### Claim 4 (depends on 1, 2, 3)

The method of any of claims 1-3, further comprising introduction of seed crystals into the fermentation broth.

### Claim 5 (depends on 4)

The method of claim 4, wherein the seed crystals are introduced before the step of separating calcium malonate crystals.

### Claim 6 (depends on 4, 5)

The method of claim 4 or claim 5, wherein the seed crystals are calcium malonate seed crystals.

### Claim 7 (depends on 6)

The method of claim 6, wherein the calcium malonate seed crystals are introduced at a concentration of at least 6 g/l.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The method of any one of claims 1-7 wherein the pH of the fermentation broth is maintained at around 5.0.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of any one of claims 1-8, further comprising fermenting a malonate-producing microorganism.

### Claim 10 (depends on 9)

The method of claim 9, wherein the microorganism is a yeast selected from Saccharomyces and Pichia.

### Claim 11 (depends on 9, 10)

The method of claim 9 or claim 10, wherein the microorganism is Pichia kudriazevii.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of any one of claims 1-11, wherein the fermentation broth comprises succinate at a concentration below 11 g/l.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The method of any one of claims 1-12, wherein the centrifugation is carried out in two centrifugation steps.

### Claim 14 (depends on 13)

The method of claim 13, wherein two centrifugation steps comprise:
a) centrifugation of the fermentation broth at high g-force;
b) resuspension of solids to a concentration of 5-25%; and
c) centrifugation of the solids at a lower g-force.

### Claim 15 (depends on 14)

The method of claim 14, wherein the lower g-force centrifugation is carried out in a hydrocyclone apparatus.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-15, wherein the ratio of malonic acid to dialkyl carbonate to sulfuric acid in the refluxing step is between 1:5:0.5 and 1:2.5:0.5.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any one of claims 1-16, wherein the distillation is performed at or near 20 torr.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The method of any one of claims 1-17, wherein the refluxing is performed for 1 to 8 hours.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any one of claims 1-18, further comprising refluxing the diester derivatives in deionized water after distillation.

### Claim 20 (depends on 19)

The method of claim 19, wherein the ratio of deionized water to the diester derivatives is 1:1.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The method of any one of claims 1-20, wherein the distillation is performed more than once.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The method of any one of claims 1-21, wherein the distillation is performed in a fractionating column.