Heterologous expression of LamA gene encoded endo-β-1,3-glucanase and CO2 fixation by bioengineered Synechococcus sp. PCC 7002

doi:10.1007/s11783-017-0910-1

Front. Environ. Sci. Eng.

2017, Vol. 11

Issue (2) : 9 https://doi.org/10.1007/s11783-017-0910-1

RESEARCH ARTICLE

Heterologous expression of LamA gene encoded endo-β-1,3-glucanase and CO₂ fixation by bioengineered Synechococcus sp. PCC 7002

Di Li¹,Swati Yewalkar²,Xiaotao Bi²(

),Sheldon Duff²,Dusko Posarac²,Heli Wang¹,Layne A. Woodfin³,Jan-Hendrik Hehemann³,Sheila C. Potter³,Francis E. Nano³

¹. School of Water Resources and Environment, China University of Geosciences, Beijing 100083, China
². Department of Chemical and Biological Engineering, University of British Columbia, Vancouve, V6T 1Z3, Canada
³. Department of Biochemistry and Microbiology, University of Victoria, Victoria, V5Z 4H4, Canada

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Abstract

Maximum growth rate of Synechococcus mutant was 0.083 h⁻¹ with 5% CO₂.

Maximum biomass concentration of Synechococcus mutant was 3.697 g·L⁻¹.

Synechococcus mutant can tolerate gas aeration with 15% CO₂.

Maximum specific activity of laminarinase was 4.325 U·mg⁻¹ dry mass.

Optimal pH and temperature of laminarinase activity were 8.0 and 70°C.

The gene for the catalytic domain of thermostable endo-β-1,3-glucanase (laminarinase) LamA was cloned from Thermotoga maritima MSB8 and heterologously expressed in a bioengineered Synechococcus sp. PCC 7002. The mutant strain was cultured in a photobioreactor to assess biomass yield, recombinant laminarinase activity, and CO2 uptake. The maximum enzyme activity was observed at a pH of 8.0 and a temperature of 70°C. At a CO2 concentration of 5%, we obtained a maximum specific growth rate of 0.083 h⁻¹, a biomass productivity of 0.42 g·L⁻¹·d⁻¹, a biomass concentration of 3.697 g·L⁻¹, and a specific enzyme activity of the mutant strain of 4.325 U·mg⁻¹ dry mass. All parameters decreased as CO2 concentration increased from 5% to 10% and further to 15% CO2, except enzyme activity, which increased from 5% to 10% CO2. However, the mutant culture still grew at 15% CO2 concentration, as reflected by the biomass productivity (0.26 g·L⁻¹·d⁻¹), biomass concentration (2.416 g·L⁻¹), and specific enzyme activity (3.247 U·mg⁻¹ dry mass).

Keywords Synechococcus sp. PCC 7002 Thermotoga maritima LamA gene Endo-β-1 3-glucanase CO₂ fixation

Corresponding Author(s): Xiaotao Bi

Issue Date: 17 March 2017

Cite this article:

Di Li,Swati Yewalkar,Xiaotao Bi, et al. Heterologous expression of LamA gene encoded endo-β-1,3-glucanase and CO₂ fixation by bioengineered Synechococcus sp. PCC 7002[J]. Front. Environ. Sci. Eng., 2017, 11(2): 9.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-017-0910-1
https://academic.hep.com.cn/fese/EN/Y2017/V11/I2/9

Fig.1 Photobioreactors setup

Fig.2 Expression of LamA-CD in Synechococcus sp. PCC 7002: Lane M (molecular weight markers), Lane A (LamA-CD purified from Escherichia coli), Lane B (cell extracts of Synechococcus LamA mutant strain), and Lane C (cell extracts of Synechococcus wild-type strain)

Fig.3 Influences of pH and temperature on the activity of recombinant laminarinase from Synechococcus LamA mutant strain. (a) Cell extracts were incubated at 70°C for 30 min with substrate (3 mg·mL^-¹ laminarin) in0.05 mol·L^-¹ citrate-phosphate buffer at pH 3.0–5.5 and in 50 0.05 mol·L^-¹ phosphate buffer at pH 6.0–9.0. The maximum enzyme activity was 4.237 U·mg^-¹ dry mass. (b) Cell extracts were incubated in 0.05 mol·L^-¹ phosphate buffer at pH 8.0 for 30 min with substrate (3 mg·mL^-¹ laminarin) at 20°C–90°C. The maximum enzyme activity was 4.446 U·mg^-¹ dry mass

Fig.4 Thermal stability of the recombinant laminarinase from Synechococcus LamA mutant strain. The cell extracts were pre-incubated at 80°C (▲), 90°C (■), and 100°C (●) in 0.05 mol·L^-¹ phosphate buffer at pH 8.0. The cell extracts were incubated at 70°C for 30 min with substrate (3 mg·mL^-¹ laminarin) in 0.05 mol·L^-¹ phosphate buffer at pH 8.0. The maximum enzyme activity was 4.298 U·mg^-¹ dry mass

Tab.1 Maximum biomass concentration (X_max, g·L^-¹), maximum specific growth rate (m_max, h^-¹), doubling time (h), maximum biomass production rate (P_max, g·L^-¹·d^-¹), and specific enzyme activity (U·mg^-¹ dry mass) for the Synechococcus wild-type and the LamA mutant strains grown in photobioreactors with 5%, 10%, and 15% CO₂

Fig.5 Growth curves of Synechococcus wild-type (□) and LamA mutant strain (■). The Synechococcus wild-type was cultivated with 5% CO₂ aeration; the LamA mutant strain was cultivated with 10% (♦) and 15% (▲) CO₂ aeration

Fig.6 Specific enzyme activity of the recombinant laminarinase from Synechococcus LamA mutant strain cultivated in the photobioreactor with 5%, 10%, and 15% CO₂ aeration

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