Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712

Wegmann, Udo; Overweg, Karin; Jeanson, Sophie; Gasson, Mike; Shearman, Claire

doi:10.1099/mic.0.062554-0

Cited by 37 publications

(47 citation statements)

References 44 publications

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“…Since the L. lactis strain that we work with has been accustomed to growing in milk, and since large amounts of milk-derived waste is generated by the dairy industry, we decided to test if dairy waste (kindly provided by ARLA Foods) could be a suitable feedstock for acetoin production. Our strain is a plasmid-cured dairy derivative, and it is unable to ferment lactose, and for this reason, we first complemented it with the lactose/protease plasmid pLP712 (Wegmann et al 2012), which resulted in strain AL001. The outcome was that 99 mM (34 g/L) lactose was completely consumed within 55 h with the formation of 157 mM (14 g/ L) acetoin (Fig.…”

Section: Development Of a Sustainable Process For Acetoin Productionmentioning

confidence: 99%

“…Samples were collected for measuring cell density (OD 600 ), lactose, and acetoin concentrations. Lactococcal plasmid Wegmann et al 2012 a The promoter strength is calculated based on the specific β-galactosidase activity, as the atpAGD gene is inserted into an operon structure together with the lacLM genes encoding a β-galactosidase…”

Section: Waste Stream Conversionmentioning

confidence: 99%

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Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

Liu

Kandasamy

Würtz

et al. 2016

Appl Microbiol Biotechnol

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Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F-ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F-ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F-ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F-ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F-ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished.

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Section: Development Of a Sustainable Process For Acetoin Productionmentioning

confidence: 99%

Section: Waste Stream Conversionmentioning

confidence: 99%

Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

Liu

Kandasamy

Würtz

et al. 2016

Appl Microbiol Biotechnol

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“…In this work, we analysed the impact of the ftsH null mutation on the life cycle of the P335 temperate phage TP712, which is able to infect and lysogenize L. lactis MG1363 (Gasson, 1983) and derivatives thereof. The complete TP712 nt sequence has been determined (GenBank accession number AY766464) essentially as described by Wegmann et al (2012).Preliminary experiments revealed that in standard plaque assays (Lillehaug, 1997), the efficiency of plaquing (EOP) of this phage on an available non-polar and in-frame L. lactis mutant lacking ftsH (Pinto et al, 2011) was reduced to 1.5610 25 when compared to its parent L. lactis NZ9000. Upon complementation with the plasmid pUK200 : : ftsH, in which ftsH from L. lactis MG1363 is under the control of the inducible nisin promoter (P nisA ), plaque formation was restored (Fig.…”

mentioning

confidence: 99%

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confidence: 99%

Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712

Roces

Wegmann

Campelo

et al. 2013

Journal of General Virology

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The temperate bacteriophage TP712 was unable to plaque on Lactococcus lactis DftsH lacking the membrane protease FtsH and complementation in trans restored the WT phenotype. Absence of ftsH did not hinder phage adsorption, phage DNA delivery or activation of the lytic cycle. Thin sections revealed that TP712 virions appeared to be correctly assembled inside the DftsH host, but were not released. These virions were infective, demonstrating that a functional host FtsH is required by TP712 to proceed effectively with lysis of the host.Large-scale dairy fermentations can be seriously compromised by bacteriophages infecting lactic acid bacteria (LAB), and particularly Lactococcus lactis, which is a commonly used starter culture for cheese making (Garneau & Moineau, 2011). Lactococcal phages are currently classified in 10 groups, eight of them belonging to the Siphoviridae family of the order Caudovirales. Those of the c2, 936 and P335 groups are the most commonly found in dairy plants (Deveau et al., 2006).A considerable effort has been made to improve and select phage-resistant starter strains based on the knowledge of resistance mechanisms developed by LAB and a better understanding of phage-host interactions (Sturino & Klaenhammer, 2006). Described mechanisms of phage resistance expand from blocking adsorption, restriction/ modification enzymes, to abortive infection systems that interfere with phage DNA replication, morphogenesis and release. Clustered regularly interspaced short palindromic repeats (CRISPRs) located in the genomes of host bacteria together with a group of associated proteins can also confer resistance to phages (Labrie et al., 2010). Recently, the response to phage infection in L. lactis has been approached by genome-wide transcriptomics (Fallico et al., 2011;Ainsworth et al., 2013). L. lactis appears to sense bacteriophage infection as a perturbation of its cell envelope and mounts a response targeted to the cell wall, activating regulators of the cell envelope stress response such as the two-component system CesSR.One of the members of the CesSR regulon in L. lactis is the ftsH gene (Martínez et al., 2007). ftsH encodes a conserved AAA (ATPase associated with various cellular activities)-type membrane protease involved in stress response and protein quality control (Ito & Akiyama, 2005;Narberhaus et al., 2009). In this work, we analysed the impact of the ftsH null mutation on the life cycle of the P335 temperate phage TP712, which is able to infect and lysogenize L. lactis MG1363 (Gasson, 1983) and derivatives thereof. The complete TP712 nt sequence has been determined (GenBank accession number AY766464) essentially as described by Wegmann et al. (2012).Preliminary experiments revealed that in standard plaque assays (Lillehaug, 1997), the efficiency of plaquing (EOP) of this phage on an available non-polar and in-frame L. lactis mutant lacking ftsH (Pinto et al., 2011) was reduced to 1.5610 25 when compared to its parent L. lactis NZ9000. Upon complementation with the plasmid pUK200 : : ftsH, ...

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“…This region is absent in pDorf4 which is stably maintained as a plasmid. This particular DNA region is also present in the L. lactis plasmid pLP712 (98.7% identity in BlastN searches at NCBI) and it has been described as a hotspot for DNA rearrangements through the activity of insertion sequence elements as several deletion derivatives of pLP712 occur at this site [32]. The presence of insertion sequences flanking the integrated pOrf4 plasmid also supports the contribution of these mobile elements that led to the integration of the pOrf4 plasmid.…”

Section: Resultsmentioning

confidence: 92%

A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

et al. 2014

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BackgroundLactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus.ResultsAttempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation.ConclusionsInserting the Lcn972 cluster into segregational unstable plasmids prevents their lost by segregation and probable could be applied as an alternative to the use of antibiotics to support safer and more sustainable biotechnological applications of genetically engineered L. lactis.

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Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712

Cited by 37 publications

References 44 publications

Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712

A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

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