MAL62 Overexpression Enhances Freezing Tolerance of Baker's

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Food and Beverage Chemistry/Biochemistry

MAL62 overexpression enhances freezing tolerance of baker’s yeast in lean dough by enhancing Tps1 activity and maltose metabolism xi sun, Jun Zhang, Zhi-Hua Fan, Ping Xiao, Shan-Na Liu, Rui-Peng Li, Wen-Bi Zhu, and Liang Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03790 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 28, 2019

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Journal of Agricultural and Food Chemistry

MAL62 overexpression enhances freezing tolerance of baker’s

1 2

yeast in lean dough by enhancing Tps1 activity and maltose

3

metabolism

4

Xi Sun1,2, Jun Zhang1,2, Zhi-Hua Fan1,2, Ping Xiao1,2, Shan-Na Liu1,2, Rui-Peng Li1,2, Wen-Bi

5

Zhu3, Liang Huang4

6 7 8

1

College of Biological Engineering, Tianjin Agricultural University, Tianjin 300384, PR China

9

2

Tianjin Engineering Research Center of Agricultural Products Processing, Tianjin 300384, PR China

10 11

3

Tianjin 300384, PR China

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Experiments and Teaching Center for Agricultural Analysis, Tianjin Agricultural University,

4

College of Agronomy and Resources Environment, Tianjin Agricultural University, Tianjin 300384, PR China

14 15 16 17



Correspondence author: Tel.: +86 22 23781298; fax: +86 22 23781300. E-mail: [email protected] (L. H.)

1

ACS Paragon Plus Environment


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Abstract

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Trehalose plays a crucial role in response to freezing stress in baker’s yeast. MAL62, a gene

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involved in adenosine diphosphoglucose-dependent trehalose synthesis pathway, can increase

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trehalose content. However, the difference between MAL62-related trehalose synthesis and

23

traditional uridine diphosphoglucose-dependent trehalose synthesis is not well understood. MAL62

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overexpression showed less effect in enhancing intracellular trehalose compared with TPS1

25

overexpression. However, MAL62 overexpression elicited trehalose synthesis before fermentation

26

with enhanced maltose metabolism and had a similar effect on cell viability after freezing.

27

Furthermore, MAL62 and TPS1 overexpression in the NTH1 deletion background further

28

strengthened freezing tolerance, and improved leavening ability. Our results suggest that the

29

enhancement in freezing tolerance by MAL62 overexpression may involve multiple pathways

30

rather than simply enhancing trehalose synthesis. The results reveal valuable insights into the

31

relationship between maltose metabolism and freezing tolerance and may help develop better yeast

32

strains for enhancing fermentation characteristics of frozen dough.

33 34

Key words: Baker’s yeast; Cell viability; Freezing tolerance; Leavening ability; MAL62; NTH1;

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TPS1

2


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Introduction

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Frozen dough technology is widely used in the bakery industry for convenience and to enable

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good quality bakery products1. However, freezing and thawing of dough induces cell oxidation

40

and apoptosis to baker’s yeast (Saccharomyces cerevisiae)2 thus leading to significantly reduced

41

leavening ability3. Yeast can respond to environmental stresses by producing protective molecules

42

such as trehalose. These molecules protect yeast cells by preserving the membrane integrity4 and

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the native conformation of proteins5. The accumulation of trehalose in baker’s yeast is induced by

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two major pathways: system I and system II. The uridine diphosphoglucose (UDPG)-dependent

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trehalose synthesis pathway (system I) contains a TPS1-encoded trehalose-6-phosphate synthase6,

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a TPS2-encoded trehalose-6-phosphate phosphatase, and a TSL1-encoded trehalose-synthesis

47

protein complex7. The adenosine diphosphoglucose (ADPG)-dependent trehalose synthesis

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pathway (system II) is linked to the utilization of maltose8-9.

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In yeast, maltose metabolism is regulated by one of the five multigene complexes: MAL1–

50

MAL4 and MAL6. Each multigene complex consists of genes that encode a maltose permease, a

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maltase, and a regulatory protein10. In lean dough, studies suggest that maltase plays a greater role

52

in leavening ability and maltose metabolism than maltose permease11-13. We previously showed

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that expression of MAL62 (a maltase encoding gene) induces trehalose accumulation14. In yeast,

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the intracellular level of trehalose is balanced by enzymatic synthesis and degradation15, both of

55

which can be induced by certain stresses16-17. Trehalose degradation is accomplished by trehalase,

56

which hydrolyzes intracellular trehalose into glucose18-19. A recent study showed that deletion of

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NTH1, which encodes neutral trehalase, results in accumulation of trehalose and increased freezing

58

resistance19.

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To further understand the protective role of trehalose in freezing resistance in baker’s yeast

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and the potential application in lean dough, we examined the possible effects of MAL62 and/or

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TPS1 overexpression in the NTH1 deletion (nth1Δ) background.

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Materials and Methods

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Strains, plasmids and growth conditions

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Table 1 lists all the plasmids and strains of bacteria and yeast used in this study. BY14a, a

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high leavening haploid strain derived from its parent strain BY14, was used as the parent strain for

66

deletion and overexpression.

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Escherichia coli (E. coli) DH5a was cultivated in Luria–Bertani (LB) medium (1% tryptone,

68

0.5% yeast extract, and 1% NaCl) at 37 °C. A total of 100 μg/mL ampicillin was added in LB

69

medium when required. Plasmids were purified using the Plasmid Mini Kit II (D6945; Omega

70

Bio-tek, Inc, Norcross, GA).

71

Yeast strains were grown in yeast extract peptone dextrose (YEPD) medium (10 g/L yeast

72

extract, 20 g/L peptone, and 20 g/L dextrose) at 30°C. Geneticin (G418) (800 mg/L) was used for

73

selection of G418-resistant transformants. Yeast cells were first grown in YEPD for 24 h at 30°C,

74

and then transferred to cane molasses medium (5 g/L yeast extract, 0.5 g/L (NH4)2SO4, 12° Brix

75

cane molasses). Cells were cultured for another 24 h with constant shaking (180 rpm), reaching a

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final OD600 of 1.8 from its initial OD600 of 0.4. Cells were centrifuged at 1,500 × g for 5 min at

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4°C, and then washed twice with sterile water at 4°C and used for fermentation experiments and

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quantitative real-time PCR (qRT-PCR). To investigate the degradation of trehalose, extracellular

79

maltose during fermentation, and cell viability after pre-fermentation-freezing and long-term

80

freezing, a modified low sugar model liquid dough (LSMLD) medium20 was used.

81

Plasmid construction and yeast transformation 4


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Yeast genomic DNA was extracted using a yeast DNA kit (Omega Bio-tek, Inc). Primers for the polymerase chain reaction (PCR) are listed in Table 2.

84

Yep-PTK (Table 1) was constructed as follows: the fragment of KpnI/BamHI KanMX was

85

amplified using pUG621 as the template, the PCR primers were Kan-U and Kan-D, and the PCR

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product was cloned into the Yep352 vector to create the plasmid Yep-K (Yep-KanMX). The

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genomic DNA of BY14a was used as template for TPS1 amplification using TPS1-U and TPS1-

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D as primers and was inserted into the pPGK122 vector resulting in plasmid pPGKT.

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The resulting PGKT fragment containing the complete PGK1 and inserted TPS1 was

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amplified from pPGKT using the primer pairs of PGK-U and PGK-D and then cloned into Yep-K

91

to produce the final plasmid, Yep-PTK. The recombinant plasmid pUC-NKAB used for NTH1

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deletion was constructed as described previously14. Plasmid pUC-NKABT was constructed by

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inserting the BamHI fragment of PGKT into plasmid pUC-NKAB for TPS1 overexpression.

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Similarly, plasmid pUC-NKABM was constructed by inserting the BamHI fragment of PGKM

95

amplified from pPGKM (containing the complete PGK1 and inserted MAL62) into plasmid pUC-

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NKAB for MAL62 overexpression.

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Yeast transformation23 as well as the NTH1 deletion strain (B-NTH1) and MAL62

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overexpression strain (B+MAL62)14 were constructed as previously described. Overexpression of

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MAL62 or TPS1 in the NTH1 deletion background was achieved by replacing the full genomic

100

copy of NTH1 with MAL62 or TPS1 using cassettes of NA-loxp-KanMX-PGK1P-MAL62 -PGK1T-

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loxp-NB or NA-loxp-KanMX-PGK1P-TPS1-PGK1T-loxp-NB, amplified with N-S and N-X,

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respectively. The recombinant strains were selected by G418 and verified using primer pairs N-S,

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K-S/PGK-U, PGK-D/MAL62-U, MAL62-D and N-S, K-S/PGK-U, PGK-D/TPS1-U, and TPS1-

104

D, respectively. KanMX was removed from the recombinant strains by Cre recombinase using the 5



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pSH-Zeocin plasmid, resulting in strains of NTH1 deletion/MAL62 overexpression (B-N+M) and

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NTH1 deletion/TPS1 overexpression (B-N+T). The transformation plasmids Yep-PMK14 and Yep-

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PTK were then transformed to B-N+T and BY14a to select the G418-resistant strains B-

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N+T+MAL62 and B+TPS1, respectively. The B-N+M strain was constructed by homologous

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recombination into the genome to make the MAL62 and TPS1 overexpression in the same genetic

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background. This is different from the B-N+MAL62 strain in our previous paper14, in which the

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MAL62 gene was overexpressed in the episomal shuttle-plasmid. Blank controls were constructed

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by transforming the empty vector Yep-K into BY14a and B-N+T strains (BY14a + K and B-

113

N+T+K).

114

Measurement of the activity of neutral trehalase

115

Activities of the neutral trehalase were measured as previously reported24. Production of 1.0

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μm of glucose per minute was defined as 1 unit of trehalase activity and the final activity was

117

calculated based on the dried weight of yeast cells. The measurement was repeated three times and

118

data were expressed as mean ± standard deviation (SD).

119

Measurement of Tps1 activity

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Activities of Tps1 were measured as previously reported25. Production of 1.0 μm of trehalose-

121

6-phosphate per minute was defined as 1 unit of Tps1 activity and the final activity was calculated

122

based on the weight of dried cells. The measurement was repeated three times and data were

123

expressed as mean ± SD.

124

Measurement of α-glucosidase activity

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Crude extracts were prepared using the Salema-Oom method to determine enzyme activities13.

126

The α-glucosidase was measured as reported previously 26. The measurement was repeated three

127

times and data were expressed as mean ± SD. 6


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Determination of expression levels of MAL62, TPS1 and NTH1

129

The expression levels of the integrated target genes (MAL62, TPS1 and NTH1) were detected

130

by quantitative real-time PCR (qRT-PCR) using the THUNDERBIRD Probe One-step qRT-PCR

131

Kit (TOYOBO). UBC6 gene was used as the reference gene27. The yeast RNAiso kit (Takara

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Biotechnol, Dalian, China) and the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real

133

Time) (Takara Biotechnol) were used to extract the cDNA. The PCR primers are listed in Table 2.

134

The PCR condition was expressed as follows: 95℃ for 30 s, 61℃ for 20 min, 95 ℃ for 30 s, 43

135

cycles at 95℃ for 5 s, 55℃ for 10 s, 74℃ for 15 s, and 72℃ for 5 min after the cycles with a

136

CFX96 real-time PCR detection system (Bio-Rad, Hercules, USA). The 2−ΔΔCt method was used

137

for quantitative analysis.

138

Measurement of the contents of intracellular trehalose

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Freshly growing cells were first washed twice with water and 0.1 g of cells was used to extract

140

trehalose using 4 mL of trichloroacetic acid (0.5 M). Trehalose was measured as previously

141

described28. The measurement was repeated three times and data were expressed as mean ± SD.

142

Extracellular maltose measurement

143

To measure the extracellular maltose, the cultured cells were filtered through a 0.45-µm-pore-

144

size cellulose acetate filter (Millipore Corp., Danvers, MA, USA), and subsequently kept at -20℃

145

until use. The equipment used in the experiment included a differential refractometer detector

146

(Waters 410 RI), Aminex HPX-87H columns (Bio-Rad, Hercules, CA, USA), and an HPLC pump

147

(Waters 515). Samples were measured at 65℃ with 5 mM H2SO4 as the mobile phase at a flow

148

rate of 0.6 ml/min29. The measurement was repeated three times and data were expressed as mean

149

± SD.

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Measurement of cell viability 7



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Yeast was cultured for 24 h in cane molasses medium and then pre-fermented in LSMLD

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medium at 30°C for 5, 10, 15, 20, and 25 min. Cells were then shifted to −20°C for 7 or 21 d.

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Trehalose content was measured before cells were frozen. Cell viability was measured after

154

freezing for 7 and 21 d. To determine the cell viability, cells that were frozen for 7 and 21 d were

155

thawed for 30 min at 30°C. Cells were then plated on YEPD plates and grew at 30°C for 2 d. Cell

156

viability was determined as (the number of colonies after freezing)/(the number of colonies before

157

freezing)×100%. The measurement was repeated three times and data were expressed as mean ±

158

SD.

159

Measurement of leavening ability

160

Leavening abilities were measured by the amount of carbon dioxide (CO2) produced by the

161

lean dough. The lean dough contained 280 g of standard flour, 4 g of salt, 9 g of fresh yeast, and

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150 mL of water. The dough was mixed at 30°C for 5 min and divided into 50-g pieces. The

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doughs were placed in a fermentograph (Type JM 451; Mekab Försäljnings AB, Nässjö, Sweden).

164

Production of CO2 was measured at 30°C for 2 h. The measurement was repeated three times and

165

data were expressed as mean ± SD.

166

To examine the effect of freeze–thaw on the leavening ability, the dough was frozen for 7 d

167

at −20°C and then thawed for 30 min at 30°C. The production of CO2 was measured while the

168

dough was at 30°C for 2 h. The measurement was repeated three times and data were expressed as

169

mean ± SD.

170

Statistical analysis

171

Data were represented as mean ± SD. Analysis of variance (ANOVA) was used to compare

172

the differences among different strains. P < 0.05 was considered statistically significant.

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Differences between strains of overexpression and the parent strain were analyzed using Student’s

174

t-test. P < 0.05 was considered statistically significant.

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Results

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TPS1 overexpression is more effective in enhancing intracellular trehalose content than

177

MAL62 overexpression

178

Tps1 is a trehalose-6-phosphate synthase, which is activated under various stress conditions30.

179

To analyze the effects of MAL62 and TPS1 overexpression on trehalose synthesis, we first tested

180

the Tps1 activity. As shown in Table 3, overexpression of TPS1 (B+TPS1, B-N+T, B-N+T+K)

181

increased the activity of Tps1 significantly (P < 0.01). Overexpression of MAL62 (B+MAL62, B-

182

N+M) also increased the Tps1 activity (P < 0.05) but was less effective compared to TPS1

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overexpression. Overexpression of both MAL62 and TPS1 in the NTH1 deletion background (B-

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N+T+MAL62) caused the highest activity of Tps1 (Table 3), suggesting that MAL62

185

overexpression alone is less effective in inducing trehalose production, but it could strengthen the

186

effect of TPS1 overexpression.

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To further confirm these results, we checked the expression levels of MAL62, TPS1 and NTH1

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in different background using qRT-PCR. As shown in Figure 1, TPS1 expression levels of B+TPS1,

189

B-N+T, B-N+T+K, B+MAL62, B-N+M and B-N+T+MAL62, were all increased, similar to the

190

trend of changes of the Tps1 activity (Table 3).

191

To further confirm these results, trehalose levels in different strains were measured and the

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growth curves of all nine strains (BY14a, BY14a+K, B+MAL62, B+TPS1, B-NTH1, B-N+M, B-

193

N+T, B-N+T+K and B-N+T+MAL62) were found to be similar. Cells entered exponential phase

194

3 hours after inoculation, and stationary phase 10 hours after inoculation (data not shown).

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Overexpression of MAL62 or TPS1, or both (B+MAL62, B+TPS1, B-N+M, B-N+T, B-N+T+K 9



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and B-N+T+MAL62), caused accumulation of trehalose starting at the late exponential stage with

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an average rate of 23.0 mg/h/g CDW. In contrast, in strains that had no MAL62 or TPS1

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overexpression (BY14a, BY14a+K, B-NTH1), trehalose started to accumulate only during

199

stationary phase with an average rate of 19.0 mg/h/g CDW (Figure 2). Notably, overexpression of

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TPS1 alone (B+TPS1, B-N+T) had a higher trehalose accumulation rate (30.5 mg/h/g CDW) than

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overexpression of MAL62 (B+MAL62, B-N+M). Consistent with the Tps1 activity and the TPS1

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expression level, overexpression of both MAL62 and TPS1 in the NTH1 deletion background (B-

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N+T+MAL62) had the highest trehalose accumulation rate.

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Relation between maltose metabolism and trehalose metabolism

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To investigate the relation between maltose metabolism and trehalose metabolism, we first

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tested the maltose fermentation ability of all strains. Results showed that the maltose metabolism

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rates of strains B+MAL62, B-N+M and B-N+T+MAL62 were significantly higher than that of

208

control (BY14a and BY14a+K) in the LSMLD medium (p < 0.01) (Figure 3A). This is in

209

agreement with the MAL62 expression levels (Figure. 1) and the activity of α-glucosidase activities

210

(Table 3). By contrast, other gene modification (sole TPS1 overexpression and/or NTH1 deletion)

211

had no significant effect on enhancing maltose metabolism.

212

We also investigated the neutral trehalase activity and the degradation rate of intracellular

213

trehalose. As shown in Table 3, the B+MAL62 strain and the B+TPS1 strain had a similar neutral

214

trehalase activity and a similar intracellular trehalose degradation rate (Figure 3B) compared to

215

their control (BY14a and BY14a+K), suggesting that overexpression of MAL62 and TPS1 did not

216

affect the trehalose degradation. However, the intracellular trehalose contents in strains with

217

overexpressing MAL62 and/or TPS1 (B+MAL62, B+TPS1, B-N+M, B-N+T, B-N+T+K and B-

218

N+T+MAL62) were significantly higher than the other strains before fermentation (Figure 3B, 10


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219

time = 0 min). These results suggest that the enhanced maltose metabolism by MAL62

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overexpression has a positive correlation with the intracellular trehalose content of yeast cells

221

before fermentation, but not important for trehalose degradation.

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Overexpression of MAL62 or TPS1 has a similar effect in increasing yeast viability after

223

freezing

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The degradation of trehalose during pre-fermentation is inevitable31. Therefore, the cell

225

viabilities of different strains were investigated to assess the possible effects of MAL62 or TPS1

226

overexpression on the freezing tolerance of yeasts after pre-fermentation and 7 d freezing. As

227

shown in Figure 3C, cell viability after 7 d freezing of all strains decreased as pre-fermentation

228

time increased. After 25 min pre-fermentation, the cell viability of the strain overexpressing both

229

MAL62 and TPS1 (B-N+T+MAL62) was the highest among all strains (P < 0.05), while the

230

viability of the control (BY14a or BY14a+K) was the lowest, decreasing from about 80% to about

231

40%. Interestingly, though the intracellular trehalose contents in the strains of B+MAL62 and B-

232

N+M were lower than the strains of B+TPS1 and B-N+T, when fermentation is finished (Figure

233

3B, time = 25 min), cell viabilities between B+MAL62 and B+TPS1 and between B-N+M and B-

234

N+T were similar (Figure 3C). These results suggest that overexpression of MAL62 or TPS1 has a

235

similar effect on the viability of yeast cells after pre-fermentation and freezing.

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Trehalose content positively correlates with cell viability

237

Residual intracellular trehalose after pre-fermentation is believed to be an important factor

238

for freezing tolerance of baker’s yeast32. To further understand the role of trehalose in freezing

239

tolerance over the long term, we examined cell viability 21 d after freezing as well as the trehalose

240

content. As shown in Figure 4, both the trehalose content and the cell viability were higher in

241

strains overexpressing MAL62, TPS1, or both (B+MAL62, B+TPS1, B-N+M, B-N+T, B-N+T+K 11



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and B-N+T+MAL62) than the parental strains (BY14a and BY14a+K) (P < 0.05). The trehalose

243

content was mostly higher in strains with higher viability, except in the sole-NTH1 deletion strain,

244

suggesting that the trehalose content positively correlates with cell viability and therefore long-

245

term freezing tolerance.

246

Overexpression of MAL62 and TPS1 with NTH1 deletion is most effective in enhancing

247

leavening ability after freezing–thaw stress

248

One important fermentation characteristic of baker’s yeast is its leavening ability, especially

249

in frozen dough33. CO2 production was measured to see if MAL62 or TPS1 overexpression affected

250

the leavening ability after freezing–thaw stress. Our results indicated that the production of CO2

251

decreased in all strains after the freezing–thaw stress (Figure 5 right). NTH1 deletion alone (B-

252

NTH1) showed little effect on enhancing fermentation ability before freezing14,

253

freezing–thaw (Figure 5). Overexpression of MAL62 (B+MAL62, B-N+M and B-N+T+MAL62)

254

significantly enhanced CO2 production either before or after freezing–thaw, compared to the parent

255

strains (P < 0.05). However, overexpression of TPS1 (B+TPS1, B-N+T and B-N+T+K) had no

256

effect on CO2 production before freezing and slightly increased the CO2 production after freezing–

257

thaw (Figure 5). Consistent with trehalose content, overexpression of both TPS1 and MAL62 in

258

NTH1 deletion (B-N+T+MAL62) had the best effect on enhancing the leavening ability after

259

freezing–thaw.

260

Discussion

34,

or after

261

Our results showed that overexpression of MAL62 or TPS1 had a similar effect to cell viability

262

after pre-fermentation-freezing and long-term freezing. Moreover, overexpression of both MAL62

263

and TPS1 in combination of NTH1 deletion showed better freezing tolerance and enhanced

264

leavening ability compared with overexpression of either MAL62 or TPS1. 12


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265

Freezing causes intracellular water molecules to form ice crystals, which damage cellular

266

structures and cause cell death35. Trehalose is a stabilizer for cellular membranes and proteins

267

under various stress conditions, such as freezing or desiccation36. In this study, we found that the

268

enhanced maltose metabolism by MAL62 overexpression was not important for trehalose

269

degradation, however, it can elicit trehalose synthesis of yeast cells before fermentation (Figures

270

3A and 3B). We further demonstrated that overexpression of MAL62 enhanced Tps1 activity

271

(Table 3), which in turn enhanced the trehalose synthesis (Figure 2). However, this enhancement

272

is not as efficient as TPS1 overexpression. One possibility is that overexpression of MAL62 could

273

just increase the Tps1 activity by partially relieving Tps1 from the catabolite repression37, and the

274

Tps1 itself, could act as a crucial pro-survival factor during apoptotic stress38.

275

In addition to Tps1 activity, MAL62 overexpression protects cells against freezing stress after

276

pre-fermentation-freezing and long-term freezing to the same extent as the TPS1 overexpression

277

(Figures 3C and 4), suggesting that the enhanced cell viability by MAL62 overexpression is not

278

solely by enhancing the Tps1 activity. In recent years, inducible MAL activator has been identified

279

to be the client protein Hsp90 and the maltose utilization in Saccharomyces cerevisiae is affiliated

280

with the molecular chaperone complex Hsp9039-40. Hsp90 is known to protects cells from

281

degradation by the proteasome pathway39 and from the damage of the free radical oxygen species

282

caused by freezing–thawing process41. Hence, enhancements in freezing tolerance by MAL62

283

overexpression may involve more pathways rather than just trehalose synthesis. A possible

284

relationship between maltose metabolism and trehalose synthesis was illustrated in Figure 6.

285

Fermentation characteristics of baker’s yeast play important roles in stress tolerance42. Our

286

study found that strains containing MAL62 overexpression (B+MAL62, B-N+M and B-

287

N+T+MAL62) have high CO2 production before or after freezing (Figure 5). A possible reason is 13



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that MAL62 overexpression may have dual functions in both promoting trehalose synthesis and

289

enhancing maltose metabolism, which is vital in fermenting dough. Conversely, TPS1

290

overexpression (B+TPS1, B-N+T and B-N+T+K) only slightly enhanced CO2 production after

291

freezing the dough possibly because of the enhanced cell viability due to increased trehalose.

292

Interestingly, the freezing tolerance and the fermentation characteristics of overexpression of

293

both MAL62 and TPS1 in the NTH1 deletion background were significantly enhanced compared

294

to that of overexpression of MAL62 (B+MAL62 or B-N+M) or TPS1 (B+TPS1 or B-N+T) after

295

the freezing–thaw stress (Figures 4 and 5) (P < 0.05). In addition, we found that NTH1 deletion

296

(B-NTH1, B-N+T, B-N+T+K, B-N+M and B-N+T+MAL62) induced a low NTH1 expression

297

level (Figure 1), and yet the neutral trehalase activity was still present (Table 3). That maybe

298

because the neutral trehalase could be encoded by multiple genes, such as NTH2 gene15,

299

However, NTH1 deletion can still help slow down the trehalose degradation in B-NTH1, B-N+T,

300

B-N+T+K, B-N+M and B-N+T+MAL62 strains. This explains why the tripartite mutant (B-

301

N+T+MAL62) provides the best enhancement for the freezing tolerance and improves the

302

fermentation characteristics44.

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Author Information

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Corresponding Author

305

Tel.: +86-022-23781298; Fax: +86-022-23781298

306

E-mail: [email protected] (L. H.)

307

Funding

308

This work was supported by the National Natural Science Foundation of China (31701569), the

309

Tianjin Municipal Special Program of Talents Development for Excellent Youth Scholars

43.

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310

(TJTZJH-QNBJRC-1-19), and the Tianjin Municipal Special Program of Talents Development

311

for Excellent Youth Scholars (TJTZJH-QNBJRC-2-12)

312

Notes

313 314

The authors declare that they have no conflict of financial interest. This paper does not contain any studies with human participants or animals performed by any of the authors.

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Page 22 of 33

Figure Captions Figure 1. qRT-PCR analysis of relative expression levels of MAL62, TPS1 and NTH1 in recombinant and control strains (BY14a and BY14a+K). Data are expressed as mean ± SD (indicated as error bars) of three independent experiments.

Figure 2. Accumulation of trehalose over 16 hours in different strains of the baker’s yeast (grown in cane molasses medium) Note: BY14a+K: BY14a carrying the empty vector Yep-K; B-N+T+K: B-N+T carrying the empty vector Yep-K; Data are expressed as mean ± SD (indicated as error bars) of three independent experiments.

Figure 3. Metabolism of maltose (A) and trehalose (B) during fermentation in LSMLD medium, and the cell viability (C) after different fermentation time (0–25 min) 7 days after frozen in LSMLD medium. Data are expressed as mean ± SD (indicated as error bars) of three independent experiments.

Figure 4. Contents of intracellular trehalose prior to being frozen and cell viability after freezing for 21 d. Data are expressed as mean ± SD (indicated as error bars) of three independent experiments.

Figure 5. Measurement of yeast CO2 production in lean dough (left: before freeze–thaw; right: after freeze–thaw). Data are expressed as mean ± SD (indicated as error bars) of three independent experiments. 22


Page 23 of 33


Figure 6. Diagram showing the simplified possible relationship between maltose metabolism and trehalose synthesis in yeast cells UDPG pathway: Traditional uridine diphosphoglucose-dependent trehalose synthesis, in which glucose is converted into glucose 6-phosphate (G6P), which together with uridine diphosphate glucose (UDPG), leads to the formation of trehalose. ADPG pathway: The alternative trehalose synthesis pathway, which has been proposed to specifically linked to maltose utilization in the strains harbor constitutive MAL gene. Trehalose can be split into two molecules of glucose by the neutral (encoded by NTH1) or the acid (encoded by ATH1) trehalase (simplified as NTH1 here). Dotted line (…) indicates the established pathway; solid line (__) indicates the pathway identified in this paper.

23



Page 24 of 33

Table 1. List of plasmids and strains used in this study Strains or plasmids

Genotype

Reference or source

Strains E. coli DH5α

Φ80 lacZ M15ΔlacU169Δ recA1 endA1 hsdR17 supE44 thi-1 gyrA relA1

YCC

BY14a

MATa

YCC

BY14a+K

MATa, Yep-K

14

B+MAL62

MATa, Yep-PMK

14

B+TPS1

MATa, Yep-PTK

This study

B-NTH1

MATa, nth1Δ:: loxP

14

B-N+M

MATa, nth1Δ:: MAL62

This study

B-N+T

MATa, nth1Δ:: TPS1

This study

B-N+T+MAL62

MATa, nth1Δ:: TPS1, Yep-PMK

This study

B-N+T +K

MATa, nth1Δ:: TPS1, Yep-K

This study

Plasmids pUG6 Yep352 Yep-K

E. coli/S. cerevisiae shuttle vector, containing Amp+,

21

loxP-kanMX-loxP disruption cassette URA3+, AmpRori control vector

Invitrogen, Carlsbad, Ca, USA

KanMX ARS URA3+, AmpRori control vector

YCC

pPGK1

bla LEU2 PGK1P-PGK1T

22

pPGKM

bla LEU2 PGK1P-MAL62-PGK1T

YCC

pPGKT

bla LEU2 PGK1P-TPS1-PGK1T

This study

pUC-NKAB

NA-loxp-KanMX-loxp-NB

YCC

pUC-NKABT

NA-loxp-KanMX-PGK1P-TPS1-PGK1T-loxp-NB

This study

pUC-NKABM

NA-loxp-KanMX-PGK1P-MAL62-PGK1T-loxp-NB

This study

Yep-PMK

bla LEU2 PGK1P-MAL62-PGK1T, KanMX

14

Yep-PTK

bla LEU2 PGK1P-TPS1-PGK1T, KanMX

This study

pSH-Zeocin

Zeor, Cre expression vector

YCC

YCC: Yeast Collection Center of the Tianjin Key Laboratory of Industrial Microbiology.

24


Page 25 of 33


Table 2. List of primers used in this study (underlining indicates the restriction sites) Primer name

Sequence 5’-3’

For recombinant construction and verification Kan-U

CGGGGTACCCAGCTGAAGCTTCGTACGC

Kan-D

CGCGGATCCGCATAGGCCACTAGTGGATCTG

MAL62-U

CCGCTCGAGATGACTATTTCTGATCATCC

MAL62-D

CCGCTCGAGTTATTTGACGAGGTAGATT

TPS1-U

CCGCTCGAGATGACTACGGATAACGCT

TPS1-D

CCGCTCGAGGGGTTCATCAGTTTTTGG

PGK-U

CGCGGATCCAAGCTTTCTAACTGATCTATCCAAAACTGA

PGK-D

CGCGGATCCAAGCTTTAACGAACGCAGAATTTTC

N-S

ATCATCATCTGTAATCGCTTCACC

K-S

CCTTTTATATTTCTCTACAGGGGCG

N-X

TACAGCGGTAAAGTTTCTATGAGCA

For qRT-PCR qMAL62-F

ACATAGTGCCCTTCACCTTG

qMAL62-R

GCGAATCGTCAGCAAATCTC

qTPS1-F

GTGGACAAGTTCACCGATG

qTPS1-R

ACTTCTGAGGCACACCTTTG

qNTH1-F

CGGTCTCGGTATTCCTCC

qNTH1-R

GCACATACGCCCTCAAAC

UBC6-F

GGACCTGCGGATACTCCTTAC

UBC6-R

TAATCGTGTGTTGGGCTTGA

25



Page 26 of 33

Table 3. Measurement of activities of α-glucosidase, Tps1, and neutral trehalase α-glucosidase activitya

Tps1 activitya

Neutral trehalase

(μmol/mg/min)

(U/g CDW)

activityb (U/g CDW)

BY14a

2.46±0.25

0.80±0.07

12.28±0.88

BY14a+K

2.45±0.22

0.83±0.10

12.26±0.81

B+MAL62

4.43±0.37**

1.06±0.10*

12.36±0.93

B+TPS1

2.45±0.23

1.20±0.07**

13.00±0.80

B-NTH1

2.45±0.21

0.82±0.07

8.68±0.74*

B-N+M

3.20±0.20**

1.05±0.10*

8.20±0.40*

B-N+T

2.45±0.20

1.10±0.06**

8.38±0.51*

B-N+T+K

2.46±0.20

1.10±0.07**

8.40±0.50*

B-N+T+MAL62

4.45±0.40**

1.30±0.08**

8.30±0.45*

Data are expressed as mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 in comparison with the parent strain. aActivities bActivity

of α-glucosidase activities and Tps1 were measured using cells from cane molasses medium.

neutral trehalase was measured using pre-fermented cells from LSMLD medium.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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For Table of Content Only

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MAL62 Overexpression Enhances Freezing Tolerance of Baker's

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