STUDY OF TREHALOSE PRODUCTION AND METABOLISM IN PROPIONIBACTERIUM SHERMANII UNDER OSMOTIC STRESS

Abstract

The current understanding of trehalose biosynthesis regulation under stress conditions is incomplete and needs further investigation. Furthermore, the knowledge of enhancement of trehalose accumulation under osmotic stress can be exploited for commercial production. Trehalose finds various pharmaceutical and industrial applications. With this view, we have investigated trehalose metabolism in the context of central carbon metabolism and glycogen and maltose accumulations under osmotic stress in a strain of dairy Propionibacterium. Similarly, suitability of crude glycerol obtained from biodiesel was studied for trehalose production. Interestingly, osmotic stress due to presence of KCl in crude glycerol was found to be reason for higher production of trehalose with crude glycerol. Therefore, two broad objectives of current study were to investigate trehalose metabolism under osmotic stress and to explore economical production of trehalose with cheap carbon source. During our search for suitable strain for this study, four strains of dairy Propionibacterium available in culture collection centres of India were procured. These strains were screened for trehalose yield under osmotic and non stress conditions. Amongst the various strains procured from culture collection centers, Propionibacterium shermanii NCIM 5137 was considered for further studies. In screening study it was observed that all procured strains of Propionibacterium produced trehalose under osmotic stress. Since trehalose yield with respect to biomass was highest in P. shermanii NCIM 5137 in osmotic and non-stress conditions (60 mg/g of biomass and 15 mg/g of biomass) hence it was used for further studies. The selected strain has the ability to accumulate trehalose under non-stress condition so this can also be used for trehalose production using cheap carbon source in absence of osmotic stress agents. Trehalose production was enhanced under osmotic stress as seen in static flask condition. So an effort was made to study the trehalose accumulation under osmotic stress in batch reactor where pH and dissolved oxygen were controlled at 6.8 ± 0.1 and 5% of saturation respectively. The highest trehalose yield obtained in osmotic stress was 160 mg/g of biomass while in non-stress conditions it was 45 mg/g of biomass. In fact, the effect of osmotic stress was more prominent on final trehalose yield obtained in stationary phase and it was 44 mg/g of biomass in osmotic stress while 15 mg/g of biomass in non-stress condition. As Cardoso et al., 2004 also reported trehalose accumulation in various other carbon sources like lactose, lactic acid, hence a similar effort was made to study trehalose production with P. shermanii NCIM 5137. It was observed that use of lactic acid didn't result in improvement of trehalose production and growth was slow. In case of carbon source lactose, marginal improvement in yield was achieved in comparison to glucose media. So, these two carbon sources were not considered for further study. In the present study, accumulation of trehalose with different carbon sources in P. shermanii NCIM 5137 was investigated. Since, use of lactic acid and lactose as carbon source didn't result in higher trehalose accumulation, hence we further used other carbon sources for trehalose production in static flask conditions and it was observed that trehalose accumulation was enhanced with disaccharides (maltose, 128.54 mg/g of biomass; sucrose, 170.47 mg/g of biomass) in comparison to monosaccharide (glucose 93.81 mg/g of biomass) while it was maximum in gluconeogenic substrate, glycerol (385 mg/g of biomass). Interestingly, maximum trehalose yield (with respect to biomass) achieved with these carbon sources were higher than the yield achieved with glucose media under osmotic stress conditions. Trehalose productions from various carbon sources like sucrose, maltose and starch are known in many microbes but accumulation of higher trehalose from glycerol is not reported in any microbe. Hence, to make trehalose production from glycerol commercially feasible, we studied the fermentative production of trehalose from crude glycerol using P. freudenreichii subsp. shermanii NCIM 5137 in batch reactor. Herein, the comparison of trehalose production in pure and crude glycerol media in batch reactor shows that maximum trehalose yield of approx 380 mg/g of biomass can be achieved and highest trehalose yield based on substrate consumed was improved three times with crude glycerol media as compared to that with pure glycerol media. Therefore, it was concluded that crude glycerol was superior as carbon source in comparison to pure glycerol. Thus an effort was made to elucidate the roles of various reported impurities of crude glycerol on enhanced trehalose accumulation in P. freudenreichii. In the present study, fatty acids like oleic acid, linoleic acid, stearic acid and palmitic acid and KCl were separately used in a chemically defined media (glycerol as carbon source) for investigating their effects on trehalose accumulation. It was observed that fatty acids have no beneficial effects on trehalose production but KCI at a concentration of 1 % has significantly improved trehalose yields. ii In summary, remarkable improvement in trehalose synthesis under osmotic stress was observed in the present study. Thus an effort was made to understand the variation in trehalose yield with respect to activities of various relevant enzymes and metabolites associated with trehalose biosynthesis. The probable role of TreS for synthesis of trehalose using glycogen as substrate under osmotic stress condition is presumed. However, this needs to be further elucidated with different sets of experiments. Another major observation was higher expression of ADP-glucose pyrophosphorylase over other nucleotide sugar synthesizing enzymes, indicating prominent role of ADP-glucose in osmotic stress condition. From this study, it became apparent that_glycogen, maltose and trehalose synthesis are inter related during osmotic stress conditions. Over expressions of ADP-glucose pyrophosphorylase and TreS was the strategy probably adopted by P. shermanii for minimizing the effect of osmotic stress. Further effort was made to analyze the biosynthesis pathway variables (enzyme activities, metabolite concentration) using multivariate approach. Although trehalose biosynthesis requires few metabolites and enzyme reactions but it seems to have a more complex metabolic regulation. The bacterial cell adapts to changing environments by changing the level of various biochemical metabolites and enzymes activities which leads to enhancement of trehalose biosynthesis.. Understanding of this complex phenomenon is not very easy without any multivariate approaches. The advantage of multivariate approach includes reduction of number of variables and simultaneous assessment of effects by various variables on trehalose biosynthesis. Hence in the present study we had shown application of one such approach known as principle component analysis.. Two principle components were extracted from the study of trehalose biosynthesis pathway under osmotic and non-stress conditions. From the score plots of principle components it was possible to discriminate osmotically stress cells. from non-stress cells. Further loading plot was able to identify pathway variables which were responsible for discrimination of osmotically stressed cells from non-stressed cells. Beside data of trehalose biosynthesis pathway obtained from cells grown in glucose, and glycerol was projected on the score plot using the developed principle components. As it was observed that osmotic stress results in higher trehalose accumulation so an effort was made to develop an osmotically sensitive mutant using EMS (Ethyl Methanesulphonate) as mutagen. From the mutation study, one mutant was selected which was osmotically sensitive (non-viable in 3% NaCl media) and was able to accumulate higher trehalose even in non-stress condition. Specifically, decrease in trehalose content of cell in the stationary phase was reduced significantly as compared to parent strain. This osmotic sensitive mutant was also found to be resistant to nisin. The efficiency of this mutant for trehalose production was evaluated in glucose (Y = 105 mg/g of biomass), sucrose (Y = 148 mg/g of biomass) and pure glycerol (Yt" = 233 mg/g of biomass) which was higher in comparison to parent strain (YM = 15, 85 and 89 mg/g of biomass in glucose, sucrose and glycerol) respectively. During static flask studies with crude glycerol as carbon source, highest trehalose yield achieved with respect to biomass was 685 mg/g of biomass and final yield was 412 mg/g of biomass, this is probably the highest yield ever reported in Propionibacterium. In a recent study, with mutant strain of Saccharomycopsis, trehalose, yield of 28% with respect to biomass (in the present study 41%) was reported using cassava starch as carbon source (Wang D-S et al., 2011). Similarly, recombinant Corynebacterium strain was able to produce trehalose with a yield of 31% of biomass with glucose as carbon source (Carpinelli et al 2006). In mutant, final trehalose yield achieved with respect to biomass was 3 times improved as compared to parent strain with crude glycerol. Likewise, trehalose yield achieved with respect to substrate consumed was increased from 21 mg/g of substrate consumed (parent strain) to 82 mg/g of substrate consumed (mutant strain). Along with this 0.42 g/g of propionic acid and 0.31 g/g of lactic acid were also obtained from crude glycerol. Importance of ADP-glucose pyrophosphorylase and GDP-glucose pyrophosphorylase over UDP-glucose pyrophosphorylase for trehalose biosynthesis was also clearly observed in the mutant as activities of these enzymes were much higher along with achievement of higher trehalose yield. In summary, it can be concluded that preference of ADP-glucose pyrophosphorylase over UDP-glucose pyrophosphorylase along with higher OtsA activity is the adopted strategy for enhanced trehalose biosynthesis in both parent and mutant strain. Since complete substrate consumption was not achieved in mutant hence an effort was made to increase biomass concentration by aerobic fermentation in flask cultures at 200 rpm using glycerol and crude glycerol (from biodiesel waste) as carbon sources. In aerobic condition, final biomass was increased ten times while trehalose titre reached 1.4 g/1 with crude glycerol as carbon source with osmotic sensitive mutant. Interestingly, substrate consumption of 94% was achieved. Although, trehalose yield with respect to biomass decreased with aeration but trehalose titre increased due to increase in biomass iv concentration under aerated conditions. The trehalose titre of 1.7 g/l was reported from recombinant E. coli with pure glycerol while in present study, final titer of 1.4 g/1 and propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were obtained with crude glycerol media. In conclusion, with respect to production, trehalose yields were increased from 1.5% of biomass (from glucose) to 39% of biomass (from crude glycerol) and 0.15 mg/g of substrate to 90 mg/g of substrate. However, trehalose titer of 1.5 g/l was also attained under aerobic condition. Similarly, new insights on trehalose metabolism under osmotic stress were obtained and major conclusions were difference in the role of different NDP-G pyrophosphorylase and probable role of TreS in trehalose synthesis from glycogen.