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.