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Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

Huang J, Xia J, Yang Z, Guan F, Cui D, Guan G, Jiang W, Li Y - Biotechnol Biofuels (2014)

Bottom Line: The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction.Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties.The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.

ABSTRACT

Background: We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel.

Results: Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%.

Conclusions: Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

No MeSH data available.


Related in: MedlinePlus

Effect of propeptide addition on cell growth and target enzyme activity. A: OD600 of recombinant strains in flask fermentation. Cell growth of control strain (zα-X33) and recombinant strain (zα-1pRML-X33) containing the propeptide sequence was much higher than that of the recombinant strain without the propeptide sequence (zα-1mRML-X33). B: Enzyme activity of recombinant strains in flask fermentation. Extracellular enzyme activity of zα-1pRML-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL). C: Extracellular protein production in fermentation supernatant detected by Western blotting. Lane 1: protein markers (top to bottom: 100, 70, 55, 40, 35, 25, 15 kDa). Lane 2: zα-1pRML-X33. Lane 3: zα-1mRML-X33. Lane 4: zα-X33. Each sample tested was 10 μL of 10 × diluted fermentation supernatant. The concentration of secreted extracellular target protein for zα-1pRML-X33 (0.15 mg/mL) was higher than for zα-1mRML-X33 (0.019 mg/mL). D: Comparison of the transcription level (by qPCR) of rml in zα-1pRML-X33 vs. zα-1mRML-X33. When the same signal peptide codons were present in the expression plasmid, adding the propeptide resulted in upregulation of target gene expression at both 48 and 96 hours.
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Fig1: Effect of propeptide addition on cell growth and target enzyme activity. A: OD600 of recombinant strains in flask fermentation. Cell growth of control strain (zα-X33) and recombinant strain (zα-1pRML-X33) containing the propeptide sequence was much higher than that of the recombinant strain without the propeptide sequence (zα-1mRML-X33). B: Enzyme activity of recombinant strains in flask fermentation. Extracellular enzyme activity of zα-1pRML-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL). C: Extracellular protein production in fermentation supernatant detected by Western blotting. Lane 1: protein markers (top to bottom: 100, 70, 55, 40, 35, 25, 15 kDa). Lane 2: zα-1pRML-X33. Lane 3: zα-1mRML-X33. Lane 4: zα-X33. Each sample tested was 10 μL of 10 × diluted fermentation supernatant. The concentration of secreted extracellular target protein for zα-1pRML-X33 (0.15 mg/mL) was higher than for zα-1mRML-X33 (0.019 mg/mL). D: Comparison of the transcription level (by qPCR) of rml in zα-1pRML-X33 vs. zα-1mRML-X33. When the same signal peptide codons were present in the expression plasmid, adding the propeptide resulted in upregulation of target gene expression at both 48 and 96 hours.

Mentions: We explored several strategies to modify RML and its heterologous expression system in order to improve enzyme activity and properties. Our first step was to examine the role of the target gene’s propeptide. We constructed two expression plasmids with carrier pPICZαA: one adding the target gene’s 70-amino-acid propeptide (prml) and one without propeptide (mrml). The recombinant plasmids were transformed separately into strain X-33. The positive strains, each containing one copy of the target gene, were termed zα-1mRML-X33 and zα-1pRML-X33. These two strains and a control strain (zα-X33, without target gene) were cultured in shaking flasks and sampled every day for cell growth and lipase activity. The screening results are shown in Figure 1. Cell growth of zα-1pRML-X33 and zα-X33 was higher than that of zα-1mRML-X33 (Figure 1A). The extracellular enzyme activity of zα-pRML-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL; Figure 1B). These findings indicate that in the absence of the propeptide, expression levels of the heterologous protein are consistent regardless of whether strain GS115 or X-33 is used as a host. Extracellular protein in fermentation supernatant was detected by Western blotting (Figure 1C). The content of secreted target protein for zα-1pRML-X33 (0.15 mg/mL) was approximately 7-fold higher than that for zα-1mRML-X33 (0.019 mg/mL).Quantitative real-time RT-PCR (qPCR) results confirmed that adding the propeptide resulted in upregulation of target gene expression (Figure 1D) at 48 and 96 hours. The propeptide evidently plays a promoting role in lipase activity and heterologous protein secretion.Figure 1


Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

Huang J, Xia J, Yang Z, Guan F, Cui D, Guan G, Jiang W, Li Y - Biotechnol Biofuels (2014)

Effect of propeptide addition on cell growth and target enzyme activity. A: OD600 of recombinant strains in flask fermentation. Cell growth of control strain (zα-X33) and recombinant strain (zα-1pRML-X33) containing the propeptide sequence was much higher than that of the recombinant strain without the propeptide sequence (zα-1mRML-X33). B: Enzyme activity of recombinant strains in flask fermentation. Extracellular enzyme activity of zα-1pRML-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL). C: Extracellular protein production in fermentation supernatant detected by Western blotting. Lane 1: protein markers (top to bottom: 100, 70, 55, 40, 35, 25, 15 kDa). Lane 2: zα-1pRML-X33. Lane 3: zα-1mRML-X33. Lane 4: zα-X33. Each sample tested was 10 μL of 10 × diluted fermentation supernatant. The concentration of secreted extracellular target protein for zα-1pRML-X33 (0.15 mg/mL) was higher than for zα-1mRML-X33 (0.019 mg/mL). D: Comparison of the transcription level (by qPCR) of rml in zα-1pRML-X33 vs. zα-1mRML-X33. When the same signal peptide codons were present in the expression plasmid, adding the propeptide resulted in upregulation of target gene expression at both 48 and 96 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4364654&req=5

Fig1: Effect of propeptide addition on cell growth and target enzyme activity. A: OD600 of recombinant strains in flask fermentation. Cell growth of control strain (zα-X33) and recombinant strain (zα-1pRML-X33) containing the propeptide sequence was much higher than that of the recombinant strain without the propeptide sequence (zα-1mRML-X33). B: Enzyme activity of recombinant strains in flask fermentation. Extracellular enzyme activity of zα-1pRML-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL). C: Extracellular protein production in fermentation supernatant detected by Western blotting. Lane 1: protein markers (top to bottom: 100, 70, 55, 40, 35, 25, 15 kDa). Lane 2: zα-1pRML-X33. Lane 3: zα-1mRML-X33. Lane 4: zα-X33. Each sample tested was 10 μL of 10 × diluted fermentation supernatant. The concentration of secreted extracellular target protein for zα-1pRML-X33 (0.15 mg/mL) was higher than for zα-1mRML-X33 (0.019 mg/mL). D: Comparison of the transcription level (by qPCR) of rml in zα-1pRML-X33 vs. zα-1mRML-X33. When the same signal peptide codons were present in the expression plasmid, adding the propeptide resulted in upregulation of target gene expression at both 48 and 96 hours.
Mentions: We explored several strategies to modify RML and its heterologous expression system in order to improve enzyme activity and properties. Our first step was to examine the role of the target gene’s propeptide. We constructed two expression plasmids with carrier pPICZαA: one adding the target gene’s 70-amino-acid propeptide (prml) and one without propeptide (mrml). The recombinant plasmids were transformed separately into strain X-33. The positive strains, each containing one copy of the target gene, were termed zα-1mRML-X33 and zα-1pRML-X33. These two strains and a control strain (zα-X33, without target gene) were cultured in shaking flasks and sampled every day for cell growth and lipase activity. The screening results are shown in Figure 1. Cell growth of zα-1pRML-X33 and zα-X33 was higher than that of zα-1mRML-X33 (Figure 1A). The extracellular enzyme activity of zα-pRML-X33 (430 U/mL) was 7.7-fold higher than that of zα-1mRML-X33 (56 U/mL; Figure 1B). These findings indicate that in the absence of the propeptide, expression levels of the heterologous protein are consistent regardless of whether strain GS115 or X-33 is used as a host. Extracellular protein in fermentation supernatant was detected by Western blotting (Figure 1C). The content of secreted target protein for zα-1pRML-X33 (0.15 mg/mL) was approximately 7-fold higher than that for zα-1mRML-X33 (0.019 mg/mL).Quantitative real-time RT-PCR (qPCR) results confirmed that adding the propeptide resulted in upregulation of target gene expression (Figure 1D) at 48 and 96 hours. The propeptide evidently plays a promoting role in lipase activity and heterologous protein secretion.Figure 1

Bottom Line: The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction.Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties.The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, 2#,Yuanmingyuan West Road, Beijing, 100193 China.

ABSTRACT

Background: We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel.

Results: Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%.

Conclusions: Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

No MeSH data available.


Related in: MedlinePlus