Acid-tolerant <i>Saccharomyces cerevisiae </i>and use thereof

Abstract

The present invention provides an acid-tolerant Saccharomyces cerevisiae strain and use thereof. By using exogenously added malic acid as a stress, an acid-tolerant mutant S. cerevisiae strain MTPfo-4 is obtained by directed evolution screening in the laboratory, which tolerates a minimum pH of 2.44. The mutant strain MTPfo-4, tolerant to multiple organic acids, has an increased tolerance to exogenous malic acid of up to 86.6 g/L. The mutant strain MTPfo-4 obtained is further identified. The mutant strain grows stably and well, and can tolerate a variety of organic acids (lactic acid, malic acid, succinic acid, fumaric acid, citric acid, gluconic acid, and tartaric acid). It also has a strong tolerance to inorganic acids (HCl and H.sub.3PO.sub.4). This is difficult to achieve in the existing research and reports of S. cerevisiae. The strain is intended to be used as an acid-tolerant chassis cell factory for producing various short-chain organic acids.

Claims

1. An acid-tolerant Saccharomyces cerevisiae, which is designated as Saccharomyces cerevisiae MTPfo-4 and deposited in the China Center for Type Culture Collection with Accession No.: CCTCC M 2020199 on Jun. 10, 2020 (Address: Wuhan University, Wuhan).

2. The Saccharomyces cerevisiae according to claim 1, wherein the lowest pH tolerated by the Saccharomyces cerevisiae is 2.44.

3. The Saccharomyces cerevisiae according to claim 1, wherein the tolerance of Saccharomyces cerevisiae to exogenous malic acid reaches 86.6 g/L.

4. The Saccharomyces cerevisiae according to claim 1, wherein the Saccharomyces cerevisiae tolerates lactic acid, malic acid, succinic acid, fumaric acid, citric acid, gluconic acid, tartaric acid, HCl, and H.sub.3PO.sub.4.

5. A microbial agent, comprising the Saccharomyces cerevisiae according to claim 1.

6. The microbial agent according to claim 5, wherein the microbial agent is a solid agent.

7. The microbial agent according to claim 5, wherein the microbial agent is a liquid agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the evolution and screening of acid-tolerant Saccharomyces cerevisiae;

(2) FIG. 2 shows the growth curves of Saccharomyces cerevisiae and its mutant under malic acid stress;

(3) FIG. 3 shows the analysis of tolerance of the mutant MTPfo-4 to other acids (pH 2.44); and

(4) FIG. 4 shows the analysis of tolerance of the mutant MTPfo-4 to other acids (pH 3.0).

(5) FIG. 5 shows the analysis of metabolic substance of the mutant MTPfo-4.

(6) FIG. 6 shows the use of the mutant MTPfo-4 in production of short-chain organic acids.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(7) The present invention will be further described below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

Example 1

Evolution and Screening of Acid-Tolerant Saccharomyces cerevisiae

(8) Starting with Saccharomyces cerevisiae CEN.PD2-1C, a mutant Saccharomyces cerevisiae strain with a tolerance to lower pH was obtained by directed evolution screening by exogenously adding different concentrations of malic acid. In the present invention, by adding different concentrations of malic acid, the pH was controlled to decrease step by step (starting from pH 6.0 at which the addition amount of exogenous malic acid was 4.6 g/L). After the cells were evolved to be able to grow at a pH level, the cells were cultured at this level for enrichment (the goal is that the OD.sub.600 of the cells at this pH is stable for 48 h, and has no significant increase between two subcultures; and the purpose is to increase the number of mutant cells and make cell growth more stable at this pH), and then evolved at a next pH level. From the initial pH 6.0 to pH 4.0, each pH span is 0.2, that is, 6.0, 5.8, 5.6 . . . 4.2, and 4.0. From pH 4.0 to pH 3.5, each pH span is 0.1, that is, 4.0, 3.9, 3.8 . . . 3.6, and 3.5. From pH 3.5 to pH 3.0, each pH span is 0.05, that is, 3.5, 3.45, 3.4 . . . 3.05, and 3.0. From pH 3.0 to pH 2.8, each pH span is 0.02, that is, 3.0, 2.98, 2.96 . . . 2.82, and 2.80. When the pH is below 2.8, each pH span is 0.01, that is, 2.8, 2.79, 2.78 . . . 2.45, and 2.44. The cells grow faster from the initial pH 6.0 to pH 4.0, and it may be possible to directly evolve from pH 4.0. However, in order to obtain a more stable acid-tolerant strain, iterative evolution from pH 6.0 is recommended in the present invention. The evolved strain obtained at each level of pH will be used as the starting strain for the next passage until a mutant that tolerates a lower pH is evolved.

(9) As shown in FIG. 1, after nearly four months of continuous directed evolution, a mutant MTPfo-4 that can tolerate 86.6 g/L malic acid (pH 2.44) was screened. After 48 hours of culture at each level of pH, the pH of the medium remains stable, and there is no increase, but a tendency to decrease. For example, after the mutant MTPfo-4 is cultured at pH 2.44 for 48 h, the pH is stabilized at 2.42-2.44.

(10) At pH 2.44, the OD.sub.600 reaches 18.5 after 48 h of culture. The results show that the mutant strain can grow stably. This is also the currently reported lowest pH that S. cerevisiae can tolerate.

(11) The screened mutant Saccharomyces cerevisiae MTPfo-4 was deposited in the China Center for Type Culture Collection with Accession No.: CCTCC M 2020199 on Jun. 10, 2020 (Address: Wuhan University, Wuhan).

Example 2

Cell Trait Analysis of Acid-Tolerant Saccharomyces cerevisiae

(12) To verify the stability of the obtained mutant S. cerevisiae strain MTPfo-4 at a low pH, the growth of MTPfo-4 and the starting strain CEN.PD2-1C were compared with the addition of different amounts of exogenous malic acid. The concentration (g/L) of the added exogenous malic acid is 0, 30, 40, 50, 60, 70, 80, 90, and 100. The cells were sampled every 12 h for consecutive 72 h. The growth curve of the strain was determined. The determination result is shown in FIG. 2. Compared with the starting strain CEN.PD2-1C, the mutant MTPfo-4 grows greatly in the presence of the exogenously added malic acid and has a strong tolerance to the exogenously added malic acid.

Example 3

Analysis of Tolerance of Acid-Tolerant Saccharomyces cerevisiae to Other Acids

(13) To analyze the tolerance of the mutant MTPfo-4 to other acids, 10 acids, including 2 inorganic acids of HCl and H.sub.3PO.sub.4, and 8 organic acids of lactic acid, malic acid, fumaric acid, succinate acid, tartaric acid, furoic acid, gluconic acid, and citric acid, were added exogenously in the present invention to analyze the acid tolerance of cells. The initial pH was controlled to 2.44. The cells were sampled every 12 h for consecutive 72 h. The acid tolerance of cells was analyzed by determining the OD.sub.600. The results are shown in FIG. 3. Compared with the starting strain CEN.PD2-1C, MTPfo-4 has a stronger tolerance to the 6 acids including HCl, H.sub.3O.sub.4, lactic acid, malic acid, citric acid, and gluconic acid. In contrast, at this pH, all 10 acids have a strong inhibitory effect on the growth of CEN.PD2-1C. Since fumaric acid, succinic acid, furoic acid, and tartaric acid also had an inhibitory effect on the mutant MTPfo-4 at pH 2.44, in the present invention, the initial pH of the four acids is adjusted to 3.0. The cells were sampled every 12 h for consecutive 72 h. The acid resistance of the cells was analyzed again by determining the OD.sub.600. The results are shown in FIG. 4. Compared with the starting strain CEN.PD2-1C, MTPfo-4 has a stronger tolerance to the four acids. Similarly, at this pH, all four acids have a strong inhibitory effect on the growth of CEN.PD2-1C.

Example 4

Analysis of Metabolic Substance of Acid-Tolerant Saccharomyces cerevisiae

(14) In order to analyze the advantages of the acid-tolerant Saccharomyces cerevisiae mutant MTPfo-4 as chassis cells, metabolomics analysis is performed on MTPfo-4 in the present invention. As compared with the starting strain CEN.PD2-1C, a variety of metabolites are affected for the mutant MTPfo-4, and the effects on the short-chain organic acids are mainly analyzed in the invention. As shown in FIG. 5, L-Lactic acid, Butyric acid, Gluconic acid and Maleic acid in the metabolites of mutant MTPfo-4 are increased significantly comparing with the starting strain (*: P≤0.05; **: P≤0.01).

Example 5

Synthesis of Organic Acids by Metabolic Modification of Acid-Tolerant Saccharomyces cerevisiae

(15) In order to better verify the potential of mutant MTPfo-4 as chassis cells to produce organic acids, the target product is synthesized by metabolic modification in the invention, for example malic acid. The synthesis pathway of malic acid in Saccharomyces cerevisiae is shown in FIG. 6A, the key gene mdh (malate dehydrogenase, selecting endogenous genes Scemdh2 and Scemdh3, the exogenous gene Aormdh being derived from Aspergillus oryzae) and pyc (pyruvate carboxylase, selecting endogenous gene Scepyc2, the exogenous gene Aorpyc being derived from Aspergillus oryzae) are integrated. First, the mdh genes of different sources are integrated and fermented in a 250 mL shake flask with a loaded liquid of 30 mL, and the fermentation medium includes glucose 20 g/L, tryptone 20 g/L, and yeast powder 10 g/L. Then the flask was cultured under 30° C. at 220 r/min for 72 h. At the end of fermentation, the yield of malic acid in the supernatant is determined by HPLC (High Performance Liquid Chromatography). 1-2 mL of fermentation broth is taken for centrifugation at 12000 rpm for 15-20 min, then the supernatant is collected to determine by HPLC the extracellular concentration of malic acid. The determination results of malic acid by HPLC in the fermentation broth are shown in FIG. 6B. As compared with the yield of 0.45 g/L for the control strain (MTPfo-4 before modification), by overexpression of mdh2, mdh3 and Aormdh, the yields of malic acid are 1.83 g/L, 3.05 g/L and 3.59 g/L respectively.

(16) Based on the significantly increased production of malic acid by overexpression of mdh3 and Aormdh, Scepyc2 and Aorpyc are overexpressed to obtain four different combinations of Scemdh3+Scepyc2, Aormdh+Scepyc2, Scemdh3+Aorpyc and Aormdh+Aorpyc. Fermentation is performed in a 250 mL of shake flask with a loaded liquid of 30 mL, the fermentation medium includes glucose 20 g/L, tryptone 20 g/L, and yeast powder 10 g/L. Then the flask was cultured under 30° C. at 220 r/min for 72 h. At the end of fermentation, the yield of malic acid in the supernatant is determined by HPLC (High Performance Liquid Chromatography). 1-2 mL of fermentation broth is taken for centrifugation at 12000 rpm for 15-20 min, and then the supernatant is collected to determine by HPLC the extracellular concentration of malic acid. The determination results of malic acid by HPLC in the fermentation broth are shown in FIG. 6B. As compared with the control strain (MTPfo-4 before modification), by overexpression of Scemdh3+Scepyc2, Aormdh+Scepyc2, Scemdh3+Aorpyc and Aormdh+Aorpyc, the yields of malic acid are 4.2 g/L, 4.55 g/L, 5.35 g/L and 6.8 g/L respectively. The above results show that, the extracellular production of overexpressed Aormdh+Aorpyc is 15.1 times higher than that of the starting strain.

(17) All the above results show that, the mutant strain MTPfo-4 has a has strong tolerance to various acids, and has the potential to produce a variety of organic acids as acid-resistant chassis cells, and thus can be further developed for the production of a variety of short-chain organic acids.

(18) The above-described embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or modifications can be made by those skilled in the art based on the present invention, which are within the scope of the present invention as defined by the claims.