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Metabolic engineering of Acinetobacter baylyi ADP1 for removal of Clostridium butyricum growth inhibitors produced from lignocellulosic hydrolysates.

Kannisto MS, Mangayil RK, Shrivastava-Bhattacharya A, Pletschke BI, Karp MT, Santala VP - Biotechnol Biofuels (2015)

Bottom Line: Pretreatment of lignocellulosic biomass can produce inhibitory compounds that are harmful for microorganisms used in the production of biofuels and other chemicals from lignocellulosic sugars.Formate was consumed during growth on acetate and by stationary phase cells, and this was enhanced in the presence of a common aromatic inhibitor of lignocellulosic hydrolysates, 4-hydroxybenzoate.Because of these encouraging results, we believe that A. baylyi ADP1 is a promising candidate for the detoxification of lignocellulosic hydrolysates for bioprocesses.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Bioengineering, Tampere University of Technology, Korkeakoulunkatu 8, Tampere, Finland.

ABSTRACT

Background: Pretreatment of lignocellulosic biomass can produce inhibitory compounds that are harmful for microorganisms used in the production of biofuels and other chemicals from lignocellulosic sugars. Selective inhibitor removal can be achieved with biodetoxification where microorganisms catabolize the inhibitors without consuming the sugars. We engineered the strictly aerobic Acinetobacter baylyi ADP1 for detoxification of lignocellulosic hydrolysates by removing the gene for glucose dehydrogenase, gcd, which catalyzes the first step in its glucose catabolism.

Results: The engineered A. baylyi ADP1 strain was shown to be incapable of consuming the main sugar components of lignocellulosic hydrolysates, i.e., glucose, xylose, and arabinose, but rapidly utilized acetate and formate. Formate was consumed during growth on acetate and by stationary phase cells, and this was enhanced in the presence of a common aromatic inhibitor of lignocellulosic hydrolysates, 4-hydroxybenzoate. The engineered strain tolerated glucose well up to 70 g/l, and the consumption of glucose, xylose, or arabinose was not observed in prolonged cultivations. The engineered strain was applied in removal of oxygen, a gaseous inhibitor of anaerobic fermentations. Co-cultivation with the A. baylyi ADP1 gcd knockout strain under initially aerobic conditions allowed the strictly anaerobic Clostridium butyricum to grow and produce hydrogen (H2) from sugars of the enzymatic rice straw hydrolysate.

Conclusions: We demonstrated that the model organism of bacterial genetics and metabolism, A. baylyi ADP1, could be engineered to be an efficient biodetoxification strain of lignocellulosic hydrolysates. Only one gene knockout was required to completely eliminate sugar consumption and the strain could be used in production of anaerobic conditions for the strictly anaerobic hydrogen producer, C. butyricum. Because of these encouraging results, we believe that A. baylyi ADP1 is a promising candidate for the detoxification of lignocellulosic hydrolysates for bioprocesses.

No MeSH data available.


Related in: MedlinePlus

The gcd knockout cassette (a) and PCR verification of the transformation (b). Names of the knockout cassette components are shown above the figure and the sites for restriction enzymes are shown below it. The samples in the lanes in B are: 1 GeneRuler™ 1 kb DNA Ladder (Thermo Scientific, Waltham, MA, USA), 2 wild-type A. baylyi ADP1, 3gcd knockout mutant of A. baylyi ADP1. (a adapted from Santala et al. [25])
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Fig6: The gcd knockout cassette (a) and PCR verification of the transformation (b). Names of the knockout cassette components are shown above the figure and the sites for restriction enzymes are shown below it. The samples in the lanes in B are: 1 GeneRuler™ 1 kb DNA Ladder (Thermo Scientific, Waltham, MA, USA), 2 wild-type A. baylyi ADP1, 3gcd knockout mutant of A. baylyi ADP1. (a adapted from Santala et al. [25])

Mentions: Acinetobacter baylyi ADP1 (DSM 24193) was used in construction of the gcd knockout strain and as a control strain in the experiments. The knockout cassette (Fig. 6a) was constructed from the one used previously by Santala et al. [25] using established molecular biotechnology methods [36]. The flanking regions of the cassette were replaced with the flanking regions for knocking out gcd (ACIAD2983, NCBI Gene ID: 2878488) by cloning them from the genome of wild-type A. baylyi ADP1 with primers having identical annealing regions to those used by de Berardinis et al. [19]. The primers used in construction of the strain are shown in Table 2. The upstream flanking region was cloned with primers U1 and U2 and the downstream flanking region with D1 and D2. Construction of the plasmid was carried out in Escherichia coli XL-1 Blue. A. baylyi ADP1 was transformed using the method of Metzgar et al. [22] using a PCR product amplified with primers C1 and C2, which annealed to the flanking regions of the cassette. These primers were used also in the verification of the transformants using genomic DNA as a template. A positive transformant gave a product of approximately 2 kb while the genomic DNA of the wild-type cells, which was used as a negative control, gave a product of approximately 3 kb (Fig. 6b).Fig. 6


Metabolic engineering of Acinetobacter baylyi ADP1 for removal of Clostridium butyricum growth inhibitors produced from lignocellulosic hydrolysates.

Kannisto MS, Mangayil RK, Shrivastava-Bhattacharya A, Pletschke BI, Karp MT, Santala VP - Biotechnol Biofuels (2015)

The gcd knockout cassette (a) and PCR verification of the transformation (b). Names of the knockout cassette components are shown above the figure and the sites for restriction enzymes are shown below it. The samples in the lanes in B are: 1 GeneRuler™ 1 kb DNA Ladder (Thermo Scientific, Waltham, MA, USA), 2 wild-type A. baylyi ADP1, 3gcd knockout mutant of A. baylyi ADP1. (a adapted from Santala et al. [25])
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4666034&req=5

Fig6: The gcd knockout cassette (a) and PCR verification of the transformation (b). Names of the knockout cassette components are shown above the figure and the sites for restriction enzymes are shown below it. The samples in the lanes in B are: 1 GeneRuler™ 1 kb DNA Ladder (Thermo Scientific, Waltham, MA, USA), 2 wild-type A. baylyi ADP1, 3gcd knockout mutant of A. baylyi ADP1. (a adapted from Santala et al. [25])
Mentions: Acinetobacter baylyi ADP1 (DSM 24193) was used in construction of the gcd knockout strain and as a control strain in the experiments. The knockout cassette (Fig. 6a) was constructed from the one used previously by Santala et al. [25] using established molecular biotechnology methods [36]. The flanking regions of the cassette were replaced with the flanking regions for knocking out gcd (ACIAD2983, NCBI Gene ID: 2878488) by cloning them from the genome of wild-type A. baylyi ADP1 with primers having identical annealing regions to those used by de Berardinis et al. [19]. The primers used in construction of the strain are shown in Table 2. The upstream flanking region was cloned with primers U1 and U2 and the downstream flanking region with D1 and D2. Construction of the plasmid was carried out in Escherichia coli XL-1 Blue. A. baylyi ADP1 was transformed using the method of Metzgar et al. [22] using a PCR product amplified with primers C1 and C2, which annealed to the flanking regions of the cassette. These primers were used also in the verification of the transformants using genomic DNA as a template. A positive transformant gave a product of approximately 2 kb while the genomic DNA of the wild-type cells, which was used as a negative control, gave a product of approximately 3 kb (Fig. 6b).Fig. 6

Bottom Line: Pretreatment of lignocellulosic biomass can produce inhibitory compounds that are harmful for microorganisms used in the production of biofuels and other chemicals from lignocellulosic sugars.Formate was consumed during growth on acetate and by stationary phase cells, and this was enhanced in the presence of a common aromatic inhibitor of lignocellulosic hydrolysates, 4-hydroxybenzoate.Because of these encouraging results, we believe that A. baylyi ADP1 is a promising candidate for the detoxification of lignocellulosic hydrolysates for bioprocesses.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Bioengineering, Tampere University of Technology, Korkeakoulunkatu 8, Tampere, Finland.

ABSTRACT

Background: Pretreatment of lignocellulosic biomass can produce inhibitory compounds that are harmful for microorganisms used in the production of biofuels and other chemicals from lignocellulosic sugars. Selective inhibitor removal can be achieved with biodetoxification where microorganisms catabolize the inhibitors without consuming the sugars. We engineered the strictly aerobic Acinetobacter baylyi ADP1 for detoxification of lignocellulosic hydrolysates by removing the gene for glucose dehydrogenase, gcd, which catalyzes the first step in its glucose catabolism.

Results: The engineered A. baylyi ADP1 strain was shown to be incapable of consuming the main sugar components of lignocellulosic hydrolysates, i.e., glucose, xylose, and arabinose, but rapidly utilized acetate and formate. Formate was consumed during growth on acetate and by stationary phase cells, and this was enhanced in the presence of a common aromatic inhibitor of lignocellulosic hydrolysates, 4-hydroxybenzoate. The engineered strain tolerated glucose well up to 70 g/l, and the consumption of glucose, xylose, or arabinose was not observed in prolonged cultivations. The engineered strain was applied in removal of oxygen, a gaseous inhibitor of anaerobic fermentations. Co-cultivation with the A. baylyi ADP1 gcd knockout strain under initially aerobic conditions allowed the strictly anaerobic Clostridium butyricum to grow and produce hydrogen (H2) from sugars of the enzymatic rice straw hydrolysate.

Conclusions: We demonstrated that the model organism of bacterial genetics and metabolism, A. baylyi ADP1, could be engineered to be an efficient biodetoxification strain of lignocellulosic hydrolysates. Only one gene knockout was required to completely eliminate sugar consumption and the strain could be used in production of anaerobic conditions for the strictly anaerobic hydrogen producer, C. butyricum. Because of these encouraging results, we believe that A. baylyi ADP1 is a promising candidate for the detoxification of lignocellulosic hydrolysates for bioprocesses.

No MeSH data available.


Related in: MedlinePlus