Construction of an Industrial Brewing Yeast Strain to Manufacture ...

772 Wang et al.
Table 3. Glucoamylase activities of TQ1, T1, and YSF5 in
fermentation tests from conical flasks (mean±SD, n=3).
Glucoamylase activity
Strain Intracellular (U/mg) Fermentation broth (U/ml)
YSF5 2.07±0.02 1.02±0.01
T1 2.36±0.09 1.49±0.03
TQ1 9.59±0.12 36.47±0.23
Fig. 3. Assay of glucoamylase (A) and dextranase (B) activities
present in the TQ1 ( ■), T1 (○), and YSF5 (△) yeast strains
during EBC tube fermentation.
Values represent the means of three replications.
synthase. The results of the PCR reactions using the
genomic DNA of YSF5 and T1 as templates are also
shown in Fig. 2, and their amplified fragment sizes were as
anticipated. This verified that the DNA fragments were
inserted into the host strain T1’s genome.
Fermentation Test and Pilot-Scale Brewing
Recombinant strain TQ1, its host strain T1, and the
parental industrial brewing yeast strain YSF5 were tested
in comparative pilot-scale fermentations. The data obtained
were analyzed by a one-way analysis of variance (ANOVA).
During EBC tube fermentation, different enzymes
activities were detected (Fig. 3). Firstly, significantly
higher levels of glucoamylase activity were detected in the
TQ1 strain, compared with T1 and YSF5, with the highest
93.26 U/ml being observed in TQ1 fermentation broth on
the 12th day of fermentation. In contrast, insignificant
glucoamylase activity was detected in both T1 and YSF5
fermentation broths through the low-level expression of
glucoamylase. However, under the control of the strong
PGK1 promoter, the SGA1 gene was highly expressed in
the recombinant strain TQ1, and via α signal sequence, its
glucoamylase could easily be secreted into the fermentation
broth. Hence, the glucoamylase activity detected in the
TQ1 fermentation broth from conical flasks exceeded that
measured intracellularly by nearly 4-fold (Table 3). This
value was higher than the heterologous glucoamylase gene
expression levels reported by Liu et al. [12]. Besidesthis,
high levels of dextranase acitivity were detected in both
TQ1 and T1 fermentations, unlike in YSF5 where none
was detected. This also indicated that the dextranase gene
was steadily expressed in strain TQ1 in despite of SGA1
expression and ADH2 disruption.
At the end of the EBC tube fermentation, the concentrations
of certain key residual saccharides were measured. Saccharides
such as glucose, sucrose, maltose, and fructose were
exhausted and not detected. The main reduction detected
was the concentration of residual maltotriose using the GC/
MS test (Table 4). The residual maltotriose concentration
measured in the TQ1 fermentation broth (0.69 mg/l) was
18.82% and 13.75% (F=20.04, P