Metabolic shifts in Lactococcus lactis:Regulation, evolution and phenotypic heterogeneity

Het onderzoek dat in dit proefschrift wordt gepresenteerd, heeft ten doel om meer te weten te komen over het suikermetabolisme, de regulering en de evolutie ervan in de melkzuurbacterie Lactococcus lactis. Speciale aandacht voor deze processen gaat uit naar de individuele cel. Het hoofddoel was om de rol van populatieheterogeniteit tijdens verschillende substraatveranderen te beoordelen. We hebben ontdekt dat een complex systeem van specifieke en globale reguleringsfactoren het gedrag van een enkele cel in de populatie bepaalt en dat de populatie cellen uit veel metabolische fenotypen bestaat. Ons evolutionair model suggereert dat de waargenomen fenotypische heterogeniteit een voorbeeld kan zijn van een bet-hedging strategie die een evolutionair voordeel kan opleveren. Dit proefschrift was ook toegewijd om de onopgeloste intrigerende fenomenen in L. lactis, zoals de groei van lactose-/cellobiose- of galactose-negatieve stammen op respectievelijk lactose, cellobiose of galactose te bestuderen. Al deze gebeurtenissen zijn al eerder waargenomen, echter zijn ze nog nooit toegeschreven aan specifieke metabolische systemen. Verschillende activatiemechanismen bleken vereist te zijn om de genclusters die plantsuikers kunnen utiliseren weer in gebruik te stellen, die vervolgens nieuwe functies konden verwerven en de cellen helpen om aan stressvolle situaties te ontsnappen. Om de adaptieve capaciteit van deze nieuwe stam met opnieuw geactiveerde lactose-utilisatievermogen verder te onderzoeken, is deze voortdurend in een chemisch gedefinieerd medium met lactose gepropageerd. Alhoewel a priori veranderingen in suikeropname- en metabole systemen voor de hand liggen, hebben wij ontdekt dat de geëvolueerde stammen hun stikstofmetabolisme aangepast hebben

Development of Lactococcus lactis Biosensors for Detection of Diacetyl

Some secondary metabolites of fermentative bacteria are desired compounds for the food industry. Examples of these compounds are diacetyl and acetaldehyde, which are produced by species of the lactic acid bacteria (LAB) family. Diacetyl is an aromatic compound, giving the buttery flavor associated with dairy products, and acetaldehyde is the compound responsible for the yogurt flavor and aroma. The quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors that facilitate the identification and quantification of certain metabolites (Mahr and Frunzke, 2016). The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aimed to develop fluorescence-based biosensors to detect diacetyl and acetaldehyde. Since the metabolic pathways for production and degradation of these compounds are present in Lactococcus lactis, the sensing mechanisms in this bacterium are expected. Thus, we identified diacetyl and acetaldehyde responsive promoters by performing transcriptome analyses in L. lactis. The characterization of the biosensors showed their response to the presence of these compounds, and a further analysis of the diacetyl-biosensors (its dynamics and orthogonality) was performed. Moreover, we attempted to produce natural diacetyl from producer strains, namely L. lactis subsp. lactis biovar diacetylactis, to benchmark the performance of our biosensors. The diacetyl-biosensors responded linearly to the amounts of diacetyl obtained in the bacterial supernatants, i.e., the increases in GFP expression were proportional to the amounts of diacetyl present in the supernatants of L. lactis subsp. lactis biovar diacetylactis MR3-T7 strain. The biosensors developed in this study may eventually be used to engineer strains or pathways for increased diacetyl and acetaldehyde production, and may facilitate the detection of these metabolites in complex food matrices

Disruption of a transcriptional repressor by an IS-element integration leads to the activation of a novel silent cellobiose transporter in Lactococcus lactis MG1363

L. lactis subsp. cremoris strains typically carry many dairy niche-specific adaptations. During adaptation to the milk environment these former plant strains have acquired various pseudogenes and insertion sequence elements indicative of ongoing genome decay and frequent transposition events in their genomes. Here, we describe the re-activation of a silenced plant sugar utilization cluster in an L. lactis MG1363 derivative lacking the two main cellobiose transporters PtcBACelB and PtcBAC upon applying selection pressure to utilize cellobiose. A disruption of the transcriptional repressor gene llmg_1239 by an IS element allows expression of the otherwise silent novel cellobiose transporter Llmg_1244 and leads to growth of mutant strains on cellobiose. Llmg_1239 was labeled CclR, for cellobiose cluster repressor.Importance Insertion sequences (IS) play an important role in the evolution of lactococci and other bacteria. They facilitate DNA rearrangements and are responsible for creation of new genetic variants with selective advantages under certain environmental conditions. L. lactis MG1363 possesses 71 copies in total of 11 different types of IS elements. This study describes yet another example of an IS-mediated adaptive evolution. An integration of IS981 or IS905 into a gene coding for a transcriptional repressor led to activation of the repressed gene cluster coding for a plant sugar utilization pathway. The expression of the gene cluster allowed assembly of a novel cellobiose-specific transporter and led to cell growth on cellobiose

Ampicillin-treated Lactococcus lactis MG1363 populations contain persisters as well as viable but non-culturable cells

Lactococcus lactis is used as cell-factory and strain selections are regularly performed to improve production processes. When selection regimes only allow desired phenotypes to survive, for instance by using antibiotics to select for cells that do not grow in a specific condition, the presence of more resistant subpopulations with a wildtype genotype severely slows down the procedure. While the food grade organism L. lactis is not often exposed to antibiotics we characterized its response to ampicillin in more detail, to better understand emerging population heterogeneity and how this might affect strain selection procedures. Using growth-dependent viability assays we identified persister subpopulations in stationary and exponential phase. Growth-independent viability assays revealed a 100 times larger subpopulation that did not grow on plates or in liquid medium, but had an intact membrane and could maintain a pH gradient. Over one third of these cells restored their intracellular pH when we induced a temporary collapse, indicating that this subpopulation was metabolically active and in a viable but non-culturable state. Exposure of L. lactis MG1363 to ampicillin therefore results in a heterogeneous population response with different dormancy states. These dormant cells should be considered in survival-based strain selection procedures.</p

Further elucidation of galactose utilization in Lactococcus lactis MG1363

Since the 1970s, galactose metabolism in Lactococcus lactis has been in debate. Different studies led to diverse outcomes making it difficult to conclude whether galactose uptake was PEP- or ATP-dependent and decide what the exact connection was between galactose and lactose uptake and metabolism. It was shown that some Lactococcus strains possess two galactose-specific systems - a permease and a PTS, even if they lack the lactose utilization plasmid, proving that a lactose-independent PTSGal exists. However, the PTSGal transporter was never identified. Here, with the help of transcriptome analyses and genetic knock-out mutants, we reveal the identities of two low-affinity galactose PTSs. A novel plant-niche-related PTS component Llmg_0963 forming a hybrid transporter Llmg_0963PtcBA and a glucose/mannose-specific PTS are shown to be involved in galactose transport in L. lactis MG1363

Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

Metabolic engineering and synthetic biology approaches have prospered the field of biotechnology, in which the main focus has been on Escherichia coli and Saccharomyces cerevisiae as microbial workhorses. In more recent years, improving the Gram-positive bacteria Lactococcus lactis and Bacillus subtilis as production hosts has gained increasing attention. This review will demonstrate the different levels at which these bacteria can be engineered and their various application possibilities. For instance, engineered L. lactis strains show great promise for biomedical applications. Moreover, we provide an overview of recent synthetic biology tools that facilitate the use of these two microorganisms even more

The evolution of gene regulation research in Lactococcus lactis

Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism. Among many other things, this has provided an unprecedented view of the major gene regulatory pathways involved in nitrogen and carbon metabolism and their overlap, and has led to the blossoming of the field of L. lactis systems biology. All of these advances have made L. lactis the paradigm of the LAB. This review will deal with the exciting path along which the research on the genetics of and gene regulation in L. lactis has trodden

Pathways to cellular supremacy in biocomputing

Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the “genetic circuit” metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of “cellular supremacy” to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found.A.G.-M. was supported by the SynBio3D project of the UK Engineering and Physical Sciences Research Council (EP/R019002/1) and the European CSA on biological standardization BIOROBOOST (EU grant number 820699). T.E.G. was supported by a Royal Society University Research Fellowship (grant UF160357) and BrisSynBio, a BBSRC/ EPSRC Synthetic Biology Research Centre (grant BB/L01386X/1). P.Z. was supported by the EPSRC Portabolomics project (grant EP/N031962/1). P.C. was supported by SynBioChem, a BBSRC/EPSRC Centre for Synthetic Biology of Fine and Specialty Chemicals (grant BB/M017702/1) and the ShikiFactory100 project of the European Union’s Horizon 2020 research and innovation programme under grant agreement 814408

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project