Medium In Shake Flasks Research Articles

Enhanced d-pantothenic acid biosynthesis by plasmid-free Escherichia coli through sodium pyruvate addition combined with glucose and temperature control strategy.

d-pantothenic acid (d-PA) is an important vitamin widely used in the feed, pharmaceutical, and food industries. This study aims to enhance the d-PA production of a recombinant Escherichia coli without plasmid and inducer induction. The fermentation medium in shake flask was optimized, resulting in a 39.50% increased d-PA titer (3.32g l-1). Subsequently, the fed-batch fermentation in a 5-l fermenter was specifically investigated. First, a two-stage temperature control strategy led to a d-PA titer of 52.09g l-1. Additionally, a two-stage glucose feeding was proposed and d-PA titer was increased to 65.29g l-1. It was also found that an appropriate amount of sodium pyruvate was beneficial to cell growth and d-PA synthesis. Finally, a two-stage glucose feeding combined with sodium pyruvate addition resulted in a substantially improved d-PA production with a titer of 72.90g l-1. The d-PA synthesis was significantly improved through the fermentation process established in this work, i.e. sodium pyruvate addition combined with the temperature and glucose control strategy. The results of this study could provide significant reference for the industrial fermentation production of d-PA.

Monitoring Yeast Cultures Grown on Corn Stover Hydrolysate for Lipid Production

Microbial oils can be used as an alternative sustainable and renewable feedstock to fossil reserves for producing lubricants and polyurethane materials. Two oleaginous yeasts were grown on non-detoxified corn stover hydrolysate supplemented with corn steep liquor and mineral medium in shake flasks. Trichosporon oleaginosus DSM 11815 displayed the highest lipid production. This strain was further cultivated in a bench bioreactor, using the same culture medium, under a batch regime. Flow cytometry was used to monitor the T. oleaginosus culture using the dual staining technique (SYBR Green and PI) for cell membrane integrity detection. Values of 42.28% (w/w) and 0.06 g/Lh lipid content and lipid productivity, respectively, were recorded for T. oleaginosus cultivated in the bench bioreactor operated under a batch regime. During the cultivation, most of the yeast cells maintained their integrity. T. oleaginosus has the potential to be used as an oil microbial source for a wide range of industrial applications. In addition, it is robust in adverse conditions such as lignocellulosic hydrolysate exposure and oxygen-limiting conditions. Flow cytometry is a powerful and useful tool for monitoring yeast cultivations on lignocellulosic hydrolysates for cell count, size, granularity, and membrane integrity detection.

Engineering Escherichia coli for selective 1-decanol production using the reverse β-oxidation (rBOX) pathway

1-Decanol has great value in the pharmaceutical and fragrance industries and plays an important role in the chemical industry. In this study, we engineered Escherichia coli to selectively synthesize 1-decanol by using enzymes of the core reverse β-oxidation (rBOX) pathway and termination module with overlapping chain-length specificity. Through screening for acyl-CoA reductase termination enzymes and proper regulation of rBOX pathway expression, a 1-decanol titer of 1.4 g/L was achieved. Further improvements were realized by engineering pyruvate dissimilation to ensure the generation of NADH through pyruvate dehydrogenase (PDH) and reducing byproduct synthesis via a tailored YigI thioesterase knockout, increasing 1-decanol titer to 1.9 g/L. The engineered strain produced about 4.4 g/L 1-decanol with a yield of 0.21 g/g in 36 h in a bi-phasic fermentation that used a dodecane overlay to increase 1-decanol transport and reduce its toxicity. Adjustment of pathway expression (varying inducer concentration) and cell growth (oxygen availability) enabled 1-decanol production at 6.1 g/L (0.26 g/g yield) and 10.05 g/L (0.2 g/g yield) using rich medium in shake flasks and bioreactor, respectively. Remarkably, the use of minimal medium resulted in 1-decanol production with 100% specificity at 2.8 g/L (0.14 g/g yield) and a per cell mass yield higher than rich medium. These 1-decanol titers, yields and purity are at least 10-fold higher than others reported to date and the engineered strain shows great potential for industrial production. Taken together, our findings suggest that using rBOX pathway and termination enzymes of proper chain-length specificity in combination with optimal chassis engineering should be an effective approach for the selective production of alcohols.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Friedelin via Genetic Manipulation.

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration (tHMG1, ERG1, ERG20, ERG9, POS5, or UPC2.1) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (bts1; rox1; ypl062w; yjl064w) displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.

Improved Production and Insulinotropic Properties of Exopolysaccharide by Phellinus igniarius in Submerged Cultures

Phellinu igniarius (P. igniarius), a basidiomycete belonging to the family Polyporaceae, is a medicinal basidiomycetous fungus belonging to the Hymenochaetaceae and is an excellent remedy with anticancer and antioxidant qualities. The mushroom has been used as traditional medicines for the treatment of cardiovascular disease, tuberculosis, liver or heart diseases, bellyache, bloody gonorrhea, and diabetes. However, the limited production and market shortage have been attributed to the slow growth and the difficult collection of the fruiting body as well as the rare natural resources. The problem can be solved through the effective approach of submerged culture to produce a high bioactivity polysaccharide of P. igniarius. The project was proposed to investigate the effect of a surfactant on the production of polysaccharide in submerged culture of P. igniarius and their insulinotropic properties. Eight different surfactants including PEG series (4000, 6000), Tween series (20, 40, 80, 85), and Span series (20, 80) all at a concentration of 0.5 g/L were supplemented in turn to the basal medium in shake flasks. Among the various surfactants tested, Tween 80 exhibited the greatest exopolysaccharide production of 128.43 mg/L, and PEG 6000 showed the maximum biomass of 6.76 mg/mL. To find the optimal Tween 80 concentration for biomass and exopolysaccharide production, different Tween 80 levels (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 g/L) were used in the medium. The maximal exopolysaccharide production of 132.76 mg/mL was achieved with the addition of 0.6 g/L of Tween 80 to the medium. The experimental results exhibited that the maximum of mycelia production in a stirred tank bioreactor was 3.01 mg/mL at Tween 80 0.2 g/L. In this study, their compounds, molecular weight, and protein content from fermentation product extracts were also tested. The average molecular weights of exopolysaccharide and intracellular polysaccharide were 1.715 × 106 Da and 4.87 × 105 Da, respectively. The protein contents of exopolysaccharide and intracellular polysaccharide were about 3.68% and 3.02%. The maximum RINm5F cell proliferations of exopolysaccharide and intracellular polysaccharide at 2 mg/mL were 142.3% and 120.07%, respectively. Cell proliferations of exopolysaccharide and intracellular polysaccharide increased with their concentrations. The maximum insulin secretion of exopolysaccharide at 2 mg/mL on RINm5F cell insulin was 0.615 μg/L.

Multilevel metabolic engineering of Bacillus licheniformis for de novo biosynthesis of 2-phenylethanol

Due to its pleasant rose-like scent, 2-phenylethanol (2-PE) has been widely used in the fields of cosmetics and food. Microbial production of 2-PE offers a natural and sustainable production process. However, the current bioprocesses for de novo production of 2-PE suffer from low titer, yield, and productivity. In this work, a multilevel metabolic engineering strategy was employed for the high-level production of 2-PE. Firstly, the native alcohol dehydrogenase YugJ was identified and characterized for 2-PE production via genome mining and gene function analysis. Subsequently, the redirection of carbon flux into 2-PE biosynthesis by combining optimization of Ehrlich pathway, central metabolic pathway, and phenylpyruvate pathway enabled the production of 2-PE to a titer of 1.81 g/L. Specifically, AroK and AroD were identified as the rate-limiting enzymes of 2-PE production through transcription and metabolite analyses, and overexpression of aroK and aroD efficiently boosted 2-PE synthesis. The precursor competing pathways were blocked by eliminating byproduct formation pathways and modulating the glucose transport system. Under the optimal condition, the engineered strain PE23 produced 6.24 g/L of 2-PE with a yield and productivity of 0.14 g/g glucose and 0.13 g/L/h, respectively, using a complex medium in shake flasks. This work achieves the highest titer, yield, and productivity of 2-PE from glucose via the phenylpyruvate pathway. This study provides a promising platform that might be widely useful for improving the production of aromatic-derived chemicals.

Isolation and characterization of a-l-rhamnosidase producing bacterium, Agrococcus sp. bkd37, from a warehouse soil and partial optimization of its culture conditions

The present study was attempted to identify and partially optimize the culture conditions of a potential α-L-rhamnosidase producing bacterium isolated from warehouse soil of Dibrugarh, Assam (India). Twenty eight bacterial strains were isolated and the isolate BKD37 showed maximum rhamnosidase enzyme activity (10.01 ± 0.001 U mL-1) in crude culture medium. The 16S rRNA gene sequencing and phylogenetic analysis of BKD37 strain showed maximum similarity with that of Agrococcus sp. The culture conditions of strain BKD37 for the production of extracellular rhamnosidase enzyme was partially optimized in a basal medium in shake flask. The maximum rhamnosidase production was achieved after 60 h incubation at 30°C and pH 6.0. The rhamnose and NH4NO3 were found to be the best carbon and nitrogen sources, respectively, in basal medium for α-L rhamnosidase enzyme production.

Production of recombinant beta-amylase of Bacillus aryabhattai

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai β-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for β-amylase production, containing glucose (7.5 g·L−1), pig bone peptone (40.0 g·L−1), Mg2+ (0.05 mol·L−1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the β-amylase yield reached 925.4 U mL−1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4 g L−1. Through this cultivation approached 30 °C with 0 g·L−1 initial glucose concentration, the maximum β-amylase activity reached 5371.8 U mL−1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.

Effect of Physical Parameters on Production of ᴅ-Xylonic Acid using Recombinant E. coli BL21 (DE3)

Abstract ᴅ-xylonic acid is a five-carbon organic acid that has gain increasing interest due to its broadly flexible application in fields such as foods, pharmaceutical and agriculture. Microbial production of ᴅ-xylonic acid can be done by using many species of Pseudomonas, Acetobacter, Gluconobacter and the latest finding was by engineered microorganism. In this study, recombinant E. coli BL21 (DE3) is used to produce ᴅ-xylonic acid from ᴅ-xylose and the effect of temperature, initial pH of medium and agitation rate on ᴅ-xylonic acid production is determined. Fermentation was carried out using super optimal broth medium in shake flasks for 24 hours. Results indicate that ᴅ-xylonic acid concentration was as high as 9.95 g L-1 with yield of 0.91 g/g ᴅ-xylose from 10 g L-1 when governed under temperature of 37°C, initial pH of medium pH 7 and agitation rate 200 rpm.

Engineering phytosterol transport system in Mycobacterium sp. strain MS136 enhances production of 9α-hydroxy-4-androstene-3,17-dione.

To enhance the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from phytosterols, a phytosterol transport system was constructed in Mycobacterium sp. strain MS136. 9-OHAD can be produced via the controlled degradation of phytosterols by mycobacteria. This involves an active transport process that requires trans-membrane proteins and ATP. A phytosterol transport system from Mycobacterium tuberculosis H37Rv was constructed in Mycobacterium sp. strain MS136 by co-expression of an energy-related gene, mceG, and two integrated membrane protein genes, yrbE4A and yrbE4B. The resultant of the Mycobacterium sp. strain MS136-GAB gave 5.7g 9-OHAD l-1, which was a 20% increase over 4.7gl-1 by the wild-type strain. The yield of 9-OHAD was increased to 6.0gl-1 by optimization of fermentation conditions, when 13g phytosterols l-1 were fermented for 84h in 30ml biotransformation medium in shake flasks. Phytosterol transport system plays an active role in the uptake and transport of sterols, cloning of the system improved the mass transfer of phytosterols and increased the production of 9-OHAD.

Improving the production of S-adenosyl-L-methionine in Escherichia coli by overexpressing metk

ABSTRACTS-adenosyl-L-methionine (SAM) has important applications in many fields including chemical therapy and pharmaceutical industry. In this study, the recombinant Escherichia coli strain was constructed for effective production of SAM by introducing the SAM synthase gene (metK). This strain produced 34.5 mg/L of SAM in basic medium in shake flask. Yeast extract, pH, and loaded volume had a significant positive effect on the yield of SAM. Their optimal values were 35 g/L, 7.5, and 30 mL, respectively. The final conditions optimized were as follows: glucose 20, g/L; peptone, 40 g/L; yeast extract, 35 g/L; NaCl, 10 g/L; MgSO4, 1.2 g/L; L-methionine, 1 g/L; rotate speed, 220 rpm; loaded volume, 30 mL; inoculation, 1%; temperature, 37°C; and initial medium, pH 7.5. The recombinant strain produced 128.2 mg/L of SAM under the above conditions in shake flask. The production of SAM in a 5 L fermentor was also investigated. The maximal biomass of the recombinant strain was 60.4 g/L after the cells were cultured for 20 hr, and the highest yield of SAM was 300.9 mg/L after induction for 8 hr in a 5 L fermentor. This study provides a good foundation for the future production and use of SAM.

Optimization of Carbon and Nitrogen Sources for Extracellular Polymeric Substances Production by Chryseobacterium indologenes MUT.2.

Bacterial Extracellular Polymeric Substances (EPS) are environmental friendly and versatile polymeric materials that are used in a wide range of industries such as: food, textile, cosmetics, and pharmaceuticals. To make the production process of the EPS cost-effective, improvements in the production yield is required which could be implemented through application of processes such as optimized culture conditions, and development of the strains with higher yield (e.g. through genetic manipulation), or using low-cost substrates. In this work, the effects of carbon and nitrogen sources were studied in order to improve the EPS production by the submerged cultivation of Chryseobacterium indologenes MUT.2. The mesophilic microorganism Chryseobacterium indologenes MUT.2, was grown and maintained in the Luria Bertani agar. The initial basal medium contained: glucose (20 g.L-1), yeast extracts (5 g.L-1), K2HPO4 (6 g.L-1), NaH2PO4 (7 g.L-1), NH4CL (0.7 g.L-1), and MgSO4 (0.5 g.L-1). For evaluating the carbon and nitrogen sources' effect on the fermentation performance, cultures were prepared in 500 mL flasks filled with 300 mL of the medium. The single-factor experiments based on statistics was employed to evaluate and optimize the carbon and nitrogen sources for EPS production in the liquid culture medium of Chryseobacterium indologenes MUT.2. The preferred carbon-sources, sucrose and glucose, commonly gave the highest EPS production of 8.32 and 6.37 g.L-1, respectively, and the maximum EPS production of 8.87 g.L-1 was achieved when glutamic acid (5 g.L-1) was employed as the nitrogen source. In this work, the culture medium for production of EPS by Chryseobacterium indologenes MUT.2 was optimized. Compared to the basal culture medium in shake-flasks and stirred tank bioreactor, the use of optimized culture medium has resulted in a 53% and 73% increase in the EPS production, respectively.

Heterologous production of α-farnesene in metabolically engineered strains of Yarrowia lipolytica

Herein, we studied the heterologous production of α-farnesene, a valuable sesquiterpene with various biotechnological applications, by metabolic engineering of Yarrowia lipolytica. Different overexpression vectors harboring combinations of tHMG1, IDI, ERG20 and codon-optimized α-farnesene synthase (OptFS) genes were constructed and integrated into the genome of Y. lipolytica Po1h. The engineered strain produced 57.08±1.43mg/L of α-farnesene corresponding to 20.8-fold increase over the initial production of 2.75±0.29mg/L in the YPD medium in shake flasks. Bioreactor scale-up in PM medium led to α-farnesene concentration of 259.98±2.15mg/L with α-farnesene to biomass ratio of 33.98±1.51mg/g, which was a 94.5-fold increase over the initial production. This first report on α-farnesene synthesis in Y. lipolytica lays a foundation for future research on production of sesquitepenes in Y. lipolytica and other closest yeast species and will potentially contribute in its industrial production.

Human papillomavirus L1 protein expressed in Escherichia coli self-assembles into virus-like particles that are highly immunogenic

HPV vaccines based on L1 virus-like particles (VLPs) provided a high degree of protection against HPVs infection. In this study, the codon optimized HPV16 L1 gene were sub-cloned into five procaryotic expression vectors (pET-28a, pET-32a, pGEX-4T-2, pE-sumo and pHSIE), and fused with different protein tags. No recombinant proteins were expressed in pET-28a-L1 and pHSIE-L1, and the proteins expressed by pET-32a-L1 plasmid with TRX-tag were in the form of inclusion body. Only SUMO-tagged and GST-tagged L1 proteins expressed by pE-Sumo-L1 or pGEX-4T-L1 were soluble. The yield of SUMO-L1 protein reached 260mg/L fermentation medium in shake flask. After SUMO tags were eliminated, a 90% purity of L1 proteins was generated by ion-exchange and Ni-NTA affinity chromatography. The purified HPV16 L1 protein self-assembled into virus-like particles (VLPs) and showed a haemagglutination activity. High titers specific and neutralizing antibodies were detected in HPV 16 L1VLPs vaccinated mice. Cytokines such as IFN-γ and IL-2 showed significant higher in VLPs vaccinated mice compared with negative control (p<0.05, p=0.055). Thus, the expression of recombinant HPV16 L1 VLPs in Escherichia coli was feasible, which could potentially be used for a VLP-based HPV vaccine.

High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin.

One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large-scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. The 6-O-sulfotransferase isoform-3 (6-OST-3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria-Bertani medium in shake flasks and inducing with isopropyl β-D-1-thiogalactopyranoside at an optical density of 0·6-0·8. The production of larger amounts of 6-OST-3 required fed-batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5-20 mg g-cell-dry weight(-1)) of active 6-OST-3 with excellent productivity (2-5 mg l(-1) h(-1)). The production of 6-OST-3 in a fed-batch fermentation on an inexpensive carbon source has been demonstrated. The ability to scale-up the production of heparin biosynthetic enzymes, such as 6-OST-3, is critical for scaling-up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal-sourced anticoagulant product currently on the market.

2,3,5,4′- Tetrahydroxystilbene-2-O-β-D-glycoside Biosynthesis by Suspension Cells Cultures of Polygonum multiflorum Thunb and Production Enhancement by Methyl Jasmonate and Salicylic Acid

Friable calli of Polygonum multiflorum Thunb have been induced in MS medium supplemented with 6-benzylaminopurine (6-BA) and kinetin (KT). Suspension cultures were initiated from friable calli by inoculating calli in liquid MS medium in shake flasks in the dark and 25 °C on an orbital shaker at 100 rpm. The maximum dry weight (DW, 7.85 g/L) and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glycoside (THSG, 56.39 mg/L) of suspension cells was obtained in MS medium after 16 days culture. Both methyl jasmonate (MeJA) and salicylic acid (SA) could increase THSG production. The most appropriate concentration of MeJA was 100 μmol/L in MS medium, in which concentration THSG content reached the maximum value of 147.79 mg/L, which represented a 162.36% increase compared to that of the control (56.33 mg/L). The most appropriate concentration of SA was 125 μmol/L in MS medium, at which concentration THSG content reached its maximum value of 116.43 mg/L, a 106.69% increase compared to that of the control (56.33 mg/L).

Large-scale production of tannase using the yeast Arxula adeninivorans

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L(-1) when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L(-1). The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L(-1). Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett–Burman design (PBD). Only Zn 2+, Mn 2+ and MoO 4 2− were the most significant components ( p < 0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn 2+ (ZnSO 4·7H 2O), Mn 2+ (MnCl 2·4H 2O), and MoO 4 2− (Na 2MoO 4·2H 2O) were 83, 42 and 135 μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14 L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25 μg DAPG/g carrier) when stored at 28 °C for 6 months. The structure of the purified DAPG was confirmed using 1H NMR and mass spectrometry.

Microbial production of meso-2,3-butanediol by metabolically engineered Escherichia coli under low oxygen condition

A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase-encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587-amino acid protein, preceded by an N-terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild-type and recombinant strains were essentially indistinguishable. A molecular mass of approximately 320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35-40 degrees C and a pH optimum at approximately 6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild-type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks.