Lactic acid bacteria (LAB) are important industrial microorganism used in the production and preservation of food-stuffs. Recently, considerable advances have been made in the genetics and molecular biology of LAB. These have resulted in the construction of food-grade gene expression systems for these bacteria. This paper aims to review the essential features for food-grade systems, food-grade selection markers, food-grade controlled gene expression and food-grade inducible signaling molecule, and recent developments on food-grade cloning and expression systems for LAB. These gene expression systems have great potential for studies on gene expression and regulation in LAB and a variety of bioprocessing application in industrial fermentations.
Food Industry ; methods ; standards ; Food Microbiology ; Gene Expression Regulation, Bacterial ; Lactococcus lactis ; genetics ; growth & development

Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co²⁺, Mn²⁺, and Zn²⁺ but was strongly inhibited by Ni²⁺and Cu²⁺. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.
Glutamyl Aminopeptidase ; Lactococcus lactis/genetics* ; Biological Transport ; Culture Media ; Glutamic Acid

Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production in Lactococcus lactis ATCC 11454 was improved by overexpression of Nisin A structural gene nisA through introducing a shuttle expression vector pMG36ek-nisA and an integrated vector pDG780-nisA into the host strain. The differences of growth profiles and Nisin A production level between the two obtained genetic engineering strains FMM1/FMM2 and the parent strain were investigated. Our results show that while the growth profile (the growth rate, biomass and pH) of FMM1 was similar to the parent strain, its Nisin A production increased 31%. In contrast, the biomass of FMM2 was notably lower than the parent strain, while its yield of Nisin A enhanced slightly. The transcription level of genes involved in Nisin A biosynthesis in both engineering strains was further detected by RT-PCR. We found that all the 11 Nisin A biosynthetic genes in FMM1 and FMM2 had a higher transcription level than those in the parent strain, and these genes exhibited more significant increasing degree of transcription level in FMM1 which hosted the autonomous replicating nisA gene. These data suggest that expression of nisA may act as a rate-limit factor in Nisin A biosynthesis. In conclusion, this work provides a new method to improve Nisin A production by increasing the transcription level of nisA, paving the way to further large-scale industrial production of Nisin A.
Bacterial Proteins ; genetics ; Genes, Bacterial ; Genetic Engineering ; Genetic Vectors ; Lactococcus lactis ; genetics ; metabolism ; Nisin ; biosynthesis ; genetics ; Transcription, Genetic

The possibility of heterologous expression of human Stearoyl-CoA Desaturase (scd1) was investigated. The scd1 encoding sequence was inserted into the pNZ8149 to generate the pNZ8149-scd1 expression plasmids. Then we introduced the pNZ8149-scd1 construct into the Lactococcus lactis NZ3900 to investigate its enzyme activity. The results show that heterologous expressed SCD1 enzyme resulted in a 92%-169% increase in the C16:1n-7 and a 53-127% increase in the C18:1n-7 (P<0.05). The SCD1 enzyme was capable of producing n-7 fatty acids in Lactococcus lactis efficiently. It also suggests that the fatty acid desaturases can be heterologous expressed in Lactococcus lactis to produce the helpful fatty acids.
Electroporation ; Humans ; Lactococcus lactis ; genetics ; metabolism ; Mutagenesis, Insertional ; Nisin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Stearoyl-CoA Desaturase ; biosynthesis ; genetics

OBJECTIVETo transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF).

METHODSThe optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF.

RESULTSDNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE.

CONCLUSIONThe recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.

Electrophoresis, Polyacrylamide Gel ; Electroporation ; Genetic Vectors ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Humans ; Lactococcus lactis ; genetics ; Recombinant Proteins

To construct a safer and more efficient gene engineering Lactococcus Lactis for expressing phenylalaine ammonia lyase (PAL) which will be benefit for PKU therapy, pal cDNA of Parsly and synthesized sequence based on Lactococcus Lactis bias codons were recombined into two Lactococcus Lactis NICE systems. The activities of the expressed PAL were detected, and the effect of Lactococcus Lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using Lactococcus Lactis bias codons in both Lactococcus Lactis NICE systems. Through which several safer andmore efficient strains of the gene engineering Lactococcus Lactis were obtained.
Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Lactococcus lactis ; genetics ; metabolism ; Phenylalanine Ammonia-Lyase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; metabolism ; Transformation, Bacterial

To construct a new high effective genetic engineering strain which can express active PAL enzyme in Lactococcus lactis (L.L), and acquire better effect on curing hyperphenylalaninemia rats, Firstly translational fusion vector and transcriptional fusion vector were constructed in E. coli MC1061, and then PAL cDNA was transformed into L.L. Two kinds of high effect strain were compared with their enzyme activity and animal experiment was carried out. The results showed: (1) Two kinds of engineering L.L. were obtained and translational fusion strain has higher level enzyme activity. (2) The amount of transcinnamic aicd reach peak when induced for 6 hours. (3) The blood phe level of the treated rats was significantly reduced compared with non-treated rats when receiving fresh p(NZ8048-PAL)1/NZ9000. The engineering L.L(translational fusion strain) can significantly reduce the blood phe level of the hyperphenylalaninemia rats, which has more superiority than pMG36e-PAL/L. L.
Animals ; Escherichia coli ; genetics ; Genetic Therapy ; Lactococcus lactis ; genetics ; Male ; Phenylalanine Ammonia-Lyase ; genetics ; Phenylketonurias ; therapy ; Polymerase Chain Reaction ; Rats ; Rats, Wistar

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.
Anaerobiosis ; Escherichia coli ; enzymology ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Glucose ; metabolism ; Industrial Microbiology ; Lactococcus lactis ; enzymology ; NAD ; metabolism ; Pentosyltransferases ; biosynthesis ; genetics ; Pyruvate Carboxylase ; biosynthesis ; genetics ; Succinic Acid ; metabolism

Nicotinamide adenine nucleotide (NADH), the key cofactor in the metabolic network, plays an essential role in biochemical reaction and physiological function of industrial strains. Manipulation of NADH availability and form is an efficient and easy way to redirect the carbon flux to the target metabolites in industrial strains. We reviewed the physiological function of NADH. Detailed strategies to manipulate NADH availability are addressed. NADH manipulation to enhance metabolic function of industrial strains was discussed and potential solutions were suggested.
Bacteria ; metabolism ; Energy Metabolism ; genetics ; physiology ; Fermentation ; Industrial Microbiology ; Lactococcus lactis ; metabolism ; NAD ; metabolism ; physiology ; Saccharomyces cerevisiae ; metabolism ; Streptococcus mutans ; metabolism

In this report, we utilized N-Acetylmuraminidase (AcmA) to develop a whole-cell catalyst of endo-beta-1, 3-1, 4-glucanase in Lactococcus lactis. The PCR-amplified full-length acmA gene from L. lactis MB191 was fused with the green fluorescent gene (gfp), followed by ligating the chimeric acmA-gfp into the Escherichia coli-L. lactis shuttle expression vector pMG36k, yielding the recombinant plasmid pMB137. SDS-PAGE analysis showed that the constitutive expression of AcmA-GFP fusion protein in the L. lactis AS1.2829 construct harboring pMB137 (named MB137), with the predicted Mr of 74 kD. Western blotting, GFP specific fluorescence intensity assays and flow cytometry analysis confirmed that AcmA-GFP was immobilized on the outer membrane, which constituted approx. 35% of the total intracellular fusion protein. Furthermore, acmA was fused with a PCR-amplified encoding fragment of the endo-beta-1, 3-1, 4-glucanase gene (gls) from Bacillus sublitis BF7658, resulting in the recombinant plasmid pMB138. By transferring pMB138 into L. lactis AS1.2829, the derived L. lactis MB138 expressing the AcmA-GLS fusion enzyme exhibited a distinct whole-cell glucanase activity (by 12 U/mL) compared to the control strain, indicating AcmA had served as a functional anchoring motif to immobilize the heterologous enzyme on the cell surface of L. lactis.
Electroporation ; Endo-1,3(4)-beta-Glucanase ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Glycoside Hydrolases ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Lactococcus lactis ; enzymology ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Recombination, Genetic