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    Enhanced production of N-acetyl-d-neuraminic acid by whole-cell bio-catalysis of Escherichia coli
    (Elsevier, 2016) Zhou, Junbo; Chen, Xianzhong; Lu, Liping; Govender, Algasan; Haiquan, Yang; Shen, Wie
    N-acetyl-d-neuraminic acid (Neu5Ac) has been considerably focused due to its promising potential appli-cations in pharmaceuticals and dairy products. A whole-cell biocatalyst process is an important tool for synthesis of pharmaceutical intermediates and fine chemicals. In this study, a whole cell process using engineered Escherichia coli strain was developed and stepwise optimization was employed for Neu5Ac production. N-acetyl-D-glucosamine 2-epimerase and Neu5Ac aldolase were overexpressed in E. coli individually and the activity ratio was optimized by varying recombinant amounts of cell biomass for syn-thesis of Neu5Ac. Moreover, substrate concentrations and ratio of pyruvate and N-acetyl-D-glucosamine (GlcNAc) and detergent concentrations were optimized to increase product synthesis. The resulting process generated 237.4 mM Neu5Ac with a yield of 40.0% mol/mol GlcNAc. Furthermore, transporter pathways involved in Neu5Ac and GlcNAc were engineered and their impact on the Neu5Ac synthesis was evaluated. Using a stepwise optimization, an overall whole-cell biocatalytic process was developed and a maximum titer of 260.0 mM Neu5Ac (80.4 g/L) with a conversion yield of 43.3% from GlcNAc was achieved. The process can be used for industrial large-scale production of Neu5Ac in terms of efficiency and economy.
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    Improvement of d-lactate productivity in recombinant Escherichia coli by coupling production with growth
    (Springer Netherlands, 2012-06) Zhou, Li; Tian, Kang-Ming; Niu, Dan-Dan; Shen, Wei; Shi, Fui-Yang; Singh, Suren; Wang, Zheng-Xiang
    Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O2-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O2-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.
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    Fine tuning the transcription of ldhA for D-lactate production
    (Springer-Verlag, 2012-03-20) Singh, Suren
    Nonlinear ion cyclotron and ion-acoustic waves have been studied in an electron–positron–ion plasma. Using Boltzmann distributions for the electrons and positrons and fluid equations for the ions, a set of nonlinear equations in the rest frame of the propagating wave is derived and numerically solved for the electric field. A scan of parameter space reveals a range of solutions for the parallel electric field, from sinusoidal to sawtooth to highly spiky waveforms. The results are compared with satellite observations.
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    Genetically switched D-lactate production in Escherichia coli
    (Elsevier, 2012-06-08) Singh, Suren
    During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative d-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ pR and pL promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013–070B (B0013-070, ldhAp::kan-cIts857-pR–pL), which could produce two-fold higher LDH activity at 42 °C than the B0013-070 strain, was created. When the genetic switch was turned off at 33 °C, strain B0013-070B produced 10% more biomass aerobically than strain B0013-070 and produced only trace levels of lactate which could reduce the growth inhibition caused by oxygen insufficiency in large scale fermentation. However, 42 °C is the most efficient temperature for switching on lactate production. The volumetric productivity of B0013-070B improved by 9% compared to that of strain B0013-070 when it was grown aerobically at 33 °C with a short thermo-induction at 42 °C and then switched to the production phase at 42 °C. In a bioreactor experiment using scaled-up conditions that were optimized in a shake flask experiment, strain B0013-070B produced 122.8 g/l d-lactate with an increased oxygen-limited productivity of 0.89 g/g·h. The results revealed the effectiveness of using a genetic switch to regulate cell growth and the production of a metabolic compound.