Bioconversions of Biodiesel-Derived Glycerol into Sugar Alcohols by Newly Isolated Wild-Type Yarrowia lipolytica Strains

The utilization of crude glycerol, generated as a by-product from the biodiesel production process, for the production of high value-added products represents an opportunity to overcome the negative impact of low glycerol prices in the biodiesel industry. In this study, the biochemical behavior of Yarrowia lipolytica strains FMCC Y-74 and FMCC Y-75 was investigated using glycerol as a carbon source. Initially, the effect of pH value (3.0–7.0) was examined to produce polyols, intracellular lipids, and polysaccharides. At low pH values (initial pH 3.0–5.0), significant mannitol production was recorded. The highest mannitol production (19.64 g L−1) was obtained by Y. lipolytica FMCC Y-74 at pH = 3.0. At pH values ranging between 5.0 and 6.0, intracellular polysaccharides synthesis was favored, while polyols production was suppressed. Subsequently, the effect of crude glycerol and its concentration on polyols production was studied. Y. lipolytica FMCC Y-74 showed high tolerance to impurities of crude glycerol. Initial substrate concentrations influence polyols production and distribution with a metabolic shift toward erythritol production being observed when the initial glycerol concentration (Gly0) increased. The highest total polyols production (=56.64 g L−1) was obtained at Gly0 adjusted to ≈120 g L−1. The highest polyols conversion yield (0.59 g g−1) and productivity (4.36 g L−1 d−1) were reached at Gly0 = 80 g L−1. In fed-batch intermittent fermentation with glycerol concentration remaining ≤60 g L−1, the metabolism was shifted toward mannitol biosynthesis, which was the main polyol produced in significant quantities (=36.84 g L−1) with a corresponding conversion yield of 0.51 g g−1.

Multiple Techno-Functional Characteristics of Leuconostoc and Their Potential in Sourdough Fermentations

In this study, the potential of Leuconostoc as non-conventional sourdough starter cultures was investigated. A screening for antifungal activities of 99 lactic acid bacteria (LAB) strains revealed high suppression of bakery-relevant moulds in nine strains of Leuconostoc with activities against Penicillium sp., Aspergillus sp., and Cladosporium sp. Mannitol production was determined in 49 Leuconostoc strains with >30 g/L mannitol in fructose (50 g/L)-enriched MRS. Further, exopolysaccharides (EPS) production was qualitatively determined on sucrose (40 g/L)-enriched MRS agar and revealed 59 EPS positive Leuconostoc strains that harboured dextransucrase genes, as confirmed by PCR. Four multifunctional Lc. citreum strains (DCM49, DCM65, MA079, and MA113) were finally applied in lab-scale sourdough fermentations (30 °C, 24 h). Lc. citreum was confirmed by MALDI-TOF MS up to 9 log CFU/g and pH dropped to 4.0 and TTA increased to 12.4. Antifungal compounds such as acetic acid, phenyllactic and hydroxyphenyllactic acids were determined up to 1.7 mg/g, 2.1 µg/g, and 1.3 µg/g, respectively, mannitol up to 8.6 mg/g, and EPS up to 0.62 g/100 g. Due to the observed multifunctionalities and the competitiveness in the natural flour microbiota present in sourdoughs, non-conventional LAB genera such as Leuconostoc seem promising for application in sourdough-based bakery products.

Deciphering the regulation of the mannitol operon paves the way for efficient production of mannitol in L. lactis

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated, and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positives, mtlF and mtlD , encoding the Enzyme IIA component (EIIA mtl ) of the mannitol phosphotransferase system (PTS), and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA , encoding the Enzyme IIBC component (EIIBC mtl ) of the mannitol PTS, mtlR , encoding a transcriptional activator, and mtlF , and a separately expressed mtlD . The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive elements ( cre ). Presence of carbon catabolite repression was demonstrated, and was shown to be relieved in stationary phase cells. The transcriptional activator MtlR ( mtlR ), in some Gram-positives, is repressed by phosphorylation by EIIA mtl , and when we knocked-out mtlF we indeed observed enhanced expression from the two promotors, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary phase cells as biocatalysts, we attained 10.1 g/L mannitol with a 55% yield, which is the highest titer ever reported for L. lactis . Summing up, the results of our study should be useful for improving the mannitol producing capacity of this important industrial organism. Importance Lactococcus lactis is the most studied species of the Lactic Acid Bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis into producing mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis , with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated, and demonstrate how this impacts on mannitol production capability. We harness this information and manage to establish efficient mannitol production, without introducing foreign genes.

Using Osmotic Stress to Stabilize Mannitol Production in Synechocystis sp. PCC6803

Background Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC 7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p, in short: a ‘mannitol cassette’), proved to be genetically unstable. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC 6803 as a model cyanobacterium. Results Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC 6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild type Synechocystis, complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

Using Osmotic Stress to Stabilize Mannitol Production in Synechocystis sp. PCC6803

Background Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO 2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC 7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase ( mtlD ) and mannitol-1-phosphatase ( m1p , in short: a ‘mannitol cassette’), proved to be genetically unstable. Results Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC 6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild type Synechocystis , complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

Using Osmotic Stress to Stabilize Mannitol Production in Synechocystis sp. PCC6803

Background Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO 2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC 7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase ( mtlD ) and mannitol-1-phosphatase ( m1p , in short: a ‘mannitol cassette’), proved to be genetically unstable. Results Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC 6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild type Synechocystis , complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of both stable mannitol production directly from CO 2 in cyanobacteria, and of the utilization of the response to salt stress as a factor that can stabilize production in a cyanobacterial cell factory.