Saccharomyces cerevisiae engineering strain for improving 2-phenylethanol synthesis capacity and construction method thereof

Abstract

The application discloses an engineering strain for improving the 2-phenylethanol synthesis capacity of Saccharomyces cerevisiae and a construction method thereof. The method is to enhance the expression of a mitochondrial citrate transporter coding gene CTP1 in a Saccharomyces cerevisiae host. An expression cassette composed of a PGK1 promoter, a CTP1 coding sequence and a PGK1 terminator is transformed into the host cell to obtain a CTP1 overexpression engineering strain. The engineering strain is used for producing 2-phenylethanol by fed-batch fermentation with L-phenylalanine as a precursor, and can improve the 2-phenylethanol accumulation and L-phenylalanine conversion level while maintaining cell growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering and fermentation engineering technology, specifically relating to an engineered strain that enhances the ability of 2-phenylethanol synthesis by enhancing the expression of the mitochondrial citrate transport protein encoding gene CTP1 in Saccharomyces cerevisiae, its construction method, and its application in the fermentation production of 2-phenylethanol. Background Technology

[0002] 2-Phenylacetyl alcohol is an aromatic alcohol compound with a rose-like aroma, widely used in food, cosmetics, fragrances, and pharmaceuticals. Compared with chemical synthesis and plant extraction, microbial fermentation for the production of 2-phenylethanol offers advantages such as milder reaction conditions, a wider range of raw material sources, and stronger natural properties. Saccharomyces cerevisiae, with its established safe use, mature genetic manipulation system, and industrial fermentation foundation, is an important host for the biomanufacturing of 2-phenylethanol.

[0003] Saccharomyces cerevisiae primarily converts L-phenylalanine to 2-phenylethanol via the Ayer pathway. This process typically involves steps such as transamination, decarboxylation, and aldehyde reduction, involving enzymes such as aromatic amino acid transaminases, phenylpyruvate decarboxylases, and alcohol dehydrogenases. Existing research has largely focused on overexpressing key enzymes in the Ayer pathway, weakening competing pathways, and optimizing precursor supplementation conditions. These strategies can enhance 2-phenylethanol accumulation, but 2-phenylethanol formation is also influenced by factors such as central carbon metabolism, TCA cycle intermediate metabolite transport, NADPH / NADP⁺ status, membrane lipid metabolism, and cell growth status.

[0004] Because 2-phenylethanol has an inhibitory effect on yeast cells, engineered strains often exhibit problems such as decreased precursor conversion capacity, reduced cell viability, and slower product accumulation during mid-to-late stage fermentation. Simply adjusting the direct reaction nodes of the Ehrlich pathway is insufficient to fully improve the overall production capacity of cells under high-product conditions. Therefore, it is necessary to identify modification targets beyond the direct reactions of the Ehrlich pathway within the whole-genome metabolic network and to verify their application value through engineered strain construction and bioreactor fermentation. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a genetically directed construction scheme that can improve the 2-phenylethanol synthesis ability of Saccharomyces cerevisiae. This scheme enhances the 2-phenylethanol accumulation ability of Saccharomyces cerevisiae in a fermentation system using L-phenylalanine as a precursor by enhancing CTP1 expression.

[0006] To address the aforementioned technical problems, this invention provides a method for improving the ability of *Saccharomyces cerevisiae* to produce 2-phenylethanol, comprising enhancing CTP1 expression in a *Saccharomyces cerevisiae* host to obtain *Saccharomyces cerevisiae* CTP1-overexpressing engineered strains. The CTP1 encodes a mitochondrial citrate transporter protein, which participates in the regulation of TCA cycle intermediate metabolite efflux and cytoplasmic precursor supply.

[0007] Specifically, an expression cassette consisting of the PGK1 promoter, CTP1 coding sequence, and PGK1 terminator was transformed into *Saccharomyces cerevisiae* cells, and positive transformants were obtained using selection markers such as KanMX. After confirmation by PCR and sequencing, *Saccharomyces cerevisiae* strains overexpressing CTP1 were obtained.

[0008] This invention also provides an engineered strain of *Saccharomyces cerevisiae* obtained by the above method, and its application in the production of 2-phenylethanol. The application includes culturing the engineered strain in a culture system containing L-phenylalanine and obtaining 2-phenylethanol through fed-batch fermentation. During fermentation, macroporous adsorption resin can be added to adsorb 2-phenylethanol, thereby reducing the inhibitory effect of free 2-phenylethanol on cells in the fermentation broth.

[0009] Compared to the starting strain, the CTP1-overexpressing strain obtained in this invention increased 2-phenylethanol accumulation during shake-flask fermentation. In a fed-batch fermentation in a 2 L bioreactor, the CTP1-overexpressing strain achieved a 2-phenylethanol yield of 14.32 g / L and an L-phenylalanine conversion rate of 0.60 g 2-phenylethanol / g L-phenylalanine. These results demonstrate that enhancing the expression of the mitochondrial citrate transport-related gene CTP1 can improve the conversion capacity of Saccharomyces cerevisiae for L-phenylalanine and improve the 2-phenylethanol accumulation level. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the construction of the CTP1 overexpression plasmid.

[0011] Figure 2 OD in shake-flask fermentation of CTP1 overexpressing engineered strain and original strain 600 Schematic diagram of the changes.

[0012] Figure 3 This is a schematic diagram showing the changes in 2-phenylethanol concentration during shake-flask fermentation of the CTP1 overexpressing engineered strain and the original strain.

[0013] Figure 4 This is a schematic diagram of the process parameters for fed-batch fermentation of CTP1 overexpressing engineered strains in a 2 L bioreactor. Detailed Implementation

[0014] The present invention will be further described below with reference to the embodiments. The following embodiments are used to illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the molecular biology operations, yeast transformation, resistance screening, and fermentation detection used can all be performed using conventional methods in the art.

[0015] Example 1: Screening for targets related to high-yield production of 2-phenylethanol based on a whole-genome metabolic network model of Saccharomyces cerevisiae

[0016] Based on the Yeast9 whole-genome metabolic network model of Saccharomyces cerevisiae, target screening was conducted to enhance the synthesis capacity of 2-phenylethanol. During model preparation, reactions related to glucose uptake, oxygen uptake, nitrogen uptake, biomass response, aromatic amino acid synthesis, L-phenylalanine conversion, and 2-phenylethanol production were examined. Subsequently, 2-phenylethanol exchange or demand reactions were enabled to allow 2-phenylethanol to be included in the model calculations. Simulation conditions used glucose as the primary carbon source, preserved cell growth constraints, and allowed related reactions such as L-phenylalanine metabolism, central carbon metabolism, mitochondrial organic acid transport, pentose phosphate pathway, and membrane lipid metabolism to participate in the calculations.

[0017] Flux balance and flux variation analyses were used to obtain a set of reactions associated with improved 2-phenylethanol synthesis. Further strain design algorithms, including IdealKnock, OptKnock, and OptForce, were used to screen for reactions and genes that may enhance 2-phenylethanol synthesis. Combining target function, criticality score, and ease of engineering construction, CTP1 was identified as the overexpression target and used for subsequent engineered strain construction and fermentation validation.

[0018] Example 2: Construction of CTP1 overexpression strain

[0019] Using Saccharomyces cerevisiae IMX581 as the starting strain, a CTP1 overexpression cassette was constructed. The overexpression cassette includes an upstream homologous arm, a PGK1 promoter, a CTP1 coding sequence, a PGK1 terminator, a KanMX selection marker, and a downstream homologous arm.

[0020] The specific operations include: amplifying the upstream and downstream homologous arms of the target integration site; amplifying the CTP1 coding sequence using the Saccharomyces cerevisiae genome as a template, with the theoretical length of the CTP1 target fragment being approximately 900 bp; amplifying the PGK1 promoter and PGK1 terminator; amplifying the KanMX selection marker; and ligating the above fragments to obtain the CTP1-OE recombinant construct.

[0021] The correctly sequenced recombinant fragment was transformed into *Saccharomyces cerevisiae* IMX581, and the transformed bacterial culture was plated on YPD solid plates containing G418. Transformants were picked for colony PCR verification. The verification fragment of the CTP1 overexpression integration site was approximately 2054 bp in size. The correctly verified strain was named IMX581-CTP1.

[0022] Example 3: Evaluation of shake-flask fermentation of CTP1 overexpression engineered strains

[0023] After activation, the starting strains IMX581 and IMX581-CTP1 were inoculated into seed culture medium, and then transferred to 250 mL shake flask fermentation medium at the same inoculation rate. The cultures were incubated at 30℃ and 220 rpm. OD was measured periodically during fermentation.600 The concentrations of 2-phenylethanol were determined to evaluate the effects of CTP1 overexpression on cell growth and product formation.

[0024] Shake flask results showed that IMX581-CTP1 could grow normally, with a maximum OD of 600 The concentration of 2-phenylethanol in IMX581-CTP1 was 15.36, showing no significant decrease compared to the starting strain. At 67 h, the concentration of 2-phenylethanol in IMX581-CTP1 reached 1.72 g / L, an increase of 22.23% compared to the starting strain. These results indicate that enhancing CTP1 expression can promote 2-phenylethanol accumulation without significantly affecting cell growth.

[0025] Based on the results of shake-flask fermentation, the CTP1 overexpressing strain was selected for fed-batch fermentation in a 2 L bioreactor for verification.

[0026] Example 4: Validation of fed-batch fermentation of CTP1 overexpression strain in a 2 L bioreactor

[0027] The IMX581-CTP1 was used for fed-batch fermentation validation in a 2 L bioreactor. The initial fermentation volume was 1 L, and pretreated macroporous adsorption resin H107 was added to the reactor for in-situ adsorption of 2-phenylethanol. During cultivation, aeration rate, stirring speed, and pH were maintained, and glucose and L-phenylalanine were added as needed based on substrate consumption. Fermentation data were obtained using a combination of offline HPLC and online near-infrared spectroscopy.

[0028] Under specific fermentation conditions, the temperature can be controlled at 30℃ and the pH at approximately 5.0. Lower aeration and stirring conditions can be used in the initial fermentation stage; after entering the feeding stage, the aeration and stirring intensity should be increased to meet the needs of cell growth and product formation. Near-infrared spectroscopy can be used to monitor the dynamic changes of glucose, L-phenylalanine, ethanol, and 2-phenylethanol, while offline HPLC results are used for calibration and confirmation.

[0029] The results showed that the yield of 2-phenylethanol from IMX581-CTP1 reached 14.32 g / L, the conversion rate of L-phenylalanine was 0.60 g 2-phenylethanol / g L-Phe, and the endpoint OD... 600 The value was 96.28. This result indicates that enhancing CTP1 expression has a clear effect on improving the 2-phenylethanol synthesis ability of Saccharomyces cerevisiae.

[0030] During fermentation, IMX581-CTP1 undergoes a process of rapid glucose consumption, phased ethanol accumulation, and continuous L-phenylalanine consumption. The CTP1 overexpression strain maintains a high 2-phenylethanol accumulation capacity in the mid-to-late stages of fermentation and achieves a high product concentration at the endpoint.

[0031] Example 5: Application of engineered strains in the production of 2-phenylethanol

[0032] The CTP1 overexpressing engineered bacteria obtained in Example 2 were used for the fermentation production of 2-phenylethanol. During fermentation, glucose was used as the main carbon source, and L-phenylalanine was used as the precursor for 2-phenylethanol synthesis. As fermentation progressed, feed was added according to the consumption of glucose and L-phenylalanine, and macroporous adsorption resin was added to adsorb 2-phenylethanol.

[0033] This application can be used for the production of 2-phenylethanol under laboratory-scale or scale-up validation conditions. The engineered strain exhibits high 2-phenylethanol yield and precursor conversion capacity in bioreactors, making it suitable for integration with subsequent feed strategy optimization, in-situ product adsorption, and online detection methods to improve the efficiency of Saccharomyces cerevisiae in producing natural aromatic alcohols.

Claims

1. A method for improving the ability of brewer's yeast to produce 2-phenylethanol, characterized in that, By enhancing CTP1 expression in the Saccharomyces cerevisiae host, an engineered Saccharomyces cerevisiae strain with improved 2-phenylethanol synthesis capacity was obtained.

2. The method according to claim 1, characterized in that, The gene is CTP1, which encodes a protein with mitochondrial citrate transport activity.

3. The method according to claim 1 or 2, characterized in that, The enhanced expression includes introducing an expression cassette containing a promoter, a target gene coding sequence, and a terminator into the Saccharomyces cerevisiae host.

4. The method according to claim 3, characterized in that, The promoter is a PGK1 promoter, and the terminator is a PGK1 terminator.

5. The method according to claim 3 or 4, characterized in that, The expression cassette was converted into Saccharomyces cerevisiae cells.

6. The method according to any one of claims 3 to 5, characterized in that, The expression box also contains a filter mark, preferably a KanMX filter mark.

7. The method according to any one of claims 1 to 6, characterized in that, The Saccharomyces cerevisiae host is Saccharomyces cerevisiae IMX581, or a Saccharomyces cerevisiae strain capable of synthesizing 2-phenylethanol from L-phenylalanine.

8. The method according to any one of claims 1 to 7, characterized in that, The engineered Saccharomyces cerevisiae strain is a CTP1 overexpression strain.

9. An engineered strain of brewing yeast, characterized in that, It is constructed by the method described in any one of claims 1 to 8.

10. The use of the engineered Saccharomyces cerevisiae according to claim 9 in the production of 2-phenylethanol.

11. A method for producing 2-phenylethanol, characterized in that, The engineered Saccharomyces cerevisiae of claim 9 is cultured, and L-phenylalanine is added to the culture system to convert L-phenylalanine into 2-phenylethanol by the engineered Saccharomyces cerevisiae.

12. The method according to claim 11, characterized in that, The culture is a fed-batch fermentation, in which glucose and L-phenylalanine are added during the fermentation process according to the consumption of glucose and L-phenylalanine.

13. The method according to claim 11 or 12, characterized in that, A macroporous adsorption resin is added to the fermentation system to adsorb 2-phenylethanol, and the macroporous adsorption resin is preferably H107 resin.

14. The method according to any one of claims 11 to 13, characterized in that, During fermentation, offline high-performance liquid chromatography and / or near-infrared spectroscopy were used to detect and monitor the concentration changes of glucose, L-phenylalanine, ethanol and 2-phenylethanol.

15. The method according to any one of claims 11 to 14, characterized in that, The fermentation was carried out at 30°C and pH 5.0, with increased aeration and stirring intensity during the feeding phase to maintain the growth of brewer's yeast and the production of 2-phenylethanol.

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