BackgroundLactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality.ResultsA high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h.ConclusionsThe work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1075-3) contains supplementary material, which is available to authorized users.

Listeria monocytogenes is a saprophytic bacterium that thrives in diverse environments and causes listeriosis via ingestion of contaminated food. RsbX, a putative sigma B ( B ) regulator, is thought to maintain the ready state in the absence of stress and reset the bacterium to the initial state in the poststress stage in Bacillus subtilis. We wondered whether RsbX is functional in L. monocytogenes under different stress scenarios. Genetic deletion and complementation of the rsbX gene were combined with survival tests and transcriptional and translational analyses of B expression in response to stresses. We found that deletion of rsbX increased survival under secondary stress following recovery of growth after primary stress or following stationary-phase culturing. The ⌬rsbX mutant had higher expression of B than its parent strain in the recovery stage following primary sodium stress and in stationary-phase cultures. Apparently, increased B expression had contributed to improved survival in the absence of RsbX. There were no significant differences in survival rates or B expression levels in response to primary stresses between the rsbX mutant and its parent strain during the exponential phase. Therefore, we provide clear evidence that RsbX is a negative regulator of L. monocytogenes B during the recovery period after a primary stress or in the stationary phase, thus affecting its survival under secondary stress. Listeria monocytogenes causes listeriosis in humans, mostly due to consumption of contaminated foods. As a saprophytic bacterium thriving in diverse environments, L. monocytogenes can survive and grow over a wide range of environmental conditions, including temperatures from Ϫ0.4 to 45°C, pH as low as 2.5, and high osmolarity (10% to 20% NaCl) (1-4). The general stressresponsive alternative sigma factor sigma B ( B ), which was first identified in Bacillus subtilis (5), plays a pivotal role in its resistance to environmental stresses (6, 7).B is coexpressed with seven of its principal regulators (regulators of sigma B rsbR, rsbS, rsbT, rsbU, rsbV, rsbW, and rsbX) in B. subtilis (8) and L. monocytogenes (9-11). Partner switching upon phosphorylation and dephosphorylation is the main regulatory mechanism of this protein cluster in response to stresses. This has been studied mostly in B. subtilis (12)(13)(14) and seldom in L. monocytogenes. In unstressed B. subtilis, RsbW sequesters B into an association that prevents it from interacting with RNA polymerase. RsbV is dephosphorylated by RsbU or RsbP in response to environmental or metabolic stress, respectively (15, 16). The dephosphorylated RsbV is capable of competing for RsbW, resulting in B liberation. The upstream proteins RsbT, RsbS, and RsbR form a complex called the "stressosome" in B. subtilis (17)(18)(19). Environmental stresses stimulate the kinase activity of RsbT, which can then be released from the stressosome available to activate RsbU (20).However, the interactions of these regulatory proteins in L. monocytogenes under stresses are not well...

Numerous lactic acid bacteria (LAB) bacteriophage genomes have been sequenced, while the functional genes are yet to be exploited. In this study, a λ Red-like recombinase operon LCABL_13040-50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome, and its recombination function was confirmed by the replacement of a 167-bp galK fragment with chloramphenicol-resistant gene (cat) in the L. casei BL23 genome. Further functional analysis showed that LCABL_13040 and LCABL_13060 were analogs to the host nuclease inhibitor (Redγ) and 5΄-3΄ exonuclease (Redα/RecE), respectively. After optimization of recombineering conditions, including induction, homology length, recovery time and double-strand DNA substrates quantity, the recombineering efficiency reached ∼2.2 × 10-7. Subsequently, combining cre-lox technology, the optimal LCABL_13040-50-60 proteins could catalyze markerless deletion of a 167-bp galK fragment and insertion of the gfp gene as well as precision point mutation of rpoB gene in the L. casei BL23 genome, suggesting the LCABL_13040-50-60 operon encoded for three recombineering proteins. Moreover, with the assistance of Redγ, the LCABL_13040-50-60 proteins also showed recombinase activity in six other L. casei strains, L. paracasei OY and L. plantarum WCSF1. All the results demonstrated that the prophage-associated recombinases LCABL_13040-50-60 have great potential to be used for genome editing in LAB.

Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. p-Amidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, β-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the β-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus.

Lactic acid bacteria (LAB) are a phylogenetically diverse group with the ability to convert soluble carbohydrates into lactic acid. Many LAB have a long history of safe use in fermented foods and are recognized as food-grade microorganisms. LAB are also natural inhabitants of the human intestinal tract and have beneficial effects on health. Considering these properties, LAB have potential applications as biotherapeutic vehicles to delivery cytokines, antigens and other medicinal molecules. In this review, we summarize the development of, and advances in, genome manipulation techniques for engineering LAB and the expected future development of such genetic tools. These methods are crucial for us to maximize the value of LAB. We also discuss applications of the genome-editing tools in enhancing probiotic characteristics and therapeutic functionalities of LAB.

BackgroundLactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment.ResultsThe pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 l-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23.ConclusionsAs reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment.

is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the and genes of ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wild-type strain, the transcription and enzymatic activity of PDH remained stable in the Δ mutant, while those of POX increased significantly in the Δ mutant. More lactate but less acetate was produced in the Δ mutant than in the wild-type and Δ mutant strains, and more HO (a product of the POX pathway) was produced in the Δ mutant. We speculated that the high levels of aerobic acetate accumulation in ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively. PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we reported that both PDH and POX worked in the aerobic conversion of lactate to acetate in ATCC 367, in dominant and secondary roles, respectively. Our findings will further develop the theory of aerobic metabolism by LAB.

BackgroundLactobacillus casei is widely used in the dairy and pharmaceutical industries and a promising candidate for use as cell factories. Recently, genome sequencing and functional genomics provide the possibility for reducing L. casei genome. However, it was still limited by the inefficient and laborious genome deletion methods.ResultsHere, we proposed a genome minimization strategy based on LCABL_13040-50-60 recombineering and Cre-lox site-specific recombination system in L. casei. The LCABL_13040-50-60 recombineering system was used to introduce two lox sites (lox66 and lox71) into 5′ and 3′ ends of the targeted region. Subsequently, the targeted region was excised by Cre recombinase. The robustness of the strategy was demonstrated by single-deletion of a nonessential ~ 39.3 kb or an important ~ 12.8 kb region and simultaneous deletion of two non-continuous genome regions (5.2 and 6.6 kb) with 100% efficiency. Furthermore, a cyclical application of this strategy generated a double-deletion mutant of which 1.68% of the chromosome was sequentially excised. Moreover, biological features (including growth rate, electroporation efficiency, cell morphology or heterologous protein productivity) of these mutants were characterized.ConclusionsTo our knowledge, this strategy is the first instance of sequential deletion of large-scale genome regions in L. casei. We expected this efficient and inexpensive tool can help for rapid genome streamlining and generation restructured L. casei strains used as cell factories.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0872-4) contains supplementary material, which is available to authorized users.