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Transcriptional analysis of Amorphotheca resinae ZN1 on biological degradation of furfural and 5-hydroxymethylfurfural derived from lignocellulose pretreatment.

Wang X, Gao Q, Bao J - Biotechnol Biofuels (2015)

Bottom Line: During the detoxification process, A. resinae ZN1 firstly reduced furfural or HMF into furfuryl alcohol or HMF alcohol, and then oxidized into furoic acid or HMF acid through furan aldehyde as the intermediate at low concentration level.Two Zn-dependent alcohol dehydrogenase genes and five AKR/ARI genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols.The genes responsible for the furfural and HMF degradation to the corresponding alcohols and acids in A. resinae ZN1 were identified based on the analysis of the genome annotation, the gene transcription data and the inhibitor conversion results.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237 China.

ABSTRACT

Background: Furfural and 5-hydroxymethylfurfural (HMF) are the two major inhibitor compounds generated from lignocellulose pretreatment, especially for dilute acid, steam explosion, neutral hot water pretreatment methods. The two inhibitors severely inhibit the cell growth and metabolism of fermenting strains in the consequent bioconversion step. The biodetoxification strain Amorphotheca resinae ZN1 has demonstrated its extraordinary capacity of fast and complete degradation of furfural and HMF into corresponding alcohol and acid forms. The elucidation of degradation metabolism of A. resinae ZN1 at molecular level will facilitate the detoxification of the pretreated lignocellulose biomass and provide the metabolic pathway information for more powerful biodetoxification strain development.

Results: Amorphotheca resinae ZN1 was able to use furfural or HMF as the sole carbon source for cell growth. During the detoxification process, A. resinae ZN1 firstly reduced furfural or HMF into furfuryl alcohol or HMF alcohol, and then oxidized into furoic acid or HMF acid through furan aldehyde as the intermediate at low concentration level. The cell mass measurement suggested that furfural was more toxic to A. resinae ZN1 than HMF. In order to identify the degradation mechanism of A. resinae ZN1, transcription levels of 137 putative genes involved in the degradation of furfural and HMF in A. resinae ZN1 were investigated using the real-time quantitative PCR (qRT-PCR) method under the stress of furfural and HMF, as well as the stress of their secondary metabolites, furfuryl alcohol and HMF alcohol. Two Zn-dependent alcohol dehydrogenase genes and five AKR/ARI genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols. For the conversion of the two furan alcohols to the corresponding acids, three propanol-preferring alcohol dehydrogenase genes, one NAD(P)(+)-depending aldehyde dehydrogenase gene, or two oxidase genes with free oxygen as the substrate were identified under aerobic condition.

Conclusions: The genes responsible for the furfural and HMF degradation to the corresponding alcohols and acids in A. resinae ZN1 were identified based on the analysis of the genome annotation, the gene transcription data and the inhibitor conversion results. These genetic resources provided the important information for understanding the mechanism of furfural and HMF degradation and modification of high tolerant strains used for biorefinery processing.

No MeSH data available.


Related in: MedlinePlus

Metabolic pathways of furfural (A) and HMF (B) degradation in A. resinae ZN1. Solid boxes (a, b) were based on the previous experimental phenomena [24]. Blue arrows were adapted from Trudgill [18] and Koopman et al. [20]. ACC, acceptor, either oxidized (ox) or reduced (red). ADH alcohol dehydrogenase, AKR aldo–keto reductase, ARI aldehyde reductse, ALDH aldehyde dehydrogenase
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Fig1: Metabolic pathways of furfural (A) and HMF (B) degradation in A. resinae ZN1. Solid boxes (a, b) were based on the previous experimental phenomena [24]. Blue arrows were adapted from Trudgill [18] and Koopman et al. [20]. ACC, acceptor, either oxidized (ox) or reduced (red). ADH alcohol dehydrogenase, AKR aldo–keto reductase, ARI aldehyde reductse, ALDH aldehyde dehydrogenase

Mentions: In recent few years, a biological detoxification method using specific microorganisms to convert furfural and HMF into non-toxic substances was proposed and the method demonstrated the unique advantages such as mild condition, low energy demand and no waste water generation [11–13]. Many biodetoxification microorganisms have been discovered and the biodetoxification mechanisms were extensively investigated [14–17]. Trudgill [18] proposed a putative degradation pathway of furfural in Pseudomonas putida F2 in 1969, and then verified by Koenig and Andreesen [19] and Koopman et al. [20]. Koopman et al. [20] extended the pathway to HMF in Cupriavidus basilensis HMF14. Zhang et al. isolated a kerosene fungus Amorphotheca resinae ZN1 [21] with fast and complete biodetoxification of almost all toxic inhibitors and has been practically applied for the high performance of ethanol, lipid, and lactic acid production [21–23]. The degradation performance of furfural and HMF by A. resinae ZN1 was investigated and a hypothesized metabolic pathway was illustrated in Fig. 1 in the previous studies [21, 24]. Furfural is quickly reduced to furfuryl alcohol, then re-oxidized into its aldehyde form (furfural) again but at a much lower and harmless concentration then oxidized into its acid form (furoic acid) under aerobic condition; furoic acid is subsequently ligated coenzyme-A into furoyl-CoA, hydroxylated into α-oxoglutaric acid and CoA, and finally α-oxoglutaric acid is metabolized via tricarboxylic acid cycle (TCA) (Fig. 1a). Similar to furfural, HMF is quickly reduced to HMF alcohol, re-oxidized to the aldehyde (HMF) under aerobic condition, then oxidized to its monocarboxylic acid (5-hydroxymethyl-furoic acid, HMF acid) and dicarboxylic acid (2, 5-furandicarboxylic acid, FDCA); FDCA is converted into furoic acid via a decarboxylation reaction, and joins into furfural catabolism (Fig. 1b). The first several steps (Fig. 1a, b) from furfural (HMF) to furfuryl alcohol (HMF alcohol) or furoic acid (HMF acid) demonstrated the primary detoxification function in A. resinae ZN1 because the two metabolites are less toxic or even non-toxic to fermenting strains [25, 26].Fig. 1


Transcriptional analysis of Amorphotheca resinae ZN1 on biological degradation of furfural and 5-hydroxymethylfurfural derived from lignocellulose pretreatment.

Wang X, Gao Q, Bao J - Biotechnol Biofuels (2015)

Metabolic pathways of furfural (A) and HMF (B) degradation in A. resinae ZN1. Solid boxes (a, b) were based on the previous experimental phenomena [24]. Blue arrows were adapted from Trudgill [18] and Koopman et al. [20]. ACC, acceptor, either oxidized (ox) or reduced (red). ADH alcohol dehydrogenase, AKR aldo–keto reductase, ARI aldehyde reductse, ALDH aldehyde dehydrogenase
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559888&req=5

Fig1: Metabolic pathways of furfural (A) and HMF (B) degradation in A. resinae ZN1. Solid boxes (a, b) were based on the previous experimental phenomena [24]. Blue arrows were adapted from Trudgill [18] and Koopman et al. [20]. ACC, acceptor, either oxidized (ox) or reduced (red). ADH alcohol dehydrogenase, AKR aldo–keto reductase, ARI aldehyde reductse, ALDH aldehyde dehydrogenase
Mentions: In recent few years, a biological detoxification method using specific microorganisms to convert furfural and HMF into non-toxic substances was proposed and the method demonstrated the unique advantages such as mild condition, low energy demand and no waste water generation [11–13]. Many biodetoxification microorganisms have been discovered and the biodetoxification mechanisms were extensively investigated [14–17]. Trudgill [18] proposed a putative degradation pathway of furfural in Pseudomonas putida F2 in 1969, and then verified by Koenig and Andreesen [19] and Koopman et al. [20]. Koopman et al. [20] extended the pathway to HMF in Cupriavidus basilensis HMF14. Zhang et al. isolated a kerosene fungus Amorphotheca resinae ZN1 [21] with fast and complete biodetoxification of almost all toxic inhibitors and has been practically applied for the high performance of ethanol, lipid, and lactic acid production [21–23]. The degradation performance of furfural and HMF by A. resinae ZN1 was investigated and a hypothesized metabolic pathway was illustrated in Fig. 1 in the previous studies [21, 24]. Furfural is quickly reduced to furfuryl alcohol, then re-oxidized into its aldehyde form (furfural) again but at a much lower and harmless concentration then oxidized into its acid form (furoic acid) under aerobic condition; furoic acid is subsequently ligated coenzyme-A into furoyl-CoA, hydroxylated into α-oxoglutaric acid and CoA, and finally α-oxoglutaric acid is metabolized via tricarboxylic acid cycle (TCA) (Fig. 1a). Similar to furfural, HMF is quickly reduced to HMF alcohol, re-oxidized to the aldehyde (HMF) under aerobic condition, then oxidized to its monocarboxylic acid (5-hydroxymethyl-furoic acid, HMF acid) and dicarboxylic acid (2, 5-furandicarboxylic acid, FDCA); FDCA is converted into furoic acid via a decarboxylation reaction, and joins into furfural catabolism (Fig. 1b). The first several steps (Fig. 1a, b) from furfural (HMF) to furfuryl alcohol (HMF alcohol) or furoic acid (HMF acid) demonstrated the primary detoxification function in A. resinae ZN1 because the two metabolites are less toxic or even non-toxic to fermenting strains [25, 26].Fig. 1

Bottom Line: During the detoxification process, A. resinae ZN1 firstly reduced furfural or HMF into furfuryl alcohol or HMF alcohol, and then oxidized into furoic acid or HMF acid through furan aldehyde as the intermediate at low concentration level.Two Zn-dependent alcohol dehydrogenase genes and five AKR/ARI genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols.The genes responsible for the furfural and HMF degradation to the corresponding alcohols and acids in A. resinae ZN1 were identified based on the analysis of the genome annotation, the gene transcription data and the inhibitor conversion results.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237 China.

ABSTRACT

Background: Furfural and 5-hydroxymethylfurfural (HMF) are the two major inhibitor compounds generated from lignocellulose pretreatment, especially for dilute acid, steam explosion, neutral hot water pretreatment methods. The two inhibitors severely inhibit the cell growth and metabolism of fermenting strains in the consequent bioconversion step. The biodetoxification strain Amorphotheca resinae ZN1 has demonstrated its extraordinary capacity of fast and complete degradation of furfural and HMF into corresponding alcohol and acid forms. The elucidation of degradation metabolism of A. resinae ZN1 at molecular level will facilitate the detoxification of the pretreated lignocellulose biomass and provide the metabolic pathway information for more powerful biodetoxification strain development.

Results: Amorphotheca resinae ZN1 was able to use furfural or HMF as the sole carbon source for cell growth. During the detoxification process, A. resinae ZN1 firstly reduced furfural or HMF into furfuryl alcohol or HMF alcohol, and then oxidized into furoic acid or HMF acid through furan aldehyde as the intermediate at low concentration level. The cell mass measurement suggested that furfural was more toxic to A. resinae ZN1 than HMF. In order to identify the degradation mechanism of A. resinae ZN1, transcription levels of 137 putative genes involved in the degradation of furfural and HMF in A. resinae ZN1 were investigated using the real-time quantitative PCR (qRT-PCR) method under the stress of furfural and HMF, as well as the stress of their secondary metabolites, furfuryl alcohol and HMF alcohol. Two Zn-dependent alcohol dehydrogenase genes and five AKR/ARI genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols. For the conversion of the two furan alcohols to the corresponding acids, three propanol-preferring alcohol dehydrogenase genes, one NAD(P)(+)-depending aldehyde dehydrogenase gene, or two oxidase genes with free oxygen as the substrate were identified under aerobic condition.

Conclusions: The genes responsible for the furfural and HMF degradation to the corresponding alcohols and acids in A. resinae ZN1 were identified based on the analysis of the genome annotation, the gene transcription data and the inhibitor conversion results. These genetic resources provided the important information for understanding the mechanism of furfural and HMF degradation and modification of high tolerant strains used for biorefinery processing.

No MeSH data available.


Related in: MedlinePlus