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Lactococcus lactis, widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ΔAcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) greater than that of original strain F44. We also demonstrated that ΔAcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids biosynthesis, carbohydrate transport and metabolism, multidrug resistance, and DNA repair proteins significantly upregulated in ΔAcrR1 using transcriptome analysis and quantitative reverse transcription-PCR assays. Additionally, AcrR1 could repress the transcription of the nisin post-translational modification gene, nisC, leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis, but presented a potential strategy to enhance industrial production of nisin., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Lactococcus lactis serves as the most extensively studied model organism and an important dairy species. Though CRISPR-Cas9 systems have been developed for robust genetic manipulations, simultaneously editing multiple endogenous loci in L. lactis is still challenging. Herein, we first report the development of a double-strand break-free, robust, multiloci editing system CRISPR-deaminase-assisted base editor (CRISPR-DBE), which comprises a cytidine (CRISPR-cDBE) and an adenosine deaminase-assisted base editor (CRISPR-aDBE). Specifically targeted by a sgRNA, CRISPR-cDBE can efficiently introduce a cytidine-to-thymidine mutation and CRISPR-aDBE can high-efficiently convert adenosine to guanosine within a 5 nt editing window. CRISPR-cDBE was validated and successfully applied to simultaneously inactivate multiple genes using a single plasmid in L. lactis strain NZ9000. Meanwhile, the temperature-sensitive plasmid of CRISPR-DBE can be cured quickly, and the continuous gene editing of L. lactis has been achieved. Furthermore, CRISPR-cDBE can also efficiently convert the targeted C to T in a nisin-producing, industrial L. lactis strain F44. Finally, we applied genome-wide bioinformatics analysis to determine the scope of gene inactivation for these base editors using different Cas9 variants and evaluated the preference of SpGn and SpRYn variants for the protospacer adjacent motif in L. lactis NZ9000. Taken together, our study provides a powerful tool for simultaneously editing multiple loci in L. lactis , which may have a wide range of industrial applications in the future.

Lactococcus lactis, a lactic acid bacterium, has been widely used in the fermented dairy products. The acid tolerance of L. lactis is of great importance to food fermentation and probiotic applications. As the first barrier of bacteria, the cell wall has a protective effect on strains under many stress conditions, whereas the regulatory mechanism has rarely been reported. Here, based on the transcription analysis of 9 cell wall or membrane-related genes of L. lactis F44 under acid stress, the transcription levels of DACB, DLTD, YLBA, HRTA, WP_080613266.1 (1610), and ERFK genes were significantly increased. We constructed 9 overexpressing strains with the cell wall or membrane-related genes, respectively. It was demonstrated that the survival rates under acid stress of DACB, DLTD, and ERFK were significantly higher than that of wild-type F44. To investigate the regulatory mechanism, a DNA pull-down assay was used to identify the transcriptional regulators of these 3 genes. It was discovered that the 2-component system (TCS) transcriptional regulator TCSR7 bound to the upstream region of DLTD involved in the teichoic acid (TA) alanylation. The combination was confirmed through an electrophoretic mobility shift assay in vitro. Reverse-transcription quantitative PCR results indicated that TCSR7 upregulated the expression of DLTD gene. In addition, the transcription level of TCSR7 increased approximately 1.8-fold (log2 fold change) under acidic conditions. In summary, this study found that TCSR7 was induced by acid stress to upregulate the transcription level of the DLT operon genes, which might increase the positive charge on the cell membrane surface to increase the acid tolerance of the strain. This study lays the foundation for the regulatory mechanism of TA alanylation under acid stress., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In Lactococcus lactis, different regulation mechanisms can be activated to overcome the effects of adverse environmental stresses. Here, a TetR family regulator LssR was demonstrated as a positive regulator in the activation of the mechanisms involved in acid and nisin tolerance of L. lactis. The deletion of lssR led to the reduction of tolerance of L. lactis NZ9000 to nisin and acid stress, and the survival rates of NZ9000 under nisin and acid stress were roughly 20-fold, 10-fold (pH 3.0, hydrochloric acid), and 8.9-fold (pH 4.0, lactic acid) of the lssR mutant NZΔlssR, respectively. Moreover, the lssR mutant NZΔlssR also displayed a lower intracellular pH stability and a changed cell surface morphology. Subsequently, transcriptome analysis revealed that genes related to the arginine deiminase pathway, the surface polysaccharides biosynthesis, carbohydrates transport and metabolism, multidrug resistance, cell repair proteins and chaperones were predominantly down transcribed in NZΔlssR. The transcript levels of the arginine deiminase pathway and the surface polysaccharides biosynthesis-associated genes under acid and nisin stresses were compared between the wild type NZ9000 and NZΔlssR using real-time fluorescence quantitative PCR. It revealed that the arginine deiminase pathway genes (arcD1C1C2T) and the surface polysaccharides biosynthesis genes (cgT, gmhB, gmhA, hddA, tagH and tarS) were proposed to be the main regulatory mechanisms of LssR in response to the acid and nisin stresses. Overall, the important role of LssR in the acid and nisin stresses response was demonstrated and the putative regulation mechanism of LssR was revealed., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Bacteria adapt to the constantly changing environment by regulating their metabolism. The global transcriptional regulator CodY is known to regulate metabolism in low-G+C Gram-positive bacteria. Systems-level identification of its direct targets by proteome and chromatin immunoprecipitation followed by sequencing (ChIP-seq) assays have rarely been reported. Here, we identified that CodY serves as an activator or a repressor of hundreds of genes involved in nitrogen metabolism, carbohydrate metabolism, and transcription through iTRAQ proteome and ChIP-seq. Combined with the electrophoretic mobility shift assay (EMSA), apart from the genes associated with amino acid biosynthesis ( ilvD , leuA , optS , ybbD , dtpT , and pepN ), genes involved in cell wall synthesis ( murD and ftsW ) and nisin immunity ( nisI ) were identified as being regulated by CodY. Moreover, it was demonstrated by nisin resistance assay that CodY activated the transcription of nisI and contributed to nisin immunity. Intriguingly, CodY showed a self-regulation through binding to the motif AAAGGTGTGACAACT in the coding sequence (CDS) region of codY , as verified by DNase I footprinting assay and MEME analysis. In addition, a novel conserved AT-rich motif, AATWTTCTGACAATT, was obtained in L. lactis F44. This study provides new insights into the comprehensive CodY regulation in L. lactis by controlling metabolism, nisin immunity, and self-expression. IMPORTANCE Lactococcus lactis, a species of lactic acid bacteria (LAB) widely used in food fermentation, has been the model strain in genetic engineering, and its application has extended from food to microbial cell factories. CodY is a global regulator in low-G+C Gram-positive bacteria. Its function and direct target genes at the genome-level are little known in L. lactis. In this study, we describe the comprehensive regulation mechanism of CodY. It widely modulated the metabolism of nitrogen and carbohydrate, cell wall synthesis, and nisin immunity in L. lactis F44, and its expression level was regulated by feedback control.

ComX can improve bacterial competence by modulating global gene expression. Although competence induction may also be a protective mechanism under stress, this has not been investigated in detail. Here, we demonstrated that ComX improved the acid tolerance and nisin yield of Lactococcus lactis, which is an important gram-positive bacterium increasingly used in modern biotechnological applications. We found that overexpression of comX could improve the survival rate up to 36.5% at pH 4.0, compared with only 5.4% and 1.1% with the wild-type and comX knockout strains, respectively. Moreover, quantitative real-time PCR results indicated that comX overexpression stimulated the expression of late competence genes synergistically with exposure to acid stress. Finally, electrophoretic mobility shift assay demonstrated the binding of purified ComX to the cin-box in the promoters of these genes. Taken together, our results reveal a regulation mechanism by which ComX and acid stress can synergistically modulate the expression of late competence genes to enhance cells' acid tolerance and nisin yield., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant ( K D ) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production. IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production., (Copyright © 2020 American Society for Microbiology.)

Lactococcus lactis encounters various environmental challenges, especially acid stress, during its growth. The cell wall can maintain the integrity and shape of the cell under environmental stress, and d-amino acids play an important role in cell wall synthesis. Here, by analyzing the effects of 19 different d-amino acids on the physiology of L. lactis F44, we found that exogenously supplied d-methionine and d-phenylalanine increased the nisin yield by 93.22% and 101.29%, respectively, as well as significantly increasing the acid resistance of L. lactis F44. The composition of the cell wall in L. lactis F44 with exogenously supplied d-Met or d-Phe was further investigated via a vancomycin fluorescence experiment and a liquid chromatography-mass spectrometry assay, which demonstrated that d-Met could be incorporated into the fifth position of peptidoglycan (PG) muropeptides and d-Phe could be added to the fourth and fifth positions. Moreover, overexpression of the PG synthesis gene murF further enhanced the levels of d-Met and d-Phe involved in PG and increased the survival rate under acid stress and the nisin yield of the strain. This study reveals that the exogenous supply of d-Met or d-Phe can change the composition of the cell wall and influence acid tolerance as well as nisin yield in L. lactis IMPORTANCE As d-amino acids play an important role in cell wall synthesis, we analyzed the effects of 19 different d-amino acids on L. lactis F44, demonstrating that d-Met and d-Phe can participate in peptidoglycan (PG) synthesis and improve the acid resistance and nisin yield of this strain. murF overexpression further increased the levels of d-Met and d-Phe incorporated into PG and contributed to the acid resistance of the strain. These findings suggest that d-Met and d-Phe can be incorporated into PG to improve the acid resistance and nisin yield of L. lactis , and this study provides new ideas for the enhancement of nisin production., (Copyright © 2020 American Society for Microbiology.)

Nisin, as a common green (environmentally friendly), nontoxic antibacterial peptide secreted by Lactococcus lactis, is widely used to prevent the decomposition of meat and dairy products and maintains relatively high stability at low pH. However, the growth of Lc. lactis is frequently inhibited by high lactic acid concentrations produced during fermentation. This phenomenon has become a great challenge in enhancing the nisin yield for this strain. Here, the shuffled strain G423 that could survive on a solid plate at pH 3.7 was generated through protoplast fusion-mediated genome shuffling. The nisin titer of G423 peaked at 4,543 IU/mL, which was 59.9% higher than that of the same batch of the initial strain Lc. lactis F44. The whole genome comparisons between G423 and F44 indicated that 6 large fragments (86,725 bp) were inserted in G423 compared with that of Lc. lactis F44. Transcriptome data revealed that 4 novel noncoding transcripts, and the significantly upregulated genes were involved in multiple processes in G423. In particular, the expression of genes involved in cell wall and membrane biosynthesis was obviously perturbed under acidic stress. Quantitative real-time PCR analysis showed that the transcription of noncoding small RNA NC-1 increased by 2.35-fold at pH 3.0 compared with that of the control (pH 7.0). Overexpression assays indicated that small RNA NC-1 could significantly enhance the acid tolerance and nisin production of G423 and F44. Our work provided new insights into the sophisticated genetic mechanisms involved in Lc. lactis in an acidic environment, which might elucidate its potential application in food and dairy industries., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Cell wall is closely related to bacterial robustness and adsorption capacity, playing crucial roles in nisin production in Lactococcus lactis. Peptidoglycan (PG), the essential component of cell wall, is usually modified with MurNAc O-acetylation and GlcNAc N-deacetylation, catalyzed by YvhB and XynD, respectively. In this study, increasing the two modifications in L. lactis F44 improved autolysis resistance by decreasing the susceptibility to PG hydrolases. Furthermore, both modifications were positively associated with overall cross-linkage, contributing to cell wall integrity. The robust cell wall rendered the yvhB/xynD-overexpression strains more acid resistant, leading to the increase of nisin production in fed-batch fermentations by 63.7 and 62.9%, respectively. Importantly, the structural alterations also reduced nisin adsorption capacity, resulting in reduction of nisin loss. More strikingly, the co-overexpression strain displayed the highest nisin production (76.3% higher than F44). Our work provides a novel approach for achieving nisin overproduction via extensive cell wall remodeling.

Lactococcus lactis encounters 3 environmental stimuli, H + , lactate, and undissociated lactic acid, because of the accumulation of lactic acid-the predominant fermentation product. Few studies have examined how these stimuli synergistically affect the bacteria. Herein, we analyzed the dissociation degree of lactic acid at different pH and investigated the cellular response to cross-stress in L. lactis ssp. lactis F44 through quantitative proteomic analysis using isobaric tags for relative and absolute quantitation of 3 groups: 0% lactic acid with pH 4.0 and 0% lactic acid with pH 5.0 for acid stress; 2% lactic acid with pH 7.0 and 3% lactic acid with pH 7.0 for lactate stress; and 2% lactic acid with pH 4.0, 2% lactic acid with pH 5.0, 3% lactic acid with pH 4.0, and 3% lactic acid with pH 5.0 for cross-stress. We observed that the metabolisms of carbohydrate and energy were inhibited, which might be due to the feedback inhibition of lactic acid. The arginine deiminase pathway was improved to maintain the stability of intracellular pH. Additionally, some differentially expressed genes associated with the general stress response, amino acid metabolism, cell wall synthesis, and regulatory systems played significant roles in stress response. Overall, we highlighted the response mechanisms to lactic acid stress and provided a new opportunity for constructing robust industrial strains., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Lactococcus lactis, a gram-positive bacterium, encounters various environmental stresses, especially acid stress, during fermentation. Small RNAs (sRNAs) that serve as regulators at post-transcriptional level play important roles in acid stress response. Here, a novel sRNA S042 was identified by RNA-Seq, RT-PCR and Northern blot. The transcription level of s042 was upregulated 2.29-fold under acid stress by Quantitative RT-PCR (qRT-PCR) analysis. Acid tolerance assay showed that overexpressing s042 increased the survival rate of L. lactis F44 and deleting s042 significantly inhibited the viability under acidic conditions. Moreover, the targets were predicted by online software and four genes were chosen as candidates. Among them, argR (arginine regulator) and accD (acetyl-CoA carboxylase carboxyl transferase subunit beta) were validated to be the direct targets activated by S042 through reporter fusion assay. The regulatory mechanism between S042 and its targets was further investigated through Bioinformatics and qRT-PCR. This study served to highlight the role of the novel sRNA S042 in acid resistance of L. lactis and provided new insights into the response mechanism of acid stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: To engineer a small nonconding RNA anti41 to enhance nisin yield by inhibiting the expression of glnR in Lactococcus lactis F44., Results: We constructed a screening library to determine appropriate artificial sRNAs and obtained a sRNA anti41 that can produce approximately three fold of the inhibitory effect on GlnR. Moreover, the transcription levels of the direct inhibitory targets of GlnR (glnP, glnQ, amtB, and glnK) were dramatically upregulated in the anti41 overexpression strain (F44-anti41), thereby confirming the inhibitory effect of anti41 on GlnR. In addition, anti41 overexpression improved the survival rate of cells by approximately three fold under acid stress, promoted cell growth, and increased nisin yield by 29.83%., Conclusions: We were able to provide a novel strategy for the construction of robust high-producing industrial strains.

To overcome the adverse impacts of environmental stresses during growth, different adaptive regulation mechanisms can be activated in Lactococcus lactis In this study, the transcription levels of eight transcriptional regulators of L. lactis subsp. lactis F44 under acid stress were analyzed using quantitative reverse transcription-PCR. Eight gene-overexpressing strains were then constructed to examine their influences on acid-resistant capability. Overexpressing ythA , a PspC family transcriptional regulator, increased the survival rate by 3.2-fold compared to the control at the lethal pH 3.0 acid shock. Moreover, the nisin yield was increased by 45.50%. The ythA -overexpressing strain FythA appeared to have higher intracellular pH stability and nisin-resistant ability. Subsequently, transcriptome analysis revealed that the vast majority of genes associated with amino acid biosynthesis, including arginine, serine, phenylalanine, and tyrosine, were predominantly upregulated in FythA. Arginine biosynthesis ( argG and argH ), arginine deiminase pathway, and polar amino acid transport ( ysfE and ysfF ) were proposed to be the main regulation mechanisms of YthA. Furthermore, the transcription of genes associated with pyrimidine and exopolysaccharide biosynthesis were upregulated. The transcriptional levels of nisIPRKFEG genes were substantially higher in FythA, which directly contributed to the yield and resistance of nisin. Three potential DNA-binding sequences were predicted by computer analysis using the upstream regions of genes with prominent changes. This study showed that YthA could increase acid resistance and nisin yield and revealed a putative regulation mechanism of YthA. IMPORTANCE Nisin, produced by Lactococcus lactis subsp. lactis , is widely used as a safe food preservative. Acid stress becomes the primary restrictive factor of cell growth and nisin yield. In this research, we found that the transcriptional regulator YthA was conducive to enhancing the acid resistance of L. lactis F44. Overexpressing ythA could significantly improve the survival rate and nisin yield. The stability of intracellular pH and nisin resistance were also increased. Transcriptome analysis showed that nisin immunity and the biosynthesis of some amino acids, pyrimidine, and exopolysaccharides were enhanced in the engineered strain. This study elucidates the regulation mechanism of YthA and provides a novel strategy for constructing robust industrial L. lactis strains., (Copyright © 2018 American Society for Microbiology.)

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H + due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).

Nisin, a polycyclic antibacterial peptide produced by Lactococcus lactis, is stable at low pH. Improving the acid tolerance of L. lactis could thus enhance nisin yield. Small non-coding RNAs (sRNAs) play essential roles in acid tolerance by regulating their target mRNAs at the post-transcriptional level. In this study, a novel sRNA, s015, was identified in L. lactis F44 via the use of RNA sequencing, qRT-PCR analysis, and Northern blotting. s015 improved the acid tolerance of L. lactis and boosted nisin yield at low pH. In silico predictions enabled us to construct a library of possible s015 target mRNAs. Statistical analysis and validation suggested that s015 contains a highly conserved region (5'-GAAAAAAAC-3') that likely encompasses the regulatory core of the sRNA. atpG, busAB, cysD, ilvB, tcsR, ung, yudD, and ywdA were verified as direct targets of s015, and the interactions between s015 and its target genes were elucidated. This work provided new insight into the adaptation mechanism of L. lactis under acid stress.

Present investigation explores the potential of defatted rice bran (DRB) serving as sole carbon source and partial nitrogen source to support Lactococcus lactis growth and nisin production. To retain the nutrients in DRB, especially protein fractions, thermal pretreatment followed by enzymatic hydrolysis without washing step was applied for saccharification. A maximum of 45.64g reducing sugar mainly containing 30.26g glucose and 5.66g xylose from 100g DRB was attained in hydrolysates of DRB (HD). A novel strategy of xylR (xylose transcriptional regulator) overexpression followed by evolutionary engineering was proposed, which significantly increased the capacity of L. lactis to metabolize xylose. Subsequently, RT-PCR results indicated that xylR overexpression stimulated expression of xylose assimilation genes synergistically with exposure to xylose. In HD medium, the highest nisin titer of the engineered strain FEXR was 3824.53IU/mL, which was 1.37 times of that in sucrose medium by the original strain F44., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nisin, one kind of natural antimicrobial peptide, is produced by certain Lactococcus lactis strains, which generally require expensive high-quality nitrogen sources due to limited ability of amino acids biosynthesis. Here we use defatted soybean meal (DSM) as sole nitrogen source to support L. lactis growth and nisin production. DSM medium composition and fermentation conditions were optimized using the methods of Plackett-Burman design and central composite design. The highest nisin production of 3879.58 IU/ml was obtained in DSM medium, which was 21.3% higher than that of commercial medium. To further increase the utilization ability of nitrogen sources, we enhanced the proteolytic function in L. lactis through rationally expressing the related enzymes, which were selected according to the compositions of amino acids and molecular weight of peptides in DSM medium. Significantly, an artificial proteolytic system consisting of a heterologous protease (NprB), an oligopeptides transporter subunit (OppA) and two peptidases (PepF and PepM) was introduced into L.lactis. The constructed strain BAFM was capable of achieving efficient biomass accumulation and nisin yield with 30% decreased amount of DSM hydrolysates, which further reduced the cost of nisin production. The strategy described here offers opportunities for low-cost L. lactis fermentation and large-scale nisin production in industry.

Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process.

Microbiological contamination and oxidative damage are the two main challenges in maintaining quality and improving shelf-life of foods. Here, we developed a Lactococcus lactis fermentation system that could simultaneously produce nisin, an antimicrobial peptide, and γ-aminobutyric acid (GABA), an antioxidant agent. In this system, we metabolically engineered a nisin producing strain L. lactis F44 for GABA production by expression of glutamate decarboxylase and glutamate/GABA antiporter. GABA biosynthesis could facilitate nisin production through enhancing acid resistance of the strain. By applying a two-stage pH-control fermentation strategy, the engineered strain yielded up to 9.12 g/L GABA, which was 2.2 times higher than that of pH-constant fermentation. Furthermore, we demonstrated the potential application of the freeze-dried fermentation product as a preservative to improve the storage performance of meat and fruit. These results suggested that the fermentation product of nisin–GABA co-producing strain could serve as a cost-effective, easily prepared, and high-performance food preservative. [ABSTRACT FROM AUTHOR]