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. 2022 May 28;32(5):663-671.
doi: 10.4014/jmb.2202.02016.

Engineering of Sulfolobus acidocaldarius for Hemicellulosic Biomass Utilization

Areum Lee  1 , Hyeju Jin  1 , Jaeho Cha  2   3
Affiliations

Engineering of Sulfolobus acidocaldarius for Hemicellulosic Biomass Utilization

Areum Lee et al. J Microbiol Biotechnol. .

Abstract

The saccharification of cellulose and hemicellulose is essential for utilizing lignocellulosic biomass as a biofuel. While cellulose is composed of glucose only, hemicelluloses are composed of diverse sugars such as xylose, arabinose, glucose, and galactose. Sulfolobus acidocaldarius is a good potential candidate for biofuel production using hemicellulose as this archaeon simultaneously utilizes various sugars. However, S. acidocaldarius has to be manipulated because the enzyme that breaks down hemicellulose is not present in this species. Here, we engineered S. acidocaldarius to utilize xylan as a carbon source by introducing xylanase and β-xylosidase. Heterologous expression of β-xylosidase enhanced the organism's degradability and utilization of xylooligosaccharides (XOS), but the mutant still failed to grow when xylan was provided as a carbon source. S. acidocaldarius exhibited the ability to degrade xylan into XOS when xylanase was introduced, but no further degradation proceeded after this sole reaction. Following cell growth and enzyme reaction, S. acidocaldarius successfully utilized xylan in the synergy between xylanase and β-xylosidase.

Keywords: Hyperthermophiles; Sulfolobus acidocaldarius; carbohydrateactive enzyme; hemicellulose.

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Conflict of interest statement

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Figures

Fig. 1
Fig. 1. S. acidocaldarius MW001 cannot utilize XOS and xylan.
(A) Growth of MW001 in the presence of hemicellulosic biomass. For the basal growth of MW001 strain, 0.2% NZ-amine and 0.02% dextrin were added to Brock’s medium. Various types of hemicellulosic biomass, including xylose (open-circle), XOS (closed-triangle), and xylan (opentriangle), were supplemented to the basal media at a concentration of 0.2% or no additive sugars were provided (closed-circle). MW001 strain was inoculated with an initial OD of 0.01, which was measured every 6 h. All experiments were conducted with triplicate and upper and lower side error bars representing maximum and minimum OD, respectively. (B) TLC chromatogram of XOS transported into S. acidocaldarius MW001. Cells were incubated with 1% XOS, and XOS transported into the cells was visualized by TLC. Xylose and XOS (X2–X5) were used as standards. S, sugar standard; 1, cell lysate after the 0 h incubation; 2, after 12 h; 3, after 48 h.
Fig. 2
Fig. 2. Expression of β-xylosidase enables S. acidocaldarius to utilize XOS.
(A) Growth of MW001/3032 in the presence of XOS and xylan. MW001/3032 strain, containing β-xylosidase, was grown in Brock’s medium supplemented with 0.2% NZ-amine and 0.02% dextrin. To compare cell growth patterns when an additional carbon source was provided, xylose (open-circle), XOS (closed-triangle), and xylan (open-triangle) or no sugar (closed-circle) was supplemented in the culture medium at a concentration of 0.2%. The initial OD of MW001/3032 was 0.01, and cell growth was measured at 6-h intervals. Error bars represent the maximum and minimum values of triplicates. (B) TLC chromatogram of XOS and xylan hydrolysis by MW001/3032. The cell-free extracts from MW001 and MW001/3032 were incubated with 5% XOS or xylan, and the reaction products were visualized by TLC. Xylose and XOS (X2–X5) served as standards. S, sugar standard; 1, no enzyme treated; 2, MW001 treated; 3, MW001/3032 treated. (C and D) Temperature and pH profiles of recombinant β-xylosidase. The recombinant β-xylosidase was purified from MW001/3032. The enzymatic reaction was conducted with 1 mM pNPX and 1 μg of the enzyme solution for 10 min in 50 mM of each buffer and at various temperatures, as described in the Materials and Methods section.
Fig. 3
Fig. 3. Expression of xylanase alone does not enable S. acidocaldarius to utilize xylan.
(A) Growth of MW001/ 1354 in the presence of XOS and xylan. The growth pattern of MW001/1354 grown without sugar supplementation (closedcircle) or with supplementation of sugars, such as xylose (open-circle), XOS (closed-triangle), and xylan (open-triangle), was compared. Brock’s medium was supplemented with 0.2% NZ-amine and 0.02% dextrin. Initial inoculation of MW001/1354 was conducted at an OD of 0.01, and cell growth was measured at 6-h intervals. Experiments were conducted in triplicates, and error bars showed the maximum and minimum OD. (B) TLC chromatogram of xylan hydrolysis by MW001/1354. The cellfree extracts from MW001 and MW001/1354 were incubated with 5% xylan, and the reaction products were visualized by TLC. Xylose and XOS (X2–X5) served as standards. S, sugar standard; 1, no enzyme treated; 2, MW001 treated; 3, MW001/1354 treated. (C and D) Temperature and pH profiles of recombinant xylanase. The recombinant xylanase was partially purified from MW001/1354. The enzyme reaction was conducted with 0.1% RBB-xylan and 10 μg of the enzyme solution for 30 min in 50 mM of each buffer and at various temperatures, as described in the Materials and Methods section.
Fig. 4
Fig. 4. Synergistic activity of xylanase and β-xylosidase in S. acidocaldarius is required to utilize xylan.
(A) Growth of LAR1-1 in the presence of hemicellulosic sugars. Each medium contains 0.2% NZ-amine and 0.02% dextrin. Cell growth in 0.2% xylose (open-circle), XOS (closed-triangle), xylan (open-triangle), or without sugar (closed-circle) was compared. Error bars represent the maximum and minimum OD values of triplicate experiment, respectively. (B) The synergetic action of β-xylosidase and xylanase toward hemicellulosic biomass. The membrane fraction of MW001/1354 and cell-free extracts from MW001/3032 were incubated with 5% XOS or xylan, and the reaction products were visualized by TLC. Lane S, xylose and XOS (X2–X5) standard; Lane C, no enzyme treated; Lane 1, MW001/1354 treated; Lane 2, MW001/3032 treated; Lane 3, LAR1-1 treated.
Fig. 5
Fig. 5. Growth of LAR1-1 in the presence of cellulosic biomass.
Each medium contains 0.2% NZ-amine and 0.02% dextrin. Cell growth in 0.2% glucose (closed-circle), COS (open-triangle), and CMC (closed-triangle) was compared. Error bars represent the maximum and minimum OD values of triplicate experiment, respectively.
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