Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica. Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

Evaluation of Structural Changes and Molecular Mechanism Induced by High Hydrostatic Pressure in Enterobacter sakazakii

The contamination of infant milk and powder with Enterobacter sakazakii poses a risk to human health and frequently caused recalls of affected products. This study aims to explore the inactivation mechanism of E. sakazakii induced by high hydrostatic pressure (HHP), which, unlike conventional heat treatment, is a nonthermal technique for pasteurization and sterilization of dairy food without deleterious effects. The mortality of E. sakazakii under minimum reaction conditions (50 MPa) was 1.42%, which was increased to 33.12% under significant reaction conditions (400 MPa). Scanning electron microscopy (SEM) and fluorescent staining results showed that 400 MPa led to a loss of physical integrity of cell membranes as manifested by more intracellular leakage of nucleic acid, intracellular protein and K+. Real-time quantitative PCR (RT-qPCR) analysis presents a downregulation of three functional genes (glpK, pbpC, and ompR), which were involved in cell membrane formation, indicating a lower level of glycerol utilization, outer membrane protein assembly, and environmental tolerance. In addition, the exposure of E. sakazakii to HHP modified oxidative stress, as reflected by the high activity of catalase and super oxide dismutase. The HHP treatment lowered down the gene expression of flagellar proteins (fliC, flgI, fliH, and flgK) and inhibited biofilm formation. These results determined the association of genotype to phenotype in E. sakazakii induced by HHP, which was used for the control of food-borne pathogens.

Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Microbial fuel cell assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes

Background: The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle.Results: MFC assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. Firstly, an acid tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV.Conclusions: A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Microbial fuel cell-assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes

Background The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid-tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle. Results MFC-assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. First, an acid-tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV. Conclusions A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Microbial fuel cell assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes

Background: The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle.Results: MFC assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. Firstly, an acid tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV.Conclusions: A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Microbial fuel cell assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes

Background: The global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle.Results: MFC assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. Firstly, an acid tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV.Conclusions: A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Microbial fuel cell assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes

Background: The global production of glycerol has been increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+.Results: Microbial fuel cells (MFC) assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. Firstly, an acid tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transfer of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol. In addition, relative constant and high value above 300 mV was obtained for at least 48 h.Conclusions: A novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.