Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

Kannan, R.; Mukundan, Gopalakrishnan; Ait-Abdelkader, Nadra; Augier-Magro, Valerie; Baratti, J.; Gunasekaran, Paramasamy

doi:10.1007/bf00305353

Cited by 27 publications

(12 citation statements)

References 37 publications

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“…Since sucrose is unable to transport through the cell membrane of Z. mobilis [19], the enzyme should be located in a position of the membrane and cell wall in contact with sucrose. From the published genome, no signal peptide for levansucrase was reported or annotated in the genome information of Z. mobilis ZM4.…”

Section: Secretive Expression By Fusion Proteinmentioning

confidence: 99%

Secretive expression of heterologous β-glucosidase in Zymomonas mobilis

Luo

Bao

2015

Bioresour. Bioprocess.

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Background: Zymomonas mobilis is an efficient ethanol fermentation strain, but its narrow substrate range limits its fermentation in lignocellulose hydrolysate. As a potential consolidated bioprocessing (CBP) stain for bioethanol production, the ability of cellulose utilization was necessary. In this study, extracellular expression of β-glucosidase on Z. mobilis was studied as the first step for construction of a practical CBP strain to reduce the use of β-glucosidase in the cellulase components. Results: The heterologous β-glucosidase from Bacillus polymyxa was expressed in the ethanologenic strain Z. mobilis (ZM4) and secreted extracellularly by an endogenous signal peptide and a fusion protein. The signal peptide SP1086 of the endoglucanase gene ZMO1086 from Z. mobilis was identified and facilitated 12 % of the endoglucanase encoded by ZMO1086 from Z. mobilis ZM4 and 16 % of the β-glucosidase encoded by bglB gene secreted out of the membrane of Z. mobilis ZM4. Another method for enhancement of the β-glucosidase secretion is to fuse the β-glucosidase encoded by bglB with the levansucrase encoded by sacB from Z. mobilis ZM4 to achieve the secretive expression. Its expression level was enhanced two times but only showed a 2 % secretion ratio in this situation. Conclusions: The SP1086 signal peptide showed an obviously secreting capacity of the β-glucosidase protein. The fusion protein with SacB also showed the secretion effect, but it was less efficient.

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Section: Secretive Expression By Fusion Proteinmentioning

confidence: 99%

Secretive expression of heterologous β-glucosidase in Zymomonas mobilis

Luo

Bao

2015

Bioresour. Bioprocess.

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“…Z. mobilis genetic modification has been reported previously with the sacC , adhB , and ndh targets for mutagenesis [19-21]. However, the existence of native plasmids [22,23] and intrinsic antibiotic resistance impedes the use of many broad-host-range plasmids [22,24,25].…”

Section: Introductionmentioning

confidence: 99%

The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors

Yang

Pelletier

et al. 2010

BMC Microbiol

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BackgroundZymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.ResultsWe generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.ConclusionsWe have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

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“…1990a;1990b) and two enzymes have been purified (Yanase et al 1992;Alt-Abdelkader 1993;O'Muilan et al 1992;Preziosi et al 1990b). Previously, we have isolated and studied the sacA (Gunasekaran et al 1990) and sacC (Kannan et al 1995) genes from Z. mobilis encoding the intracellular sucrase and the extracellular sucrase, respectively. In this study, we report the cloning, expression, characterization and sequencing of sacB gene encoding the extracellular levansucrase.…”

Section: Introductionmentioning

confidence: 99%

The sacB and sacC genes encoding levansucrase and sucrase form a gene cluster in Zymomonas mobilis

et al. 1995

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The Zymomonas mobilis gene sacB that encodes the extracellular levansucrase was cloned and expressed in Escher~chia coli. The gene product exhibited both sucrose hydrolysis activity and levan forming capability. Sub-cellular fractionation ofE. coli carrying pLSS41 revealed that about 95% of the total sucrase activity was detected in the cytoplasmic fraction. The levansucrase gene was overexpressed (about hundred fold) in E. coti under T7 polymerase expression system. Nucleotide sequence analysis of this gene revealed an open reading frame of 1269 bp long coding for a protein of 423 amino acids with a molecular mass of 46.7 KDa. The deduced amino acid sequence was identical to the N-terminal amino acids of protein A51 ofZ. mobilis ZM4. Therefore, the product ofsacB is levansucrase. This is the first extracellular enzyme of Z. mobilis sequenced which does not possess a signal sequence.This gene is located 198 bp upstream ofsacC gene encoding for the extracellular sucrase forming a gene cluster INTRODUCTION

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Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

Cited by 27 publications

References 37 publications

Secretive expression of heterologous β-glucosidase in Zymomonas mobilis

Secretive expression of heterologous β-glucosidase in Zymomonas mobilis

The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors

The sacB and sacC genes encoding levansucrase and sucrase form a gene cluster in Zymomonas mobilis

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