Effect of pathway manipulation on the metabolism
As previously described, BTCC3 (wild type) strain did not produce lactic
acid at a detectable level due to the lack of a metabolic pathway for
lactic acid production from sugar. Here, we inserted an exogenousLDH gene and disrupted several PDC genes. Figure 2
s hows how this pathway adjustment affected the product accumulation. As
expected, the insertion of LDH genes enhanced the production of lactic
acid in all mutant strains. LX1 strain (PDC1 –,LDH +) generated a 20-fold higher amount of
lactic acid compared with the results using LX5 strain
(PDC5 –, LDH +). Our
results also revealed that the generation of lactic acid elevated the
accumulation of ethanol and glycerol owing to the fact that both mutant
strains exhibited higher levels of production compared with the levels
from wild-type strains.
Moreover, two PDC genes were knocked out to generate LA1 strain
(PDC1 –, PDC5 –,LDH +). This strain exhibited a 1.9-fold
increase in lactic acid generation and a 1.8-fold decrease in ethanol
accumulation compared with LX1 (PDC1 –,LDH +) without a noticeable drop-in glucose
uptake rate (Supplementary Figure S1 ). Surprisingly, the
concentration of lactic acid produced from two copies of the LDHgene-harboring strain, namely LA15 (PDC1 –,LDH +, PDC5 –,LDH +) and LA1 (PDC1 –,LDH +, PDC5 –), showed
no significant differences (P < 0.05).
The effect of promoter strength on the production of lactic acid in this
strain was also examined. In addition to the LA1 strain
(PDC1 –, PDC5-,PTDH3 -LDH +) that
possesses LDH gene integrated to a constitutive promoter, LA2
strain (PDC1 –, PDC5-,PPDC1 -LDH +) containing aLDH gene with glucose-dependent promoter was also constructed.
Interestingly, our result revealed that the LA2 strain produced 43.23
g·L-1 of lactic acid (approximately two times higher
than LA1). This result indicated that the use of a constitutive promoter
might not be suitable for our strain, although this type of promoter is
commonly used in numerous experiments.