Improvement of Multiple-Stress Tolerance and Lactic Acid Production in Lactococcus lactis NZ9000 under Conditions of Thermal Stress by Heterologous Expression of Escherichia coli dnaK

Abstract

ABSTRACT The effects of nisin-induced dnaK expression in Lactococcus lactis were examined, and this expression was shown to improve stress tolerance and lactic acid fermentation efficiency. Using a nisin-inducible expression system, DnaK proteins from L. lactis (DnaK Lla ) and Escherichia coli (DnaK Eco ) were produced in L. lactis NZ9000. In comparison to a strain harboring the empty vector pNZ8048 (designated NZ-Vector) and one expressing dnaK Lla (designated NZ-LDnaK), the dnaK Eco -expressing strain, named NZ-EDnaK, exhibited more tolerance to heat stress at 40°C in GM17 liquid medium. The cell viability of NZ-Vector was reduced 4.6-fold after 6 h of heat treatment. However, NZ-EDnaK showed 13.5-fold increased viability under these conditions, with a very low concentration of DnaK Eco production. Although the heterologous expression of dnaK Eco did not effect DnaK Lla production, heat treatment increased the DnaK Lla level 3.5- and 3.6-fold in NZ-Vector and NZ-EDnaK, respectively. Moreover, NZ-EDnaK showed tolerance to multiple stresses, including 3% NaCl, 5% ethanol, and 0.5% lactic acid (pH 5.47). In CMG medium, the lactate yield and the maximum lactate productivity of NZ-EDnaK were higher than the corresponding values for NZ-Vector at 30°C. Interestingly, at 40°C, these values of NZ-EDnaK were not significantly different from the corresponding values for the control strain at 30°C. Lactate dehydrogenase (LDH) activity was also found to be stable at 40°C in the presence of DnaK Eco . These findings suggest that the heterologous expression of dnaK Eco enhances the quality control of proteins and enzymes, resulting in improved growth and lactic acid fermentation at high temperature.