Weon-Taek Seo

Influence of agitation speed on production of curdlan by Agrobacterium species

Abstract Influence of dissolved oxygen level on production of curdlan by Agrobacterium species was investigated. Preliminary shake flask experiments showed that both cell growth and curdlan production were higher at a smaller volume of medium (50–100 ml in 500 ml flasks). As culture volume increased from 100 ml to 300 ml, both cell concentration and curdlan production decreased, indicating that higher oxygen transfer is required for a higher production of curdlan. Time profiles of cell concentration and curdlan production in a 5-liter jar fermentation at different agitation speeds, ranging from 300 rpm to 700 rpm, supported the fact of higher production of curdlan at higher oxygen transfer rate observed in shake flask cultures. At a higher agitation speed (600 rpm), the highest curdlan production (64.4 g/l) was obtained in 120 h of a batch fermentation. However, curdlan production was not improved at the higher agitation speed (700 rpm). For the mass production of curdlan, fermentation was performed in a 300-liter fermenter under the condition where the same volumetric oxygen transfer coefficient was obtained as in 5-liter jar fermentation. As high as 9.28 kg of curdlan with a final concentration of 58 g/l was obtained in 120 h batch cultivation, enlarging the potential in the industrial production of curdlan.

Production of vanillin by metabolically engineered Escherichia coli

E. coli was metabolically engineered to produce vanillin by expression of the fcs and ech genes from Amycolatopsis sp. encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively. Vanillin production was optimized by leaky expression of the genes, under the IPTG-inducible trc promoter, in complex 2YT medium. Supplementation with glucose, fructose, galactose, arabinose or glycerol severely decreased vanillin production. The highest vanillin production of 1.1 g l−1 was obtained with cultivation for 48 h in 2YT medium with 0.2% (w/v) ferulate, without IPTG and no supplementation of carbon sources.

Enhanced Vanillin Production from Recombinant E.coli Using NTG Mutagenesis and Adsorbent Resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl‐CoA synthase) and ech (enoyl‐CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5α harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin‐resistant mutant of NTG‐VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD‐2 resin absorption. The vanillin production of NTG‐VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild‐type strain. When 50% (w/v) of XAD‐2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG‐VR1 was 2.9 g/L, which was 2‐fold higher than that obtained with no use of the resin.

Enhanced production of b-carotene from Blakeslea trispora with Span 20

Span 20 at 10 g/l induced dispersed mycelial growth of Blakeslea trispora, and significantly increased b-carotene production to 2.16 g/l. Without surfactant only 0.15 g b-carotene/l was produced. Other non-ionic surfactants composed of the same hydrophilic part as Span 20 and the hydrophobic fatty acids of Spans, including lauric acid (C 12 ), neither induced dispersed growth of the microorganism nor increased b-carotene production.

Production of vanillin from ferulic acid using recombinant strains ofEscherichia coli

Vanillin is one of the worlds principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with PBAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.

Enhanced synthesis of trisporic acid and β-carotene production in Blakeslea trispora by addition of a non-ionic surfactant, Span 20

When a non-ionic surfactant, Span 20, was added to a mated culture of Blakeslea trispora, β-carotene production increased 15 fold compared to that without the surfactant, reaching 2.3 g/l. The marked increase of β-carotene was closely related to the titer of trisporic acid, a strong stimulator of β-carotene synthesis. With Span 20, the microorganism produced 15 times more trisporic acid (8.8 mg/g dry cell weight) than without the surfactant (0.6 mg/g dry cell weight). Therefore, it was concluded that Span 20 considerably enhanced the production of trisporic acid, which subsequently stimulated β-carotene synthesis.

Increased β-carotene synthesis in Blakeslea trispora under strong alkaline culture condition

SummaryA high yield of β-carotene, 48 mg/l, was obtained by cultivation of a strain of Blakeslea trispora under a strong alkaline culture condition (pH 10), which was significantly higher than those obtained under milder pH conditions (3.26–7.80 mg/l at pH 4 to 8). The formation of small pellets at pH 10 was observed, whereas a single large cluster was formed for the pHs ranged from 4 to 8. Span 20 at 0.1 to 1% in the culture medium changed cellular morphology to micro-pellets regardless of the culture pH. The same trend of β-carotene production, depending on the alkalinity of the culture broth, was also observed in the presence of 1% of Span 20 yielding much higher productivity. The amounts of β-carotene yielded at pH 10 and 11 were 92.0 and 100.9 mg/l, respectively, which were about two-times higher than those obtained at pH 4 to 9.