In a recent report, it was reported that the wild-type E. coli W3110 cells first consumed glucose rapidly with a rate of 1.37 g/gDCW/h, followed by galactose consumption with a rate of 0.04 g/gDCW/h in both sugars co-fermentation . This indicates that galactose consumption was strictly inhibited by the CCR mechanism in the W3110 strain (Fig. 1). In this study, to see the effect of the CCR mechanism on consumption rate under sole carbon source fermentation, we determined the specific sugar consumption rate in W3110 cultures supplemented with either 6 g/L glucose or 6 g/L galactose (Fig. 2a, b). In the glucose fermentation, the wild-type W3110 strain grew up to OD600 of 1.52 from 5.33 g/L carbon source in 48 h cultures (Fig. 2a), which is very similar to the value obtained in the galactose fermentation (Fig. 2b). However, we found differences in the maximum specific growth rate and the specific sugar consumption rate between glucose- and galactose-fermentations using the W3110 strain (Table 1). The wild-type W3110 strain showed increases in growth and sugar consumption rates in glucose fermentation by 1.41 and 1.37 times, respectively, compared to values in galactose fermentation (Table 1). This result indicates that, although the two carbon sources are not competing conditions, the wild-type W3110 strain favors glucose as a feedstock rather than galactose under the same situation.
Next, we examined the galR and galS double mutant, GR2 strain, to see the effect of deregulation of repressor-specific CCR on the consumption of glucose and galactose as a sole carbon source (Fig. 2c, d). Galactose operon repressors encoded by the galR and galS genes are involved in the CCR mechanism by binding to the operator locus (Fig. 1). In the previous report, under the glucose and galactose co-fermentation (each 4 g/L), the E. coli GR2 cells showed a similar specific glucose consumption rate of the W3110 strain . However, in the same co-fermentation, the GR2 strain achieved a 3.38 times increase in specific galactose consumption rate . In this study, in 6 g/L glucose fermentation, the GR2 strain showed an increase in the specific glucose consumption rate compared with the W3110 strain, but no significant change in the maximum specific growth rate (Table 1). In 6 g/L galactose fermentation, the GR2 strain showed an increase in the specific galactose consumption rate and the maximum specific growth rate compared with the wild-type strain (Table 1). These indicate that the deregulation of repressor-specific CCR positively affected central metabolism in both cultures supplemented with glucose and galactose as the sole carbon source. Especially, the double mutation of galR and galS genes was more sensitive in galactose fermentation than glucose fermentation. Nevertheless, glucose was still favored as the carbon source by the E. coli GR2 strain, in which the GalS and GalS repressor-specific CCR was deregulated.
To see the effect of additional gene knockout of either pfkA or pfkA/zwf in the GR2 strain on sugar consumption, we examined the GR2P and GR2PZ strains, in which three (galR, galS, and pfkA) and four (galR, galS, pfkA, and zwf) genes were knocked out, respectively (Fig. 3). Interestingly, GR2P strain showed similar maximum specific growth rates of 0.2989 /h and 0.3057 /h in glucose- and galactose-fermentations, respectively. These were resulted from a high similarity between the maximum specific sugar consumption rates on glucose (0.5566 g/gDCW/h) and galactose (0.5234 g/gDCW/h). It seems that the growth of the GR2P strain was delayed by blocking the Embden-Meyerhof-Parnas (EMP) pathway, regardless the type of carbon source. Thus, in the fermentation using E. coli GR2P strain, it was also revealed that glucose was not further preferred as a sole carbon source compared to galactose. In the GR2P strain, glucose and galactose are primarily catabolized through the pentose phosphate pathway (PPP) at the node of glucose-6-phosphate . In the fermentations using GR2PZ strain, in which the PPP was further blocked by the zwf gene knockout together with disruption of galR, galS, and pfkA genes, the growth was further retarded. However, in such fermentation using GR2PZ strain, the preference for glucose as a carbon source was restored as like to wild-type and GR2 strain. In this study, we characterized the wild-type E. coli W3110 strain and its mutants GR2 (galR- and galS-), GR2P (galR-, galS-, and pfkA-), and GR2PZ (galR-, galS-, pfkA-, and zwf-) in sole carbon source fermentation supplemented with either glucose or galactose. In all strains tested in this study except GR2P, glucose was primarily preferred as a sole carbon source rather than galactose. The GR2P strain showed no difference between fermentation using glucose and galactose as a sole carbon source, especially in sugar consumption and cell growth rates. Our study provides critical information for flexible managing in the replacement of feedstocks depending on the industrial supply and demand situation of carbon sources in fermentation using the CCR engineered E. coli strains.In this study, we characterized the wild-type E. coli W3110 strain and its mutants GR2 (galR– and galS–), GR2P (galR–, galS–, and pfkA–), and GR2PZ (galR–, galS–, pfkA–, and zwf–) in sole carbon source fermentation supplemented with either glucose or galactose. In all strains tested in this study except GR2P, glucose was primarily preferred as a sole carbon source rather than galactose. The GR2P strain showed no difference between fermentation using glucose and galactose as a sole carbon source, especially in sugar consumption and cell growth rates. Our study provides critical information for flexible managing in the replacement of feedstocks depending on the industrial supply and demand situation of carbon sources in fermentation using the CCR engineered E. coli strains.
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