Evolutionary engineering of polyphenol resistance in lactic acid bacteria

The aim of this study was to obtain robust starter cultures for more efficient olive fermentations by developing stress-resistant lactic acid bacteria, more preferably Lactobacillus plantarum strains by using evolutionary engineering approach. Since resistance mechanisms in lactic acid bacteria involve numerous genes and many regulators, it would be difficult to obtain the desired resistant phenotypes by direct genetic engineering or over-expressing certain genes. Thus, evolutionary engineering could be a good alternative strategy to obtain the desired phenotypes. In this approach, the resulting strains are not considered as genetically modified organisms which makes them more acceptable for the food industry. It was generally thought that lactic acid bacterial starter cultures for olive fermentations were not robust enough due to the presence of olive polyphenols and mainly oleuropein in fermentation brines. In the first part of this thesis, this hypothesis was tested by determining the resistance of various strains to stress factors that are commonly encountered during olive fermentations. The results revealed that NaCl, and not oleuropein, was mainly responsible for the decreased growth rates of lactic acid bacteria during olive fermentations. For the evolutionary engineering studies, a L. plantarum strain (T129) isolated from table olive-producing companies in the Marmara Region was obtained from TUBITAK MAM Food Institute collection and a L. plantarum type strain DSMZ 10492 isolated from olive-processing brines in Portugal was obtained from DSMZ Culture Collection. These strains were treated with EMS (ethyl methane sulfonate) to obtain two different, genetically diverse initial populations for selection. The NaCl resistance of these populations was increased by applying increasing levels of NaCl stress during successive batch cultures of selection. Mutant individuals were selected from the final mutant populations that resisted 10 % (w/v) NaCl. The survival rates of mutant individuals were compared with those of the wild-type at 10% (w/v) NaCl and 5 g/l oleuropein-containing 10% (w/v) NaCl. Most of the mutant individuals survived both NaCl and oleuropein stress, unlike their wild-type strains. The cross-resistance of selected mutant individuals against commonly encountered stress factors during olive fermentations were determined to confirm the suitability of the mutants as robust olive starter cultures. One mutant strain derived from L. plantarum DSMZ 10492 (D2) and one mutant strain derived from the L. plantarum T129 (T8) were chosen for further studies. The NaCl resistance of the mutant strains during five successive cultivations in non-selective (stress-free) media were determined and the results confirmed that NaCl resistance was not an adaptation, but had a heritable genetic background. These genetically stable strains could therefore be used as starter cultures for olive fermentations without losing their gained resistance. Another aim of this thesis study was to obtain lactic acid bacteria that could be used as probiotic cultures for olive fermentation. Such a starter culture would not only ensure a standardized olive fermentation, but also provide added health benefits to its user. Thus, lactic acid bacteria need to be engineered to increase their survival during the passage through the upper digestive tract. It is difficult, however, to select a single stress condition to obtain efficient probiotic mutants. Ingested microorganisms are faced with a series of stress factors during their passage through the gastrointestinal tract. Time-based decrease in pH and the presence of gastric enzymes are major stress factors in the stomach. While peristaltic movement of the small intestine prevents microbial colonization, weakened bacteria are faced with pancreatic enzymes, bile salts and nutrient deprivation in different sections of the small intestine. Thus making use of an in vitro model of the upper gastrointestinal tract was the novel evolutionary engineering strategy to select for more viable, robust and efficient probiotic strains. It was hypothesised that mutant individuals with high survival rates would be enriched during the consequent passages through the in vitro model. By this strategy, it would be possible to isolate an individual with increased survival. The TNO in vitro model of the stomach and small intestine (TIM-1) is a computer-controlled dynamic model that accurately mimics the dynamic conditions in the upper parts of the human digestive system. TIM-1 consists of four compartments: the stomach, duodenum, jejunum and ileum. It mimics body temperature, peristalsis, intestinal passage, specific pH conditions, enzyme and electrolyte levels as close as possible to the physiological conditions. Therefore, the EMS-mutagenized rifampicin-resistant mutant population of T129 was introduced to this system for five successive times. At the end, an individual strain, J1, was selected with a survival rate close to 100%. The genetic stability of this strain was determined by reintroducing this strain to the TIM-1 system after stress-free cultivation. The survival in the upper gastrointestinal tract was preserved which showed that the mutant strain was genetically stable. Finally the genetic background of the increased survival of the J1 strain was investigated by comparing the whole genome sequences of the wild type T129 and The mutant strain J1. It was confirmed that all mutations were either G to A or C to T, as expected typically for EMS mutagenesis. The whole genome was annotated and the genes that were affected by these mutations were determined as: phosphoribosylformylglycinamidine synthase subunit PurL, glutamine ABC transporter, permease protein, DNA repair protein RecN, ribosome small subunitdependent GTPase A, molecular chaperone DnaK, DNA-directed DNA polymerase III, alpha chain PolC-type, phosphatidate cytidylyltransferase cell surface protein, Cardiolipin synthetase and ATP-dependent zinc metalloprotease FtsH. This revealed that changes in cellular metabolism, DNA repair, and cell wall composition might possibly have led to the increased survival of the mutant strain J1. To conclude, novel selection strategies were successfully applied in this study to obtain stress-resistant and robust L. plantarum strains with improved probiotic properties for potential use in olive fermentations. Further transcriptomic and proteomic research would be necessary to understand the complex molecular basis of the desirable phenotypes.