Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis

Abstract

Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.

FIG. 1.

Activity of the native cellulolytic proteins in Z. mobilis and E. coli strains. Z. mobilis strains ATCC 39676, ZM4, and CP4 and E. coli strain BL21(DE3) were studied. (A) Coomassie blue-stained polyacrylamide gel. (B) Carboxymethyl cellulose (CMC) zymogram. (C) Patched colonies of Z. mobilis and E. coli growing on an RMG-CMC plate, and the same plate with the cells removed and stained with Congo red to reveal areas of CMC degradation.

FIG. 2.

Expression of E1 and GH12 in E. coli strains BL21(DE3) and Rosetta 2. Coomassie blue-stained polyacrylamide gel of protein lysates from E. coli strains BL21(DE3) and Rosetta 2 harboring plasmids pJL101 (E1 lanes), and pJL103 (GH12 lanes) with (+) or without (−) protein induction with 1 mM IPTG.

FIG. 3.

Expression of various E1 constructs in multiple strains of Z. mobilis. Protein lysates from Z. mobilis strains ATCC 39676, CP4, and ZM4 transformed with the plasmids pZB188 (control lanes), pJL113 (P_tac-E1 lanes), p25143 [P_tac-E1 (c/o) lanes], and pJL110 (P_pdc-E1 lanes) were run identically on two independent 12% polyacrylamide gels supplemented with 0.12% carboxymethyl cellulose (CMC). (A) A PVDF membrane stained with amido black to show total protein. (B) Immunoblot probed with an anti-E1 antibody. (C) A CMC zymogram performed on the second of two duplicate polyacrylamide gels to show cellulolytic activity. E1 activity is represented by the top band, and cellulolytic activity endogenous to Z. mobilis can be seen by the bottom band.

FIG. 4.

Expression, solubility analysis, and activity of E1 and GH12 in Z. mobilis strain ATCC 39676. Protein lysates from Z. mobilis strain ATCC 39676 transformed with pZB188 (control [ctrl]), p25144 (GH12 lanes), and p25143 (E1 lanes) were run identically on two independent 12% polyacrylamide gels supplemented with 0.12% carboxymethyl cellulose (CMC). (A) One of the duplicate polyacrylamide gels was stained with Coomassie blue to show total protein. The locations of the GH12 and E1 proteins are indicated by black arrows in the gel. (B) A CMC zymogram performed on the second of two duplicate polyacrylamide gels designed to show cellulolytic activity.

FIG. 5.

Extracellular secretion and subcellular localization of E1 in Z. mobilis strain ATCC 39676. (A) Amido black-stained PVDF membrane showing total (T) and extracellular medium (Ex) protein lysate fractions to show the total protein load. (B) Anti-E1 immunoblot of the membrane in panel A. (C) Amido black-stained PVDF membrane showing total protein load of protein lysates derived from Z. mobilis expressing multiple versions of E1. Cp, cytoplasmic fraction; Pp, periplasmic fraction. (D) Anti-E1 immunoblot of the membrane in panel C. (E) Relative quantification of E1 activity against methylumbelliferyl cellobiopyranoside (MUC) in periplasmic, cytoplasmic, and extracellular fractions. The relative total activity of equivalent whole-cell lysates from the indicated strains is shown below the bar graph to highlight differential expression between the strains.

FIG. 6.

Extracellular secretion and subcellular localization of GH12 in Z. mobilis strain ATCC 39676. Whole-cell protein lysates and periplasmic and cytoplasmic fractions derived from Z. mobilis strain ATCC 39676 transformed with plasmids pZB188 (Control lanes), p25144 (GH12 lanes), and pJL116 (Z130-GH12 lanes) were run identically on two independent 12% polyacrylamide gels supplemented with 0.12% carboxymethyl cellulose (CMC). (A) Coomassie blue-stained gel to show total protein. (B) A CMC zymogram performed on the second of two duplicate polyacrylamide gels designed to show cellulolytic activity. (C) Relative quantification of GH12 activity against methylumbelliferyl cellobiopyranoside (MUC) in periplasmic, cytoplasmic, and extracellular fractions. The relative total activity of equivalent whole-cell lysates from the indicated strains is shown below the bar graph to highlight differential expression between the strains. (D) Z. mobilis strain ATCC 39676 transformed with plasmids pZB188 (Control), p25143 (E1), p25144 (GH12), pJL111 (Z130-E1), pJL112 (Z331-E1), and pJL116 (Z130-GH12) were spotted onto an agar plate containing 2% glucose and 0.12% CMC. After 18 h of anaerobic growth at 30°C, the plates were photographed (top panel), and the cells were washed off. The plate was subsequently stained with 0.2% Congo red, destained with 1 M NaCl, and photographed again to show CMC degradation (bottom panel). (E) Growth curve analysis of Z. mobilis strain ATCC 39676 in RMG medium harboring the following plasmids: pZB188 (control) (closed diamonds), p25143 (E1) (open diamonds), pJL111 (Z130-E1) (closed triangles), pJL112 (Z331-E1) (open triangles), p25144 (GH12) (closed circles), and pJL116 (Z130-GH12) (open circles).