Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

Gurunathan, Sangiliyandi; Gunasekaran, Paramasamy

doi:10.1590/s1516-89132004000300001

Cited by 3 publications

(3 citation statements)

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“…However, levansucrase mRNA transcript levels were not quantified in these studies. Notably, (41) observed levansucrase mRNA transcripts in Z. mobilis cultures on both glucose and fructose. Perhaps, different mechanisms govern levansucrase expression in different species, which employ EPS for different functions in their different ecological habitats/niches.…”

Section: Discussionmentioning

confidence: 99%

Molecular inactivation of exopolysaccharide biosynthesis inPaenibacillus polymyxaDSM 365 for enhanced 2,3-butanediol production

Okonkwo

Ujor

Ezeji

2018

Preprint

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25Formation of Exopolysaccharides (EPS) during 2,3-butanediol (2,3-BD) fermentation by 26 Paenibacillus polymyxa decreases 2,3-BD yield, increases medium viscosity and impacts 2,3-BD 27 downstream processing. Therefore, additional purification steps are required to rid the fermentation 28 broth of EPS prior to 2,3-BD purification, which adds to the production cost. To eliminate EPS 29 production during 2,3-BD fermentation, we explored a metabolic engineering strategy to disable the 30 EPS production pathway of P. polymyxa, thereby increasing 2,3-BD yield and productivity. The 31 levansucrase gene which encodes levansucrase, the enzyme responsible for EPS biosynthesis in P. 32 polymyxa, was successfully disrupted. The resulting P. polymyxa levansucrase null mutant showed 33 34% and 54% increases in growth with 6.4-and 2.4-folds decrease in EPS formation in sucrose and 34 glucose cultures, respectively. The observed decrease in EPS formation by the levansucrase null 35 mutant may account for the 27% and 4% increase in 2,3-BD yield, and 4% and 128% increases in 36 2,3-BD productivity when grown on sucrose and glucose media, respectively. Genetic stability of 37 the levansucrase null mutant was further evaluated. Interestingly, the levansucrase null mutant 38 remained genetically stable over fifty generations with no observable decrease in growth and 2,3-39 BD formation with or without antibiotic supplementations. Collectively, our results show that P. 40 polymyxa levansucrase null mutant has potential for improving 2,3-BD yield, and ultimately, the 41 economics of large-scale microbial 2,3-BD production. 42 43 44 polysaccharide polymerase 45 46 47 48 49Considering the finite nature of fossil fuels, recurrent instability in oil price and the 50 environmental concerns associated with oil consumption, there is an urgent need to develop 51 sustainable alternatives to fossil fuels and their derivatives. Over the past few decades, significant 52 attention has been devoted to the development of alternative sources of fuels and chemicals. 2,3-53 Butanediol (2,3-BD) is an industrial platform chemical that is generated via cracking of petroleum-54 derived hydrocarbons (e.g. butenes). 2,3-BD has wide industrial applications. For instance, 2,3-BD 55 can be used as a feedstock chemical in the production of 1,3-butadiene (1,3-BD), the monomer from 56 which synthetic rubber is produced (1, 2). Also, 2,3-BD can be used as a feedstock for producing 57 methyl ethyl ketone (MEK), a fuel additive which has a higher heat of combustion than ethanol, and 58 as a solvent from which resins and lacquers can be produced (1, 2). Additionally, 2,3-BD has 59 massive potential as a feedstock for the synthesis of a host of numerous pharmaceuticals, cosmetics, 60 paints, and food preservatives (3, 4). 61 Several microorganisms have been shown to possess the metabolic machinery to convert 62 carbohydrates to 2,3-BD. However, 2,3-BD is produced via a mixed acid fermentation pathway 63 where other products such as ethanol, acetoin, lact...

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Section: Discussionmentioning

confidence: 99%

Molecular inactivation of exopolysaccharide biosynthesis inPaenibacillus polymyxaDSM 365 for enhanced 2,3-butanediol production

Okonkwo

Ujor

Ezeji

2018

Preprint

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“…In fact, sucrose-mediated induction of levansucrase gene expression has been suggested by other authors (19,20), although transcript levels were not quantified. Furthermore, levansucrase mRNA transcripts were observed in Zymomonas mobilis grown on both glucose and fructose (21), indicating that different mechanisms may govern levansucrase expression among species, which use EPS in functionally distinct ways that are adapted to their diverse ecological niches. Further studies are required to better delineate the role of sucrose in levansucrase gene expression in P. polymyxa.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation

Okonkwo

Ujor

Cornish

et al. 2020

Appl Environ Microbiol

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The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa. The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges. IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

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“…Among them, the SacC sucrase activity accounts for more than 70% of the total sucrase activity, which mainly hydrolyzes sucrose into monosaccharides, but the intracellular sucrase SacA has extremely low hydrolysis efficiency of sucrose. The levansucrase SacB has sucrose-hydrolyzing and levan-generating activity (Gurunathan & Gunasekaran, 2004). Ananthalakshmy et al overexpressed the sacB gene in Z. mobilis B14023, which increased the extracellular levansucrase activity to 187 U/ml (Ananthalakshmy & Gunasekaran, 1999).…”

Section: Introductionmentioning

confidence: 99%

Lowering whole cost for sugarcane‐based ethanol production by engineered Zymomonas mobilis

Dai

Liu

et al. 2021

GCB Bioenergy

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Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

Abstract: The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number).

Cited by 3 publications

References 31 publications

Molecular inactivation of exopolysaccharide biosynthesis inPaenibacillus polymyxaDSM 365 for enhanced 2,3-butanediol production

Molecular inactivation of exopolysaccharide biosynthesis inPaenibacillus polymyxaDSM 365 for enhanced 2,3-butanediol production

Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation

Lowering whole cost for sugarcane‐based ethanol production by engineered Zymomonas mobilis

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