EP4028535A1

EP4028535A1 - <smallcaps/>? ? ?acinetobacter soli? ? ? ? ?process for production of 2-phenylethanol by a selectedstrain and uses thereof

Info

Publication number: EP4028535A1
Application number: EP20745298.8A
Authority: EP
Inventors: Luísa PEIXE; Filipa LEDO; Maria REIS; Cristiana TORRES
Original assignee: Universidade do Porto; Universidade Nova de Lisboa
Current assignee: Universidade do Porto; Universidade Nova de Lisboa
Priority date: 2019-09-12
Filing date: 2020-07-09
Publication date: 2022-07-20
Also published as: PT115777B; PT115777A; WO2021048645A1

Abstract

The present invention relates to a process of naturally producing 2-PE by using Acinetobacter soli bacterium. For this purpose, A. soli strains selected from environmental isolates were cultured in a bioreactor under average fermentation conditions. A. soli strain ANG 344B (Accession Number 1593) is particularly able to improve the production of 2-PE resulting in high yields of said compound. This compound is responsible for the scent of roses having a high commercial price when compared with the synthetically produced compound. It can be used in food, cosmetic and pharmaceutical sectors as organoleptic enhancer, antibacterial and antifungal agent. Moreover, 2-PE is also a valuable alcohol that can be used as a next generation biofuel, and as a raw material for its derivatives in pharmaceutical and fine chemical industries. Therefore, the present invention is in the field of microbiology, chemistry and biotechnology and has application in the food, cosmetic, pharmaceutical and fuel industries.

Description

DESCRIPTION

Process for production of 2-phenylethanol by a selected Acinetobacter soli strain and uses thereof

TECHNICAL DOMAIN OF THE INVENTION

The present invention relates to a process for production of 2-phenylethanol (2-PE) by an Acinetobacter soli microorganism cultured in a low-cost media and average fermentation conditions.

This process results in high yields of said compound that can be used in food, cosmetic and pharmaceutical sectors as organoleptic enhancer, antibacterial and antifungal agent. Moreover, 2-PE is also a valuable alcohol for the next generation biofuel and is used as a raw material for its derivatives in pharmaceutical and fine chemical industries.

The present invention also relates to an Acinetobacter soli strain that was selected from environmental isolates. This strain is useful in the production of 2-PE and was deposited under the Budapest Treaty regulations, having received the Identification Reference Number ANG 344B given by the Depositor, and Accession Number 1593 given by the IDA, the University of Latvia (MSCL).

Therefore, the present invention is in the field of microbiology, chemistry and biotechnology and has application in the food, cosmetic, pharmaceutical and fuel industries. BACKGROUND OF THE INVENTION

Compound 2-Phenylethanol (2-PE) is the responsible for the scent of roses and is the most used fragrance in perfume and cosmetics with an estimated world market of about 10,000 t per year, according to data from 2010. The majority of 2-PE currently used is produced chemically. However, its quality is greatly reduced due to the toxic reagents used in the synthesis (e.g., petrochemicals such as benzene, styrene oxide, or propylene) and in the purification process, that may result in the formation of undesirable by-products that affect the 2-PE organoleptic characteristics.

Due to this, natural 2-PE is preferred rather than the one resulting from chemical synthesis. Natural 2-PE has a market volume of 0.5-1 t per year being sold at market prices of 1000 euros per kilogram, whereas the same compound from chemical synthesis presents a market price of only 3.5 euros per kilogram. Moreover, consumers also tend to prefer natural aromas due to the belief that are healthier and environmentally friendly, and thus is important to provide new natural sources for this compound production.

Some microorganisms may produce 2-PE and 2-phenethyl acetate (2-PEA) as a result of their natural metabolisms, which include the Shikimate and the Ehrlich pathways. Among these microorganisms we may find mainly yeasts, like Saccharomyces cerevisiae, fungi like Aspergillus niger, and bacteria like Enterobacter sp. Additionally, some strains of S. cerevisiae, Enterobacter sp. and Escherichia coll were subjected to genetic modifications for 2-PE and 2-PEA high yield production. However, worldwide consumers have also concerns regarding the use of genetic modified organisms (GMO) and tend to prefer GMO-free products.

The present invention overcomes the prior art problems mentioned above by providing a process to produce to produce 2-PE and 2-PEA, by a naturally occurring bacterium belonging to Acinetobacter soli species isolated from environment and in particular a specific strain of A. soli for improving the production of said compounds.

SUMMARY OF THE INVENTION

The present invention relates to a process for production of 2-phenylethanol (2-PE) by Acinetobacter soli bacterium cultured in a low-cost media and average fermentation conditions and to a specific strain of A. soli.

Therefore, in one aspect, the present invention relates to an Acinetobacter soli strain ANG 344B with Accession Number 1593 given by the IDA, the University of Latvia (MSCL), according to claim 1.

This strain is particularly advantageous in the production of high yields of 2-PE and 2-PEA.

In a second aspect, the present invention relates to a process of producing 2-PE by using Acinetobacter soli bacterium, according to claim 2. This process allows to obtain high yields of 2-PE and 2-PEA in a simple, natural and environmentally friendly way without the need to use oils or genetically modified organisms.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to a process for production of 2-phenylethanol (2-PE) by a microorganism of Acinetobacter soli genus and to a specific Acinetobacter soli strain selected from natural isolates for improving the 2-PE production.

1. Acinetobacter soli microorganism

Acinetobacter is a genus of Gram-negative bacteria belonging to the wider class of Gammaproteobacteria, order Pseudomonadales and family Moraxellaceae. Acinetobacter species are oxidase-negative, exhibit twitching motility, and occur in pairs under magnification. These microorganisms are strictly aerobic, non-fermentative, Gram-negative bacilli,

Acinetobacter genus is characterized by a ubiquitous distribution in nature and a versatile metabolism reflected in its capacity to use different carbon sources to grow, depending on the ecological niche. As a result, Acinetobacter strains have been explored for biotechnological applications such as the degradation of different organic, inorganic and heavy metals pollutants. Important bio-products from Acinetobacter strains have been also explored, such as polysaccharides, polyesters, and lipases. In addition, bioemulsifiers produced by different Acinetobacter strains and already commercially available. Some examples of these are emulsan produced by Acinetobacter calcoaceticus RAG-1, A. calcoaceticus BD4 RAG- 1, biodispersan produced by Acinetobacter calcoaceticus A2, and alasan produced by Acinetobacter radioresistens KA53, which are currently used in microbial enhanced oil recovery and biodegradation of toxic compounds.

When performing laboratory screenings aiming to detect antibiotic resistant bacteria from environmental sources it was surprisingly noticed a rose scent from a group of agar plates, where bacterial strains from river water samples were grown. After several steps of isolation and purification processes, it was possible to relate said scent to a particular Acinetobacter sp. strain.

Species identification was initially performed by partial sequencing the rpoB (b subunit of RNA polymerase) gene. Strain characterization also involved whole genome sequencing that allowed the identification of different enzymes involved in the Shikimate pathway as important for the rose scent production. It is of note the uniqueness of those genes among Acinetobacter genus, i.e. do not present identity to any other bacterial genus. The most closely related genome to this one is of A. soli strain GFJ2 (genbank accession number NZ_CP016896.1). Nevertheless, a relevant enzyme to the Shikimate pathway was absent, namely the aromatic-amino-acid transaminase involved in phenylalanine metabolism (EC 2.6.1.57).

Preliminary studies using a bioreactor showed that 2-PE was produced as a primary metabolite, using L-Phe as the main nitrogen source. Therefore, with the aim of improving the production of 2-PE via non-genetically manipulated bacteria this specific: strain of Acinetobacter soli was further analysed and surprisingly presented the highest 2-PE production yield described among wild-type bacteria.

In the scope of the present invention, this strain was deposited under the Budapest Treaty regulations, having received the Identification Reference Number ANG 344B given by the Depositor, and Accession Number 1593 given by the IDA, the University of Latvia.

2 . Cultivation of A. soli

The cultivation medium for growing A. soli in average conditions comprises a carbon source, a nitrogen source, which is typically provided by yeast extract, L-phenylalanine a precursor of 2-PE, and inorganic salts.

Several types of carbon source are adequate for growing A. soli, including food and industrial wastes or by-products, such as for example cheese whey, fruit pulps or fruit peels. However, the preferential carbon source is glucose, and the optimum concentration is in the range of 5 and 25 g/L, preferably 10 and 20 g/L.

In the scope of the present invention, fruit pulp or fruit peels, adequate as glucose source for A. soli cultivation are peach, pineapple, apple, grapes and banana.

When peach, pineapple, apple, grapes and banana are present in the cultivation medium their concentration may vary in order to obtain a glucose concentration between 5 and 25 g/L of glucose, preferably 10 and 20 g/L.

Nitrogen sources adequate for growing A. soli include ammonium salts, such as ammonium chloride or ammonium sulphate or urea. A preferable nitrogen source is provided by yeast extract with a concentration variable in the range of 0.4 and 2.0 g/L.

L-phenylalanine is preferably added as the precursor of 2-PE, more preferably with a minimum concentration of 0.5 g/L. The conversion time was 3-26 hours.

3. 2-phenylethanol

Phenethyl alcohol, or 2—phenylethanol, is a primary alcohol compound of formu1a I (C6H₅CH2CH2) having a phenethyl group attached to OH.

Formula I

It is a colourless liquid that is slightly soluble in water (2 ml/100 ml H2O), but miscible with most organic solvents. It occurs widely in nature, being found in a variety of essential oils such as in extract of rose, carnation, hyacinth, Aleppo pine, orange-blossom, ylang-ylang, geranium, neroli and champaca, thus being frequently used as fragrance in perfume and cosmetics. Several microorganisms, such as yeast and bacteria are able of producing 2-PE and 2-phenethyl acetate (2-PEA) as a result of their natural metabolisms, although A. soli was never described as one of them.

2 . Process for the production of 2-PE

The process of the present invention comprises the production of 2-phenylethanol by cultivating the strain Acinetobacter soli ANG 344B in conventional culture media as described above or other culture media comprising food and industrial wastes or by-products, such as for example cheese whey, fruit pulps or fruit peels as source of glucose as strategy to reduce the 2-PE production costs, and in parallel valorizing food waste components.

The bioreactor cultivation is initiated by the inoculation of the selected microorganism in the aqueous nutrient medium. Aeration is a preferred condition, in particular aeration with compressed air. The volume of inoculum should be within the range of 5 to 20% of the total reaction medium volume.

The temperature is controlled between the range of 20°C and 40°C, preferably in the range of 25°C and 35°C, and the pH value is controlled in the range of 5.0 and 9.0, preferably between 6.5 and 7.5.

The air flow rate can be kept between 0.5 and 2 vvm (volume of gas per volume of reactor). The dissolved oxygen concentration is controlled by the automatic variation of the stirring speed between 100 and 2000 rpm, preferably between 300 and 1000 rpm. Aromatic compound production is concomitant with the cellular growth, when cellular growth slows down or ends due to, for example, nitrogen limitation, the 2-phenylethanol production also stops.

The synthesis of the aromatic compounds of the invention may be carried out in a batch, fed-batch, repeated fed-batch or in a continuous process. These alternative bioreactor operations modes enable to optimize the process, since new inoculum and bioreactor preparation are eliminated.

The 2-phenyethanol is extracted from the fermentation broth by the conventional methods existents in the art, namely pervaporation and vacuum distillation.

EXAMPLES

Example 1. Cultivation of Acinetobacter soli (ANG344B) in conventional culture media

A culture of Acinetobacter soli (ANG344B) was inoculated in 2 L of culture media with the composition described in Table 1. The bioreactor (BioStat B-Plus, Sartorius) was operated under controlled conditions: temperature at 30°C; pH 7.00±0.05 with automatic addition of NaOH 5M and HC1 2M and a constant aeration of 1 vvm. The dissolve oxygen decreased gradually from 99% to 30% during the first 6 h and was controlled at 30% by the automatic control of the stirring speed between 300 and 1000 rpm. Table 1 - Composition of the culture medium.

At the end of 13h of cultivation run, cellular growth ended and a CDW (cell dry weight) concentration of 4.80 g/L. The cultivation run ended 24h after the start up.

It was obtained a production of 2.60 g/L of 2-phenylethanol. The productivity attained was 0.2 g/L.h and the yield from L- phenylalanine was 0.7 g/g.

Example 2. Cultivation of Acinetobacter soli (ANG344B) in alternative culture media

In a strategy to reduce the 2-PE production costs, and in parallel valorizing food waste components, 2-PE production was assayed using banana peel to substitute the glucose in the medium.

For this purpose, an alternative to the conventional culture medium was prepared, which included banana peel extract, L-phe (5 g/L), Na₂HP0₄ (4 g/L), K₂HP0₄ (1 g/L), NaCl (0,2 g/L), MgS04.7H20 (0,2 g/L), CaC12.H20 (0,05 g/L), and yeast extract (5 g/L). Banana peel extract was obtained by the following procedure:

- banana peel (812,02 g) + 672 mL of water. Banana peels and water were mixed in a blender and the mixture was hydrolyzed in autoclave for 40 min (121°C, 1 bar). After, mixture was centrifuged at 10000 rpm during 30 min, 4°C and the supernatant was recovered. The pH was adjusted to 7 with NaOH (pH=4.993) and finally, filtered with paper filter (20 pm).

A. soli ANG 344B was grown in 200 mL of culture medium at 30°C, with agitation (200 rpm) during 96 h, A total of 2,74 g/L of L-Phe was used.

The 2-PE production reached a total of 1,043 g/L, with a yield of 0.38,

Example 3. Production of 2-PE by different microorganisms

Wild-type and modified bacteria have been characterized in different optimized culture media for accessing the 2-PE production (1-6) - COMP results. Acinetobacter soli ANG344B MSCL 1593 was cultivated in the same conditions as of Example 1 - INV results. Table 2 below shows the obtained results in comparison with available ones in the literature.

Table 2 - 2-PE microorganism production

Acinetobacter soli ANG344B MSCL 1593 presented a 2-PE production concentration higher than those reported in literature for other wild type bacteria (Table 2). On the other hand, the 2-PE concentration achieved (2600 mglu¹) is in the range of genetically modified bacteria (285 - 5320 mglu¹), which were optimized to increase greatly the 2-PE production, whereas the 2-PE concentration (INV results) was achieved under non-optimized conditions in a batch bioreactor. References of Table 2

1- Zhang H, Cao M, Jiang X, Zou H, Wang C, Xu X, Xian M. 2014. De-novo synthesis of 2-phenylethanol by Enterobacter sp. CGMCC 5087. BMC Biotechno, 14:30.

2- Jollivet N, Bezenger MC, Vayssier Y et al. 1992. Production of volatile compounds in liquid cultures by six strains of coryneform bacteria. Appl Microbiol Biotechnol, 36: 790.

3- Deetae P, Spinnler HE, Bonnarme P, Helinck S. 2009. Growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp. during ripening in a cheese model medium. Appl Microbiol Biotechnol. 82(1):169-77.

4- Kang Z, Zhang C, Du G, Chen J. 2014. Metabolic Engineering of Escherichia coli for Production of 2-phenylethanol from Renewable Glucose. Appl Biochem Biotechnol. 172(4):2012-21.

5- Wang P, Yang X, Lin B, Huang J, Tao Y. 2017. Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol. Metabolic Engineering. 44: 143-149.

6- Hwang JY, Park J, Seo JH, Cha M, Cho BK, Kim J, Kim BG. 2009. Simultaneous synthesis of 2-phenylethanol and L- homophenylalanine using aromatic transaminase with yeast Ehrlich pathway. Biotechnol. Bioeng. 102 (5), 1323-1329

Claims

1. A bacteria of Acinetobacter soli species, strain ANG 344B with Accession Number 1593 (MSCL).

2. A bacteria according to claim 1 for producing 2-PE and/or 2-PEA.

3. A process for producing 2-PE and/or 2-PEA comprising the culture of an Acinetobacter soli strain ANG 344B, as described in any of the claims 1 or 2, in an adequate culture medium.

4. A process according to claim 3 wherein the volume of A. soli inoculum is in the range of 5 to 20% of the total reaction medium volume.

5. A process according to any of the claims 3 or 4 wherein the culture media comprises L-phenylalanine, preferably in a concentration of more than 0.5 g/L, more preferably in a concentration of 0.5 g/L and 10 g/L, even more preferably in a concentration of 0.5 and 5 g/L.

6. A process according to any of the claims 3 to 5 wherein the culture media comprises glucose as carbon source, preferentially in a concentration of 5 and 25 g/L, preferably 10 and 20 g/L, and yeast extract as nitrogen source, preferably in a concentration of 0.4 and 2.0 g/L.

7. A process according to any of the claims 3 to 6 wherein the culture media comprises food and industrial wastes or by-products as carbon source.

8. A process according to any of the claims 3 to 5 wherein the carbon source in the culture media consists in food and industrial wastes or by-products.

9. A process according to any of the claims 7 or 8 wherein the food and industrial wastes or by-products comprise cheese whey, fruit pulps and/or fruit peels as carbon source.

10. A process according to any of the claims 3 to 9 wherein the Acinetobacter soli is cultivated under aeration conditions, preferably 0.5 and 2 vvm (volume of gas per volume of reactor).

11. A process according to any of the claims 3 to 10 wherein the Acinetobacter soli is cultivated in a batch under continuous fed-batch process.

12. A process according to any of the claims 3 to 11, wherein the 2-PE is extracted by pervaporation and vacuum distillation.