C. Sso1354 promotes the consumption of longer cellodextrins

1. Co-ordinates 1132569 – 1226862 ~90kb 1936 1936 1936 1936 1936 1936 1934 1934 1934 1934 1934 1934 1937 1937 1937 1937 1937 1937 1933 1933 1933 1933 1933 1933 1931 1931 1931 1931 1931 1931 1932 1932 1932 1932 1932 1932 1945 1945 1945 1945 1945 1945 1945 1945 1945 1945 1945 1945 1945 1938 1938 1938 1938 1938 1938 1939 1939 1939 1939 1939 1939 1943 1943 1943 1943 1943 1943 1941 1941 1941 1941 1941 1941 1942 1942 1942 1942 1942 1942 1948 1948 1948 1948 1948 1948 1948 1948 1948 1948 1948 1948 1948 1957 1957 1957 1957 1957 1957 1957 1957 1957 1957 1957 1957 1957 1946 1946 1946 1946 1946 1946 1946 1946 1946 1946 1946 1946 1946 1949 1949 1949 1949 1949 1949 1951 1951 1951 1951 1951 1951 1952 1952 1952 1952 1952 1952 1949 1949 1949 1949 1949 1949 1949 1953 1953 1953 1953 1953 1953 1951 1951 1951 1951 1951 1951 1951 1954 1954 1954 1954 1954 1954 1952 1952 1952 1952 1952 1952 1952 1953 1953 1953 1953 1953 1953 1953 1954 1954 1954 1954 1954 1954 1954 1956 1956 1956 1956 1956 1956 1956 1956 1956 1956 1956 1956 1956 1959 1959 1959 1959 1959 1959 1960 1960 1960 1960 1960 1960 1961 1961 1961 1961 1961 1961 1947 1947 1947 1947 1947 1947 1947 1947 1947 1947 1947 1947 1947 1958 1958 1958 1958 1958 1958 1958 1958 1958 1958 1958 1958 1958 1317 1317 1317 1317 1317 1317 7675 7675 7675 7675 7675 7675 1362 1362 1362 1362 1362 1362 1363 1363 1363 1363 1363 1363 1321 1321 1321 1321 1321 1321 1322 1322 1322 1322 1322 1322 1359 1359 1359 1359 1359 1359 1359 1359 1365 1365 1365 1365 1365 1365 1320 1320 1320 1320 1320 1320 1351 1351 1351 1351 1351 1351 1351 1351 1318 1318 1318 1318 1318 1318 1319 1319 1319 1319 1319 1319 1324 1324 1324 1324 1324 1324 1325 1325 1325 1325 1325 1325 1328 1328 1328 1328 1328 1328 1350 1350 1350 1350 1352 1352 1352 1352 1350 1350 1350 1350 1353 1353 1353 1353 1352 1352 1352 1352 1355 1355 1355 1355 1353 1353 1353 1353 1356 1356 1356 1356 1357 1357 1357 1357 1355 1355 1355 1355 1356 1356 1356 1356 1360 1360 1360 1360 1357 1357 1357 1357 1361 1361 1361 1361 1360 1360 1360 1360 1361 1361 1361 1361 1327 1327 1327 1327 1327 1327 1354 1354 1354 1354 1354 1354 1354 1354 Hyperthermoacidophiles for biomass deconvolution: Cellodextrins hydrolysis and transport in an extremophile Sulfolobus solfataricus P2 1305 1303 1318 1361 1386 1385 98/2 Co-ordinates 2041280 – 2047854 ~7kb IS1190 ISC1229 2258 ISC1173 2261 2262 2257 ISC1217 Scale: ~1kb for the 98/2 region ~22.4 kb for the P2 region The length of the two regions are drawn to scale but not length of each ORFs Genes unique to 98/2 Present in both 31 aa protein in 98/2 – 79% identical to Sso1305 from P2 Region deleted in 98/2 Region duplicated in P2 *Sreedevi Madhusoodhanan, Landon Peterson, Karl Dana, Dr. Paul Blum NCESR, NUenergy and School of Biological Sciences, University of Nebraska – Lincoln MFS (Ssol_2261) – Major facilitator superfamily_1 CYC (Ssol_2262) – Cyclase pblum1@unl.edu B. Region of duplication in SsoP2 at the genomic environment of Sso1354 E. Construction of cellodextrins transporter Sso3053 cassette deletion and complementation in Sso98/2 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. A 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. Sequencing results showing the deletion region in Sso98/2 Vs SsoP2 B C D E F.Sso3053 is essential for cellodextrins transport C. Sso1354 promotes the consumption of longer cellodextrins A B A C 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. Introduction 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. B C D D. Role of intracellular beta glucosidase, putative cellodextrins transporter and Sso1354 A. A proposed model for the hydrolysis and transport of cellodextrins in Sulfolobus solfataricus 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. B A Conclusions Sso1354 is present at a duplication region in SsoP2 strain Sso1354 is naturally absent in Sso98/2 strain Sso1354 helps in the hydrolysis of longer cellodextrins Sso3053 is required for cellodextrins transport and partially required for maldextrins transport in Sso. Sso2847 is not required for glucose transport in Sso. 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. Sso1354 hydrolyzes longer cellodextrins upto G9. Beta glucosidase and cellodextrins transporter assists in the consumption of cellodextrins upto G8.