Cremoris NZ9000 Research Articles

Characterization of a Panel of Constitutive Promoters from Lactococcus cremoris for Fine-Tuning Gene Expression.

Lactococcus cremoris (homotypic synonym: Lactococcus lactis) is receiving increasing attention as a prominent vehicle for the delivery of live vaccines. This can hardly be achieved without developing tools for the genetic manipulation of L. cremoris, and the paucity of studies on L. cremoris endogenous promoters has attracted our attention. Here, we report the discovery and characterization of 29 candidate promoters identified from L. cremoris subsp. cremoris NZ9000 by RNA sequencing analysis. Furthermore, 18 possible constitutive promoters were obtained by RT-qPCR screening from these 29 candidate promoters. Then, these 18 promoters were cloned and characterized by a reporter gene, gusA, encoding β-glucuronidase. Eventually, eight endogenous constitutive promoters of L. cremoris were obtained, which can be applied to genetic manipulation of lactic acid bacteria.

Heterologous expression and purification of the phage lysin-like bacteriocin LysL from Lactococcus lactis LAC460.

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34mg of protein to be used in future functional studies.

The gene cluster associated with strong biofilm-formation capacity by histamine-producing Lentilactobacillus parabuchneri encodes a sortase-mediated pilus and is located on a plasmid

Histamine is a biogenic amine synthesized through the enzymatic decarboxylation of the amino acid histidine. It can accumulate at high concentrations in foods through the metabolism of certain bacteria, sometimes leading to adverse reactions in consumers. In cheese, histamine can accumulate at toxic levels; Lentilactobacillus parabuchneri has been identified the major cause of this problem. Previous studies have shown some L. parabuchneri strains to form biofilms on different surfaces, posing a contamination risk during cheese production, particularly for cheeses that are processed post-ripening (e.g., grating or slicing). The food contamination they cause can result in economic losses and even foodborne illness if histamine accumulates in the final product. The aim of the present work was to identify the genes of L. parabuchneri involved in biofilm formation, and to determine their function. The genomes of six strains with different biofilm-production capacities (strong, moderate and weak) were sequenced and analysed. A cluster of four genes , similar to those involved in sortase-mediated pilus formation, was identified in the strong biofilm-producers, suggesting it to have a role in surface adhesion. Cloning and heterologous expression in Lactococcus cremoris NZ9000 confirmed its functionality and involvement in adhesion and, therefore, in biofilm formation. PacBio sequencing showed this cluster to be located on a 33.4 kb plasmid, which might increase its chances of horizontal transmission. These findings provide insight into the genetic factors associated with biofilm formation in histamine-producing L. parabuchneri, and into the risks associated with this bacterium in cheese production.

Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae

Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.

Improvement of γ-aminobutyrate biosynthesis by genetically engineered Lactococcus lactis

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). Biosynthesis of GABA by lactic acid bacteria (LAB) has particularly attracted attention, as LAB are generally recognized as safe organisms. In this work, based on identification of biosynthetic gene cluster for GABA in Lactococcus lactis subsp. lactis CV56, the GadB has been heterologously expressed in Escherichia coli, purified and biochemically characterized. The recombinant enzyme existed as a homodimer under native conditions with a molecular mass of 115.03 kDa, and exhibited maximal activity at 50 °C and pH 4.7. The Km value of GadB was 3.9 mM for L-glutamate, with a limiting rate (Vmax) of 15.1 U/mg. Subsequently, the genetic engineering technique has been employed to increase the levels of expression of key GAD system genes from strain CV56 in the model LAB-L. lactis subsp. cremoris NZ9000. The fermentation results revealed that the coexpression of gadC and gadB made much more contribution to the overall yield of GABA than overexpression of any individual gene. Moreover, a novel two-stage pH control strategy for the selected strain L. lactis NZ9000/pNZ8148-gadBC was developed, and 25.61 g/L of GABA was finally achieved in the batch fermentation.

Comparative proteomic analysis of four biotechnological strains Lactococcus lactis through label-free quantitative proteomics.

Summary Lactococcus lactis is a bacteria with high biotechnological potential, where is frequently used in the amino acid production and production of fermented dairy products, as well as drug delivery systems and mucosal vaccine vector. The knowledge of a functional core proteome is important extremely for both fundamental understanding of cell functions and for synthetic biology applications. In this study, we characterized the L. lacits proteome from proteomic analysis of four biotechnological strains L. lactis: L. lactis subsp. lactis NCDO2118, L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000 and L. lactis subsp. cremoris MG1363. Our label‐free quantitative proteomic analysis of the whole bacterial lysates from each strains resulted in the characterization of the L. lactis core proteome that was composed by 586 proteins, which might contribute to resistance of this bacterium to different stress conditions as well as involved in the probiotic characteristic of L. lactis. Kegg enrichment analysis shows that ribosome, metabolic pathways, pyruvate metabolism and microbial metabolism in diverse environments were the most enriched. According to our quantitative proteomic analysis, proteins related to translation process were the more abundant in the core proteome, which represent an important step in the synthetic biology. In addition, we identified a subset of conserved proteins that are exclusive of the L. lactis subsp. cremoris or L. lactis subsp. lactis, which some are related to metabolic pathway exclusive. Regarding specific proteome of NCDO2118, we detected ‘strain‐specific proteins’. Finally, proteogenomics analysis allows the identification of proteins, which were not previously annotated in IL1403 and MG1363. The results obtained in this study allowed to increase our knowledge about the biology of L. lactis, which contributes to the implementation of strategies that make it possible to increase the biotechnological potential of this bacterium.

Expression of manB Gene from Escherichia coli in Lactococcus lactis and Characterization of Its Bifunctional Enzyme, Phosphomannomutase.

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and 50°C, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.

One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis

GDP-l-fucose is an l-fucose donor to synthesize fucosylated compounds such as human milk oligosaccharides or Lewis antigen. In this study, we used Lactococcus lactis subsp. cremoris NZ9000 to express 4 enzymes, ManB, ManC, Gmd, and WcaG and produced GDP-l-fucose by using one-pot synthesis method with mannose-6-phosphate as substrate and the enzymes as biocatalyst. For preparation of enzyme mixture, 4 genes (manB, manC, gmd, and wcaG) cloned from Escherichia coli were transformed into L. lactis strains using pNZ8008 and the recombinant cell lysates were obtained after cultivation. When mannose-6-phosphate was used as the substrate, the consecutive reactions with ManB, ManC, Gmd, and WcaG resulted in the successful production of GDP-l-fucose (0.13mM). When GDP-d-mannose was used as the substrate, it was entirely converted to GDP-l-fucose (0.2mM; 0.12g/L) via 2 enzymatic reactions mediated by Gmd and WcaG. This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria.

Production of bioactive ginsenoside Rg3(S) and compound K using recombinant Lactococcus lactis

BackgroundGinsenoside Rg3(S) and compound K (C-K) are pharmacologically active components of ginseng that promote human health and improve quality of life. The aim of this study was to produce Rg3(S) and C-K from ginseng extract using recombinant Lactococcus lactis. MethodsL. lactis subsp. cremoris NZ9000 (L. lactis NZ9000), which harbors β-glucosidase genes (BglPm and BglBX10) from Paenibacillus mucilaginosus and Flavobacterium johnsoniae, respectively, was reacted with ginseng extract (protopanaxadiol-type ginsenoside mixture). ResultsCrude enzyme activity of BglBX10 values comprised 0.001 unit/mL and 0.003 unit/mL in uninduced and induced preparations, respectively. When whole cells of L. lactis harboring pNZBglBX10 were treated with ginseng extract, after permeabilization of cells by xylene, Rb1 and Rd were converted into Rg3(S) with a conversion yield of 61%. C-K was also produced by sequential reactions of the permeabilized cells harboring each pNZBgl and pNZBglBX10, resulting in a 70% maximum conversion yield. ConclusionThis study demonstrates that the lactic acid bacteria having specific β-glucosidase activity can be used to enhance the health benefits of Panax ginseng in either fermented foods or bioconversion processes.

Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis

Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.

Controlled enterolysin A-mediated lysis and production of angiotensin converting enzyme-inhibitory bovine skim milk hydrolysates by recombinant Lactococcus lactis

Cloning of the enterolysin A (EnlA) gene (enlA) from Enterococcus faecalis DAC9 into the pMSP3545-derived pMLG2-protein expression vector encoding EnlA under control of the inducible PnisA promoter permitted the controlled release and heterologous expression of mature EnlA by Lactococcus lactis subsp. cremoris NZ9000 and Lactococcus lactis subsp. lactis IL1403. The nisin-induced expression of enlA by L. lactis NZ9000 (pMLG2) and L. lactis IL1403 (pMLG2), grown in GM17 or bovine skim milk (BSM), caused a noticeable reduction of the optical density (OD600) of the cultures and death of the growing cells. However, a high angiotensin converting enzyme (ACE)-inhibitory activity (ACE-IA) was only observed in the BSM-derived hydrolysates of L. lactis IL1403 (pMLG2) after 48 h-induction with nisin. Analysis of these hydrolysates by reversed phase high performance liquid chromatography-tandem mass spectrometry permitted the identification of major peptide fragments with known ACE-IA or sharing at least three C-terminal residues with those displaying ACE-IA.

Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase

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 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. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD(+) ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l(-1) glucose and produced 14.08 g l(-1) succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased.

Nisin-controlled extracellular production of apidaecin in Lactococcus lactis

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee (Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals.

Chimeras of Mature Pediocin PA-1 Fused to the Signal Peptide of Enterocin P Permits the Cloning, Production, and Expression of Pediocin PA-1 in Lactococcus lactis

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SPentP) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SPentP:pedA) and pMPP14i (SPentP:pedA+pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti–PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SPEntP is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.

Cloning, production and expression of the bacteriocin enterocin A produced by Enterococcus faecium PLBC21 in Lactococcus lactis

Replacement of the leader sequence of enterocin A (EntA), a bacteriocin produced by Enterococcus faecium PLBC21, by the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by E. faecium P13, permitted production of EntA in Lactococcus lactis. Chimeras encoding the EntP signal peptide (SP( entP )) fused to mature EntA (entA), with or without its immunity gene (entiA), were cloned into the expression vector pMG36c to generate the recombinant plasmids, pMPA15 (SP( entP ):entA) and pMPA10i (SP( entP ):entA + entiA). Transformation of competent L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000 with the recombinant plasmids permitted production of EntA by the transformed cells, and the co-production of nisin A and EntA by the L. lactis subsp. lactis DPC5598 transformants. Mature EntA fused to SP(EntP) is the minimum requirement for synthesis, processing and secretion of biologically active EntA in L. lactis. The production of EntA by most recombinant L. lactis hosts was larger than in the E. faecium control strains. All L. lactis derivatives showed antimicrobial activity against Listeria spp., and L. lactis (pMPA15) displayed the highest antilisterial effect.

Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidative-stress resistance of the host

This study describes how a metabolic engineering approach can be used to improve bacterial stress resistance. Some Lactococcus lactis strains are capable of taking up glutathione, and the imported glutathione protects this organism against H 2O 2-induced oxidative stress. L. lactis subsp. cremoris NZ9000, a model organism of this species that is widely used in the study of metabolic engineering, can neither synthesize nor take up glutathione. The study described here aimed to improve the oxidative-stress resistance of strain NZ9000 by introducing a glutathione biosynthetic capability. We show that the glutathione produced by strain NZ9000 conferred stronger resistance on the host following exposure to H 2O 2 (150 mM) and a superoxide generator, menadione (30 μM). To explore whether glutathione can complement the existing oxidative-stress defense systems, we constructed a superoxide dismutase deficient mutant of strain NZ9000, designated as NZ4504, which is more sensitive to oxidative stress, and introduced the glutathione biosynthetic capability into this strain. Glutathione produced by strain NZ4504(pNZ3203) significantly shortens the lag phase of the host when grown aerobically, especially in the presence of menadione. In addition, cells of NZ4504(pNZ3203) capable of producing glutathione restored the resistance of the host to H 2O 2-induced oxidative stress, back to the wild-type level. We conclude that the resistance of L. lactis subsp. cremoris NZ9000 to oxidative stress can be increased in engineered cells with glutathione producing capability.

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.

Heterologous leaky production of transglutaminase in Lactococcus lactis significantly enhances the growth performance of the host.

This study describes a novel strategy to improve the growth performance of Lactococcus lactis by heterologous production of food-grade transglutaminase. The mtg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into L. lactis subsp. cremoris NZ9000. The leaky expression of the mtg gene from the nisA promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of L. lactis, which is an important step for the purposes of producing proteins of commercial interest using L. lactis as a host.

Cloning and expression of an oligopeptidase, PepO, with novel specificity from Lactobacillus rhamnosus HN001 (DR20).

Oligopeptidases of starter and nonstarter lactic acid bacteria contribute to the proteolytic events important in maturation and flavor development processes in cheese. This paper describes the molecular cloning, expression, and specificity of the oligopeptidase PepO from the probiotic nonstarter strain Lactobacillus rhamnosus HN001 (DR20). The pepO gene encodes a protein of 70.9 kDa, whose primary sequence includes the HEXXH motif present in certain classes of metallo-oligopeptidases. The pepO gene was cloned in L. rhamnosus HN001 and overexpressed in pTRKH2 from its own promoter, which was mapped by primer extension. It was further cloned in both pNZ8020 and pNZ8037 and overexpressed in Lactococcus lactis subsp. cremoris NZ9000 from the nisA promoter. The purified PepO enzyme demonstrated unique cleavage specificity for alpha(s1)-casein fragment 1-23, hydrolyzing the bonds Pro-5-Ile-6, Lys-7-His-8, His-8-Gln-9, and Gln-9-Gly-10. The impact of this enzyme in cheese can now be assessed.