C. Sso1354 promotes the consumption of longer cellodextrins

Hyperthermoacidophiles for biomass deconvolution: Cellodextrins hydrolysis and transport in an extremophile Sulfolobus solfataricus

*Sreedevi Madhusoodhanan, Landon Peterson, Karl Dana, Dr. Paul BlumNCESR, NUenergy and School of Biological Sciences, University of Nebraska – Lincoln

pblum1@unl.edu

A. A proposed model for the hydrolysis and transport of cellodextrins in Sulfolobus solfataricus

B. Region of duplication in SsoP2 at the genomic environment of Sso1354

1959 1960 1961

1362 1363 1365

1959 1960 1961

1362 1363 13651350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 19581945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511317 7675 1318 1319 1320 1321 1322 1324 1325 1327 1328

1931 1932 1934 1936 1937 1938 1939 19431933 1941 1942

1317 7675 1318 1319 1320 1321 1322 1324 1325 1327 1328

1931 1932 1934 1936 1937 1938 1939 19431933 1941 1942 1959 1960 1961

1362 1363 1365

1959 1960 1961

1362 1363 13651350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 19581945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

1351

1959 1960 1961

1362 1363 1365

1959 1960 1961

1362 1363 13651350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 19581945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 19581945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511350 1352 1353 1354 1355 1356 1357 1359 1360 1361

1945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 19581945 1946 1947 1948 1949 1951 1952 1953 1954 1956 1957 1958

13511317 7675 1318 1319 1320 1321 1322 1324 1325 1327 1328

1931 1932 1934 1936 1937 1938 1939 19431933 1941 1942

1317 7675 1318 1319 1320 1321 1322 1324 1325 1327 1328

1931 1932 1934 1936 1937 1938 1939 19431933 1941 1942

1317 7675 1318 1319 1320 1321 1322 1324 1325 1327 1328

1931 1932 1934 1936 1937 1938 1939 19431933 1941 1942

1317 7675 1318 1319 1320 1321 1322 1324 1325 1327 1328

1931 1932 1934 1936 1937 1938 1939 19431933 1941 1942

Sequencing results showing the deletion region in Sso98/2 Vs SsoP2

MFS (Ssol_2261) – Major facilitator superfamily_1CYC (Ssol_2262) – Cyclase

P2

98/2

Co-ordinates 1132569 – 1226862 ~90kb Co-ordinates 2041280 – 2047854 ~7kb

1303

1305 1318 136

11386

1385

2257

2261ISC1229 ISC1173 IS119022622258

ISC1217

Present in both

Region deleted in 98/2 Region duplicated in P2

Genes unique to 98/2 31 aa protein in 98/2 – 79% identical to Sso1305 from P2

Scale: ~1kb for the 98/2 region ~22.4 kb for the P2 regionThe length of the two regions are drawn to scale but not length of each ORFs

D. Role of intracellular beta glucosidase, putative cellodextrins transporter and Sso1354

F. Sso3053 is essential for cellodextrins transport

A C

B

A B

E. Construction of cellodextrins transporter Sso3053 cassette deletion and complementation in Sso98/2

A

BC

E

D

A

DC

B

Cellulosic ethanol is an emerging solution for biofuels that will make strong contributions to American domestic energy needs. This study focuses on the development of process-compatible enzymes and organisms to convert biomass derived lignocellulose feedstocks into biofuels and value added chemicals. The Crenarchaeote Sulfolobus solfataricus (Sso) is an extreme thermoacidophile and is known to have three GHF12 endoglucanases. The primary focus of the research is on one of the three endoglucanases [Sso1354]. Apart from Sso1354, the other two enzymes Sso2534 and Sso1949 that are reported as endoglucanases turned out to be nonfunctional on longer cellodextrins during the in vivo analysis. The competitive functions of the intracellular beta-glucosidase and the putative cellodextrins transporter in the process of cellodextrins uptake and hydrolysis is also considered in this study.

A. The construction of Sso3053 cassette knockout in Sso98/2 by circular plasmid transformation. B. PCR validation if the Sso3053 deletion using Sso3053 specific internal primers. 1. DNA ladder, 2. no DNA control, Sso98/2, Sso3053 knockout. C. pKlacS+Sso3053 with natural promoter construct for complementation into PBL2028 strain. D. PBL2028 strain genotype showing deletion of genes from Sso3017 through Sso3050 that includes lacS as well as insertion in the Sso3053 gene. E. PCR validation using the Sso3053 internal primers showing an insertion in PBL2028. 1. DNA ladder, no DNA control, Sso98/2, PBL2028.

A The longer cellodextrins consumption pattern of various strains in PBL2092 (Endoglucanase triple knock out) spent media B. HPLC analysis results of the spent of various strains in PBL2092 (Endoglucanase triple knock out) spent 3.

A. Lanes: 1, Glucose; 2, Cellodextrins mix; 3, spent media from Sso 98/2; 4, spent media from Sso P2; 5, spent media from Sso 98/2 complemented by Sso1354; 6, Non inoculated media. B. pKlacS construct with Sso1354+ native promoter insert for complementation into Sso98/2 strain. C. The HPLC analysis results of the spents after 3 cycles of Sso98/2, Sso98/2+Sso1354 and SsoP2 in cellodextrins.

Growth in A. BSG – Glucose minimal media, B. BSM – Maltose minimal media, C. BSp+1:4CD (v/v), D. HPLC analysis results of the spent in BSp+1:4CD (v/v) by the three strains.

Abstract

Extremely thermoacidophilic microbes such as Sulfolobus solfataricus use soluble cellodextrins as sole carbon and energy sources. In this study, endoglucanases and transporters required for this process were identified using a combination of comparative genomics and genetics in assays that coupled substrate utilization to growth. S. solfataricus strain-specific genomic differences indicated that strain 98/2 lacks endoglucanase Sso1354 while two other endoglucanases are shared includingSso1949 and Sso2534. Plasmid-based expression of Sso1354 in strain 98/2 conferred the ability to rapidly hydrolyze longer oligosaccharides including cellohexaose (G6) through cellonanaose (G9). Protein transporters required for cellodextrin uptake were identified through mutagenesis and complementation of an ABC transporter cassette including a putative oligosaccharide binding protein, Sso3053. In addition, Sso3053 ablation compromised growth on glucose while inactivation of the glucose transporter, Sso2847, had no impact. These data demonstrate that Sulfolobus solfataricus has redundant mechanisms for soluble cellodextrin catabolism comprised of both uptake and extracytoplasmic hydrolytic properties.

Introduction

Sso1354 hydrolyzes longer cellodextrins upto G9. Beta glucosidase and cellodextrins transporter assists in the consumption of cellodextrins upto G8.

Conclusions

Sso1354 is present at a duplication region in SsoP2 strainSso1354 is naturally absent in Sso98/2 strainSso1354 helps in the hydrolysis of longer cellodextrinsSso3053 is required for cellodextrins transport and partially required for maldextrins transport in Sso.Sso2847 is not required for glucose transport in Sso.