Hsp from Lactobacillus plantarum Expression in Lactococcus lactis MG1363

. Small heat shock proteins are protective proteins produced by organisms under thermal stress. They are widely present in living organisms. Here, Hsp18 , Hsp18.55 and Hsp19.5 genes were cloned from Lactobacillus plantarum and heterologous expressed in Lactococcus lactis , and their potential functions under ethanol stress were investigated. The results showed that the recombinant strain over expressing Hsp19.5 gene had stronger stress resistance, which provided a basis for further study of the survival ability of other microorganisms under ethanol stress.


Introduction
As a safe food-grade host bacterium expressing foreign proteins [1] , Lactococcus lactis has developed from food bacteria to microbial cell factories and is used to produce industrial products [2] . In recent years, the molecular biology of L. lactis has developed rapidly [3,4] . Through genetic engineering technology, foreign gene were introduced into L. lactis and expressed, so that the strain had beneficial characteristics and abundant metabolites [5][6][7] . However, in actual production, L. lactis faces many survival challenges that affect the physiological functions of cells and their survival rate [8][9][10] . One of the biological responses to environmental stress is the synthesis of small heat shock proteins (sHSPs) [11] , which prevent or reduce the destruction of other proteins by stress [12] and assist in the refolding or degradation of stress-damaged proteins [13] . Basic expression of Hsps is essential for normal protein folding, maintenance of signal transduction, and normal development [14] . The induction levels of different heat shock genes are regulated by perceptive sublethal factors. Small heat shock protein Lo18 is an important participant in the stress response of Oenococcus oeni, which prevents thermal aggregation of proteins and plays a crucial role in membrane quality control [15] . Because L. lactis itself does not contain Hsp genes, expression of the small heat shock protein Lo18 from O. oeni improved the tolerance of L. lactis to heat and acid conditions [16] . Then, the small heat shock protein gene from Streptococcus thermophilus was expressed in L. lactis ML23, and the results showed that the expression of Hsps inserted in the food-grade vector pMG made L. lactis ML23 resistant to environmental stress [17] . It is resistant to environmental stresses. Fiocco et al. [18] overexpressed three heat shock protein genes (Hsp18.5, Hsp18.55 and Hsp19. 3) in Lactobacillus plantarum WCFS1 strain and found that overexpression of Hsp18.55 and Hsp19.3 genes improved the solvent tolerance of L. plantarum WCFS1. In this study, Hsp18.5, Hsp18.55 and Hsp19.3 genes were cloned from L. plantarum, and were heterologous expressed in L. lactis by connecting with shuttle plasmids, to study the role of the three genes in L. plantarum ethanol tolerance.

Strains, Plasmids and Primers
The plasmids and strains used in this study are listed in Table 1. The primers used in this study are listed in Table  2. L. lactis and L. plantarum were purchased from the Center for Microbial Culture, Escherichia coli. coli competence Fast-T1 was purchased from Vazyme Biotech Co., Ltd, and L. lactis competence was made in our laboratory. Erythromycin was purchased from Beijing Solarbio Science & Technology Co., Ltd. PrimeSTAR GXL DNA Polymerase was purchased from Takara Biomedical Technology (Beijing) Co., Ltd. ClonExpress II One Step Cloning Kit, FastPure Bacteria DNA Isolation Mini Kit, and FastPure Gel DNA Extraction Mini Kit were purchased from Vazyme Biotech Co., Ltd. All chemicals were used at analytical grade.

Culture Conditions
L. plantarum was grown in MRS medium and E. coli was grown in Luria-Bertani (LB) medium. L. lactis was grown in GM17 (M17 medium with added glucose) medium. L. plantarum was incubated at 28℃. E. coli recovery conditions was 1 mL LB liquid medium at 37°C for 1 h with 200 rpm, and then was spread on LB agar plates with 400 mg/L erythromycin for growth. L. lactis was resuscitated by incubation in 1 mL GM17 liquid medium at 30°C for 2 h. After that was spread on GM17 agar plates with 5 mg/L erythromycin.

Construction and Transformation of Recombinant Plasmids
All three DNA fragments were amplified from L. plantarum genome by polymerase chain reaction (PCR) using high fidelity DNA polymerase. The target gene was ligated to the pMG36e linear vector using homologous recombinant polymerase and transformed into the E. coli competence Fast-T1. The positive colonies were screened with 400 mg/L erythromycin. The recombination plasmid were extracted from the positive colonies and electrotransformed into L. lactis, and the recombinant colonies were screened at GM17 agar plates with 5 mg/L erythromycin.

Ethanol Tolerance Test of Recombinant L.
lactis The recombinant L. lactis and wild-type L. lactis were inoculated into GM17 liquid medium and incubated until the medium were turbid. After that, the inoculums were transferred into 50 mL of liquid medium with different ethanol concentrations at 2% (v/v) inoculum, and incubated anaerobically at 30℃ in a constant temperature incubator. Hsp18.5, Hsp18.55 and Hsp19.3 and Construction of Recombinant Plasmids The Hsp18.5, Hsp18.55 and Hsp19.3 genes were amplified by PCR using the genome of L. plantarum as a template. The amplified fragments were cloned into the L. lactis pMG36e expression plasmid by homologous recombinase and were transformed into E. coli Fast-T1. The positive colonies were screened by colony PCR using the specific primers form the three fragments ( Table 2). The results were shown in Fig.1. The eight recombinant strains with pMG36e-Hsp18.5, four recombinant strains with pMG36e-Hsp18.55 and nine recombinant strains with pMG36e-Hsp19.3 were obtained. Finally, these recombinant plasmids form strains were verified by DNA sequencing.

Construction of Recombinant L. lactis
The three successfully sequenced plasmids pMG36e-Hsp18.5, pMG36e-Hsp18.55 and pMG36e-Hsp19.3 were electro-transformed into L. lactis MG1363, and the positive colonies were screened by colony PCR using the specific primers from the three fragments (Table 2) and the size of the products was shown in Fig.2 Fig.3, with increasing ethanol concentration in the medium, and the growth rate of the three recombinant L. lactis MG1363 strains showed a decreasing trend, which was the similar to the wild strain under ethanol stress. However, the three recombinant strains displayed growth much better than wild type at the same ethanol concentration. For example, the growth of wild type can be severely inhibited at 7.0% ethanol concentration (Fig.3a). But the growth of Hsp18.5 expressing and Hsp18.55 expressing strains were severely inhibited at 8.0% ethanol concentration (Fig.3b, Fig.3c). Furthermore, the growth of Hsp19.3 expressing strains had a higher growth activity than the other two recombinant strain at each ethanol concentration. Therefore, sHsp overexpression exactly improved the ethanol tolerance of host bacteria L. lactis MG1363 and Hsp19.3 was more effective than the two Hsps to ethanol tolerance of host bacteria.

Conclusion
Adverse condition usually leads to protein misfolding or unfolded protein aggregation in organisms, which severely hinders the cells from exercising normal functions and thus affects the growth and development of organisms. Inducing stress protein expression is therefore a methodological strategy to combat adverse environmental conditions. In the harsh environment of wine, a variety of microorganisms have difficulty surviving, but O. oeni and L. plantarum possess a high ability to survive. Studies on O. oeni have shown that the O. oeni genome contains the Hsps sequence encoding the heat shock protein Lo18, which interacts with the cell membrane and regulates the physical state of lipids under heat shock conditions [15] . It has been found that the genome of L. plantarum also contains Hsps, but the tolerance of L. plantarum overexpressing strains to cold, heat, acid and butanol is different, which may be related to the different functions of Hsps. Because L. lactis itself does not contain heat shock protein genes, and the growth of L. lactis was inhibited under ethanol stress. Therefore, heterologous expression of heat shock proteins in L. lactis is helpful to improve the understanding of heat shock proteins. Based on the experimental results, it is reasonable to assume that the expression or not of the heat shock protein gene and the amount of expression will play a rather important role for the fermentation industry, and it provides new ideas to solve the bottlenecks encountered in the fermentation process.