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Getting value from the waste: recombinant production of a sweet protein by Lactococcus lactis grown on cheese whey.

ABSTRACT: BACKGROUND:Recent biotechnological advancements have allowed for the adoption of Lactococcus lactis, a typical component of starter cultures used in food industry, as the host for the production of food-grade recombinant targets. Among several advantages, L. lactis has the important feature of growing on lactose, the main carbohydrate in milk and a majoritarian component of dairy wastes, such as cheese whey. RESULTS:We have used recombinant L. lactis NZ9000 carrying the nisin inducible pNZ8148 vector to produce MNEI, a small sweet protein derived from monellin, with potential for food industry applications as a high intensity sweetener. We have been able to sustain this production using a medium based on the cheese whey from the production of ricotta cheese, with minimal pre-treatment of the waste. As a proof of concept, we have also tested these conditions for the production of MMP-9, a protein that had been previously successfully obtained from L. lactis cultures in standard growth conditions. CONCLUSIONS:Other than presenting a new system for the recombinant production of MNEI, more compliant with its potential applications in food industry, our results introduce a strategy to valorize dairy effluents through the synthesis of high added value recombinant proteins. Interestingly, the possibility of using this whey-derived medium relied greatly on the choice of the appropriate codon usage for the target gene. In fact, when a gene optimized for L. lactis was used, the production of MNEI proceeded with good yields. On the other hand, when an E. coli optimized gene was employed, protein synthesis was greatly reduced, to the point of being completely abated in the cheese whey-based medium. The production of MMP-9 was comparable to what observed in the reference conditions.

SUBMITTER: Boumaiza M

PROVIDER: S-EPMC6094915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.

Project description:BackgroundCheese ripening is a complex, time consuming and expensive process, which involves the generation of precursors from carbohydrates, proteins and fats and their subsequent conversion into a wide range of compounds responsible for the flavour and texture of the cheese. This study aims to investigate production of cheese aroma compounds outside the cheese matrix that could be applied for instance as food supplements in dairy or non-dairy products.ResultsIn this study, aroma formation by a dairy Lactococcus lactis was analysed as a function of the growth medium [milk, hydrolysed micellar casein isolate (MCI) and chemically defined medium (CDM)] and the cultivation conditions (batch culture, retentostat culture and a milli-cheese model system). In the retentostat cultures, the nutrient supply was severely restricted resulting in low growth rates (~ 0.001 h-1), thereby mimicking cheese ripening conditions in which nutrients are scarce and bacteria hardly grow. In total 82 volatile organic compounds were produced by the bacteria. Despite the use of a chemically defined medium, retentostat cultures had the biggest qualitative overlap in aroma production with the milli-cheese model system (36 out of 54 compounds). In the retentostat cultures, 52 known cheese compounds were produced and several important cheese aroma compounds and/or compounds with a buttery or cheese-like aroma increased in retentostat cultures compared to batch cultures and milli-cheeses, such as esters, methyl ketones, diketones and unsaturated ketones. In cultures on CDM and MCI, free fatty acids and their corresponding degradation products were underrepresented compared to what was found in the milli-cheeses. Addition of a mixture of free fatty acids to CDM and MCI could help to enhance flavour formation in these media, thereby even better resembling flavour formation in cheese.ConclusionsThis study demonstrates that retentostat cultivation is the preferred method to produce cheese flavours outside the cheese matrix by mimicking the slow growth of bacteria during cheese ripening.

Project description:BACKGROUND:Recently, it has been indicated that anaerobic co-digestion of waste activated sludge with other waste streams at wastewater treatment plants is a promising strategy for enhancing methane production and materials recovery. The enhanced methane production can be used as a renewable source of energy in wastewater treatment plants. It can also reduce the amount of greenhouse gas emission in landfilling of the waste streams. RESULTS:According to the results obtained in this study, anaerobic co-digestion of waste activated sludge with mixed fruit waste and cheese whey improves methane production and the quality of digested sludge in comparison to the anaerobic digestion of waste activated sludge individually. It was indicated that carbon/nitrogen ratio (C/N) in the mixture of waste activated sludge, fruit waste and cheese whey improved considerably, leading to better anaerobic organisms' activity during digestion. With assessing the activity of protease and cellulase, as the main enzymes hydrolyzing organic matter in anaerobic digestion, it was indicated that co-digestion of waste activated sludge with mixed fruit waste and cheese whey enhances the activity of these enzymes by 22 and 9% respectively. At the end of digestion, the amount of cumulative methane production significantly increased by 31% in the reactor with 85% waste activated sludge and 15% mixed fruit waste and cheese whey, compared to the reactor with 100% waste activated sludge. In addition, chemical oxygen demand (COD) and volatile solid (VS) in digested sludge was improved respectively by 9 and 7% when mixed fruit waste and cheese whey was used. CONCLUSIONS:This study revealed that mixed fruit waste and cheese whey is potentially applicable to anaerobic digestion of waste activated sludge, as fruit waste and cheese whey have high C/N ratio that enhance low C/N in waste activated sludge and provide a better diet for anaerobic organisms. This is of significant importance because not only could higher amount of renewable energy be generated from the enhanced methane production in wastewater treatment plants, but also capital costs of the companies whose waste streams are being transported to wastewater treatments plants could be reduced considerably.

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Getting value from the waste: recombinant production of a sweet protein by Lactococcus lactis grown on cheese whey.

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