Isolation and identification of yeast strains
A total of 25 yeast strains were obtained from the samples collected in China (Table 1).S. cerevisiae is the main yeast among the isolated strains. Observation of yeast colonies showed that the colony morphology was generally round ridges, milky white, opaque, neat edges, and no wrinkles on the surface. The Wickerhamomyces anomalus colony has irregular edges and a large number of wrinkles on the surface. Through colony morphology analysis, microscopic cell observation and molecular identification, 19 strains of S.cerevisiae, 2 strains of Wickerhamomyces anomalus, 2 strains of P.pastoris, 1 strain of Tolerula spores, 1 strain of Candida anglica were identified. According to ITS sequence analysis, those with a similarity greater than 95% belong to the same yeast genus, and the genetic relationship of each strain was shown by drawing a phylogenetic tree. The S.cerevisiae CWY132 stored in this laboratory shows a close genetic relationship with the SC001, SC003 and SC009 isolated from Sichuan brewed samples. They were more closely related to S.cerevisiae JYC2558 in GenBank (Fig. 1). LSC-1 from CICC and SC008 isolated from Gansu homemade sourdough starters has high homology, while S.C-1 isolated from Zhejiang Quzhou rice wine is not closely related to S.C-2 from Zhejiang Shaoxing wine, the same province (Fig. 1).
Table 1
Source numbers and names of isolated yeast strains
source
|
Strain number
|
Strain name
|
Laboratory preservation
|
CWY132、NGER
|
Saccharomyces cerevisiae
|
C-U
|
Candida utilis
|
C-L
|
Yarrowia lipolytica
|
Purchased from CICC
|
LSC-1、LSC-2、LSC-3
|
Saccharomyces cerevisiae
|
Zhejiang Shaoxing Rice Wine Koji
|
S.C-1
|
Saccharomyces cerevisiae
|
Zhejiang Quzhou rice wine koji
|
S.C-2
|
Saccharomyces cerevisiae
|
Soil separation
|
SC001、SC002、SC003
|
Saccharomyces cerevisiae
|
Sichuan Liquor Brewing Koji
|
SC004、SC005、SC006、SC007
|
Saccharomyces cerevisiae
|
Gansu folk dough fermentation raw materials
|
SC008
|
Saccharomyces cerevisiae
|
LQ001、LQ002
|
Wickerhamomyces anomalus
|
LQ003
|
Candida anglica
|
LQ004
|
Torulaspora delbrueckii
|
Downstream Fermented Products of Sichuan Soy Sauce Factory
|
SC009
|
Saccharomyces cerevisiae
|
Guangdong Biotech Company develops bacteria preparations
|
SC010
|
Saccharomyces cerevisiae
|
LQ006
|
Pichia pastoris
|
Downstream fermentation products of Sichuan vinegar factory
|
LQ005
|
Pichia pastoris
|
Evaluation of the Performance of Isolated Strains
The yield of 2-PE, tolerance to 2-PE and high temperature of the 25 isolated yeast strains were tested and evaluated. The industrial S.cerevisiae strain CWY132 in our laboratory was used as a reference. The results showed that the ability of different strains to convert L-Phe and synthesize 2-PE was significantly different. As shown in Table 2, there are 7 yeast strains with 2-PE yields between 1–2 g/L, and 4 yeast strains with yields below 1 g/L. The 2-PE synthesis capacity of S.cerevisiae is greater than other yeast species. Among them, the yields of 2-PE of the five S.cerevisiae strains LSC-1, SC002, SC003, SC004 and SC005 were stable above 3.4 g/L, which were at least 10% higher than the industrial strain CWY132 (Table 2). This result proved that S.cerevisiae has a natural advantage in the synthesis of 2-PE, compared with other unconventional yeasts. These results further confirmed that the production of 2-PE by yeasts depends on the yeast strains as described previously [13, 38].
Table 2
2-PE and ethanol production of isolated yeast strains
Strains
|
2-PE(g/L)
|
Molar conversion rate (%)
|
Ethanol(g/L)
|
CWY132
|
3.12 ± 0.03a
|
85.1 ± 0.03
|
0.37 ± 0.11
|
NGER
|
2.02 ± 0.05
|
55.1 ± 0.05
|
0.54 ± 0.05
|
C-U
|
1.50 ± 0.01
|
41.0 ± 0.01
|
0.54 ± 0.09
|
C-L
|
0.52 ± 0.09
|
14.2 ± 0.09
|
0.48 ± 0.05
|
LSC-1
|
3.41 ± 0.05b
|
95.0 ± 0.05
|
0.63 ± 0.07
|
LSC-2
|
1.39 ± 0.02
|
37.9 ± 0.02
|
0.43 ± 0.12
|
LSC-3
|
0.49 ± 0.03
|
13,4 ± 0.03
|
0.46 ± 0.05
|
S.C-1
|
1.77 ± 0.07
|
48.3 ± 0.07
|
0.64 ± 0.04
|
S.C-2
|
0
|
0
|
0
|
SC001
|
3.08 ± 0.11
|
84.03 ± 0.11
|
0.35 ± 0.01
|
SC002
|
3.41 ± 0.04b
|
93.04 ± 0.04
|
0.39 ± 0.03
|
SC003
|
3.44 ± 0.09b
|
93.86 ± 0.09
|
0.38 ± 0.03
|
SC004
|
3.46 ± 0.11b
|
95.77 ± 0.11
|
0.62 ± 0.11
|
SC005
|
3.48 ± 0.05b
|
96.04 ± 0.05
|
0.66 ± 0.08
|
SC006
|
3.32 ± 0.06
|
90.58 ± 0.06
|
0.40 ± 0.13
|
SC007
|
1.57 ± 0.08
|
42.84 ± 0.08
|
0.57 ± 0.12
|
SC008
|
3.33 ± 0.04
|
90.86 ± 0.04
|
0.37 ± 0.05
|
SC009
|
3.00 ± 0.09
|
81.85 ± 0.09
|
0.65 ± 0.06
|
SC010
|
3.13 ± 0.11
|
85.40 ± 0.11
|
0.39 ± 0.04
|
LQ006
|
1.87 ± 0.02
|
51.02 ± 0.02
|
0.87 ± 0.04
|
LQ001
|
2.62 ± 0.08
|
71.49 ± 0.08
|
0.61 ± 0.12
|
LQ002
|
2.26 ± 0.08
|
61.66 ± 0.08
|
0.58 ± 0.13
|
LQ003
|
1.62 ± 0.03
|
44.20 ± 0.03
|
0.41 ± 0.15
|
LQ004
|
0.15 ± 0.01
|
4.10 ± 0.01
|
0.58 ± 0.08
|
LQ005
|
0.23 ± 0.02
|
6.28 ± 0.02
|
0.92 ± 0.19
|
Values are mean ± standard error. Statistical significance was analysed through one-way ANOVA. |
With CWY132 as a control, the five high-yield S.cerevisiae strains LSC-1, SC002, SC003, SC004 and SC005 were tested to evaluate their growth performance. The results showed that there were no significant differences in the growth rates among these five strains, but their growth rate were all significantly higher than CWY132, and the maximum growth was also higher (Fig. 2). These results demonstrated that these strains with high yield of 2-PE also had good growth performance. As the growth rate of LSC-1 was higher than the other four strains, this strain was selected as the parental candidate strain for protoplast fusion.
Through gradient dilution and drop plate method, the strains tolerance to 2-PE was evaluated. Three strains of S.cerevisiae NGER, S.C-1 and LSC-1 were screened out, which showed good tolerance phenotype to 2-PE. When the concentration of 2-PE was lower than 3.00 g/L, the growth of these three yeasts and CWY132 were not significantly inhibited. But when the concentration was increased to 3.40 g/L, these three yeast strains grew significantly better than CWY132. When the 2-PE concentration in plate reached 3.60 g/L, only strain NGER showed growth at cell concentration of 107/mL (Fig. 3a), indicating this strain has good traits underlying the ability to 2-PE tolerance. Our results are similar to those previously reported, that is, 4.0 g/L 2-PE can completely inhibit the growth of tested yeast cells [19].
The performance of these strains with good 2-PE tolerance at high temperature was also tested further. The results showed that at 40°C the growth of CWY132 was significantly inhibited, whereas the other three yeast strains still showed growth. When the temperature was increased to 41°C, only strain S.C-1 grew after incubation for 48h (Fig. 3b), indicating that S.C-1 resist the stress of high temperature very well.
Studies have shown that thermo-tolerance is a good indicator of other stress tolerance. For example, strains with higher temperature tolerance also have stronger resistance to oxidative and osmotic stress[39]. It was found that a thermo-resistant strain Ye9-612 had a constant growth rate at 30°C to 40°C, but the growth rate of the non-heat-resistant strain Ye9-596 (the offspring of the same parent) was severely affected[28]. The multiple stress resistance phenotype also exists in fungi and bacteria [40, 41].
In conclusion, using the 2-PE industrial strain CWY132 as a control, we obtained the LSC-1 strain with stronger 2-PE synthesis ability, the NGER strain with significantly improved tolerance to 2-PE, and the S.C-1 with better high temperature tolerance. These strains served as parent strains for subsequent cell fusion.
Optimization of Conditions for Protoplast Preparation
In protoplast preparation by enzymatic hydrolysis, the effect of enzyme concentration and treatment time on the preparation rate and regeneration rate of protoplasts was explored. We set the snailase concentration of 20, 30, 40, 50, 70, 100 and 140 mg/mL, respectively. The results showed that when treatment by snailase was 70 mg/mL for 100 min, the preparation rate was close to 75%~85%, and the regeneration rate was close to 10 ~ 18% (Fig. 4a; Fig. 4b). Further, we improved the protoplast preparation by addition of driselase. At a concentration of 2%, the protoplast preparation rate of LSC-1 was close to 95% after 30 minutes of treatment with driselase (Fig. 4c).
Highly tolerant fusants produced by the first round of protoplast fusion
S. cerevisiae NGER and S.C-1 were used as the parental strains to construct the first round protoplast fusion through inactivating parental protoplasts to obtain the strains capable of enhancing 2-PE related characters. The inactivation rate was close to 100% at the condition of that NGER protoplasts were exposed to a water bath at 60 ℃ for 2 min and S.C-1 protoplasts were irradiated at a distance of 20 cm under a 25W UV lamp for 15 min(Fig. 4d, Fig. 4e).There are 20 fusants were obtained. One fusant RH1-4 showed similar growth performance to NGER but with increased 2-PE tolerance at a concentration of 3.40 g/L in drop plate (Fig. 5a). Next, we compared the growth performances of RH1-4, NGER, S.C-1 and CWY132 incubated at 40°C. The results showed that RH1-4 has the same thermal resistance as S.C-1 (Fig. 5b), demonstrating that the fusion strain RH1-4 has obtained both the traits of high temperature and 2-PE stress tolerance from parent NGER and S.C-1.
RH1-4 also showed increased 2-PE production of 2.11 g/L, increased by 11%~24% compared with two parental strains (Fig. 5c). The results showed that the stress resistance of the fusant RH1-4 was improved, thereby increasing the yield of 2-PE. The high tolerance of RH1-4 is very valuable for further improving the 2-PE synthesis ability in S. cerevisiae.
Construction of 2-PE high-yield fusants through the second round of protoplast fusion
S.cerevisiae RH1-4 and LSC-1 were used as parent strains for the second round of protoplast fusion to obtain a strain with high tolerance to 2-PE and increased yield. There are about 100 fusant strains were obtained and classified into grades A to C according to 2-PE tolerance, as follows: A: close to the level of LSC-1; B: between LSC-1 and RH1-4; C: close to the level of RH1-4(Fig. 6a). Contrary to the expected result, only a small number of fusants in the C grade yielded 2-PE close to RH1-4 of 2.11 g/L, and most of them were lower than RH1-4, which was close to the yield of first round parent SC-1 of 1.67g/L(Fig. 6b). Although the tolerance of grade B fusants did not reach the expected level as RH1-4, their yields were relatively higher. Among them, one strain RH2-16 produced 2-PE of 4.05 g/L, increased by 18.8% compared with the parent LSC-1.The other selected strain RH2-26, also showed a high 2-PE yield of 3.96 g/L, an increase of 16.5%.(Fig. 6b). The results of transferring the excellent tolerance traits of RH1-4 to LSC-1 through the second round of fusion show that this method is very effective in further improving 2-PE yield. These results demonstrates that direct screening of yeast strains with multiple traits from nature and pyramid of traits through protoplast fusion is a practical method for yeast breeding. This method is more simple and efficient, compared with genome shuffling which needs mutant library construction and recursive fusion[42]. This approach would have a good application prospect in microbial breeding.
Identification of fusants and determination of their genetic stability
To test the genetic stability as well as to eliminate the effect of reverse mutation, fusants of interest were transferred at least 5 generations and the 2-PE and ethanol production capacity was detected and compared. The result demonstrated that the fusants showed good genetic stability. In order to confirm the fusants at the level of cell biology, the DNA content of the fusants and parent cells was determined. The results indicated that the DNA content of NGER, SC-1 and LSC-1 cells were 0.71, 0.67 and 1.08 µg per 107 cells, respectively. The DNA content of fusants RH1-4 and RH2-16 was higher, reaching 1.05 and 1.49 µg per 107 cells (Table 3). The DNA content of the fusants was greater than the DNA content of two parent strains, but was less than its sum, which was consistent with the characteristics of heterozygoma, confirming the preparation of the fusion strain. Fusants of interest were subcultured and the synthesis ability of 2-PE remained stable, the yield of RH2-16 reached the highest 4.31 g/L (Table 4). The comprehensive comparison of 2-PE yield, tolerance and other traits of parent and fusant strains are shown in Table 5.
Table 3
Strains
|
DNA content µg/107
|
NGER
|
0.71 ± 0.09
|
S.C-1
|
0.67 ± 0.14
|
RH1-4
|
1.05 ± 0.11
|
LSC-1
|
1.08 ± 0.07
|
RH2-16
|
1.49 ± 0.13
|
RH2-26
|
1.56 ± 0.05
|
Table 4
Production of 2-PE of fusant in subculture
Strain number
|
generations
|
2-Phenylethanol(g/L)
|
Ethanol(v/v%)
|
RH1-4
|
1
|
2.47 ± 0.21
|
0.26 ± 0.12
|
2
|
2.32 ± 0.09
|
0.31 ± 0.08
|
3
|
2.35 ± 0.13
|
0.28 ± 0.01
|
4
|
2.47 ± 0.07a
|
0.36 ± 0.03
|
5
|
2.47 ± 0.15
|
0.38 ± 0.03
|
LSC-1
|
1
|
3.39 ± 0.04
|
0.36 ± 0.17
|
2
|
3.46 ± 0.07
|
0.45 ± 0.03
|
3
|
3.41 ± 0.07
|
0.51 ± 0.05
|
4
|
3.53 ± 0.03b
|
0.65 ± 0.11
|
5
|
3.43 ± 0.12
|
0.61 ± 0.13
|
RH2-16
|
1
|
3.98 ± 0.08
|
0.46 ± 0.2
|
2
|
3.92 ± 0.09
|
0.61 ± 0.03
|
3
|
4.31 ± 0.02c
|
0.74 ± 0.06
|
4
|
3.99 ± 0.06
|
0.77 ± 0.04
|
5
|
4.01 ± 0.05
|
0.74 ± 0.13
|
RH2-26
|
1
|
3.88 ± 0.02
|
0.47 ± 0.01
|
2
|
3.87 ± 0.12
|
0.53 ± 0.11
|
3
|
3.83 ± 0.08
|
0.57 ± 0.12
|
4
|
3.88 ± 0.01d
|
0.55 ± 0.07
|
5
|
3.75 ± 0.27
|
0.59 ± 0.02
|
Analysis of statistical significance was performed through one-way ANOVA. |
Table 5
Summary of phenotypes of parents and fusants
category
|
Strain number
|
Tolerance to 2-PE(g/L)
|
2-PE yield(g/L)
|
Maximum cell concentration that can grow at 40°C
|
The first round fusion
|
Parent 1
|
NGER
|
3.6
|
2.02
|
105
|
Parent 2
|
S.C-1
|
3.4
|
1.77
|
104
|
Fusants
(Parent 1)
|
RH1-4
|
3.6
|
2.11
|
104
|
The second round fusion
|
Parent 2
|
LSC-1
|
3.4
|
3.48
|
105
|
Fusants
|
RH2-16
|
3.6
|
4.31
|
105
|
Inokuma et al [43] performed transcriptome analysis on the diploid yeast strain with good stress tolerance and xylose utilization obtained by rearranging the spore genome, and found genes that affect the strain's tolerance to multiple stresses. It helped to understand the multiple tolerance mechanism of yeast to high temperature and acid. Therefore, the RH2-16 in this study with good traits should be an excellent candidate strain for analysis of character and research on mechanisms underlying 2-PE metabolic pathway regulation.
2-PE production of DI-CRISPR-edited strains
In order to further use genetic engineering technology to increase the 2-PE yield of fusion strain, we tested the effect of overexpression of 2-PE synthesis related genes by using DI-CRISPR with RH2-16 as the starting strain. The strains with high copy numbers of the genes in Ehrlich pathway, aromatic amino acid aminotransferase (ARO8), phenylpyruvate decarboxylase (ARO10) and alcohol dehydrogenase (ADH2), were successfully constructed.
A total of 7 strains expressing these genes combinations were obtained(Fig. 7), ↑ARO8, ↑ARO10, ↑ADH2, ↑ARO8+↑ARO10, ↑ARO8+↑ADH2, ↑ARO10+↑ADH2 and ↑ARO8+↑ARO10+↑ADH2, and the yield of 2-PE of these strains were determined(Table 6). The 2-PE yield of the starting strain RH2-16 is 3.92 g/L. In the genetic manipulated strains, RH-ER-3 and RH-ER-1-3 showed yield of 3.91 g/Land 3.88 g/L respectively, close to the starting strain RH2-16. However, the yield of other strains decreased significantly. Among them, the RH-ER-2 strain was 3.49 g/L, a decrease of 12%. The results show that this method cannot effectively increase the production of 2-PE, and even reduces the yield. The most likely explanation for this result is that high concentration 2-PE is toxic to yeast cells. Therefore, improving the tolerance of 2-PE is the bottleneck in the breeding of S. cerevisiae to overcome the increase of 2-PE.
Table 6
2-PE yield of strains manipulated by DI-CRISPR
Number
|
Strains
|
Feature
|
2-PE yield(g/L)
|
|
RH2-16
|
WT
|
3.92 + 0.08a
|
1
|
RH-ER-1
|
↑ARO8
|
3.71 + 0.12c
|
2
|
RH-ER-2
|
↑ARO10
|
3.49 + 0.13d
|
3
|
RH-ER-3
|
↑ADH2
|
3.91 + 0.08baa
|
4
|
RH-ER-1-2
|
↑ARO8+↑ARO10
|
3.81 + 0.03b
|
5
|
RH-ER-1-3
|
↑ARO8+↑ADH2
|
3.88 + 0.06ba
|
6
|
RH-ER-2-3
|
↑ARO10+↑ADH2
|
3.56 + 0.11dc
|
7
|
RH-ER-1-2-3
|
↑ARO8+↑ARO10+↑ADH2
|
3.59 + 0.09dcb
|
Analysis of 2-PE production statistical significance was performed through one-way ANOVA. |