Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

Abstract

Background: Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed.

Results: To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called "promoter-based gene assembly and simultaneous overexpression" (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.

Conclusion: Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.

Figure 1

The cellulose utilization pathways and cellulase cocktail formulation. (A) The fungal cellulose utilization systems in yeast during simultaneous saccharification and fermentation of cellulose. (B) The filter paper activity (FPA) assay of potential enzyme synergistic effects with the crude enzyme of KR5. Several different commercial cellulases, including EG (EglA and EGIII), CBH (CBHI and CBHII) and BGL (purified NpaBGS), were used for cocktails with the supernatants of KR5 by mixing equivalent proportions of volume individually. One unit of FPA is defined as one μmol reducing sugar released from filter paper in one minute. The cellodextrin transport pathway includes a cellodextrin transporter (CDT) and intracellular cellulases (EG, CBH, and BGL). The sugar catabolism pathway present in yeasts includes hexose transporters (HXT) and lactose permease (LP). In SSF, pretreatment and extracellular cellulase cocktail process may both be used.

Figure 2

The growth curve and fluorescence microscopy photograph of the engineered yeast strains. (A) Three recombinant strains KY3-NpaBGS-CDT, KR5 and KR7 that expressed green fluorescent protein in cytoplasm. KR5 was employed as a control. (B) and (C) the CDT gene fusion with GFP gene was expressed on the cell membrane of KY3-NpaBGS-CDT and KR7, respectively. The growth curve was conducted by the engineered yeast strains using cellobiose (D) and cellodextrin (E) as the solo substrate, respectively.

Figure 3

Genomic integration of seven gene cassettes in KR7. Each of the seven gene cassettes contains an independent promoter, the alpha factor, a gene coding region, a terminator, and a 55 bp fragment homologous to its neighboring cassette. (A) The gene cassettes assembled in the predesignated order, kanMX, egIII, cbhI, eglA, cbhII, cdtI-gfp, and npabgs, and are shown in red, orange, yellow, green, blue, dark blue, and purple, respectively. (B) The seven gene cassettes were amplified by PCR for transformation with seven primer pairs and the PCR products resulted in seven specific amplicons: 1: kanMX, 2: egIII, 3: cbhI, 4: eglA, 5: cbhII, 6: cdtI-gfp, and 7: npabgs. (C) The order of the gene cassettes was confirmed by PCR with seven internal primer pairs and the PCR products resulted in eight specific amplicons: 1: Lac4-kanMX, 2: kanMX-egIII, 3: egIII-cbhI, 4: cbhI-eglA, 5: eglA-cbhII, 6: cbhII-cdtI-gfp, 7: cdtI-gfp-npabgs, and 8: npabgs-Lac4.

Figure 4

Quantitative PCR analysis of the seven gene cassettes in KR7. (A) Relative copy numbers of inserted genes. (B) Relative mRNA levels of cellulase genes inserted in KR7. The relative ratios of the seven genes and their transcripts are shown in comparison to the endogenous alg9 gene in KR7 at 40°C in YPAD culture. The promoter names of the individual genes were given in the brackets.

Figure 5

Cellulolytic enzyme assays of engineered yeasts. (A) CBH relative activity with PASC as the sole substrate. (B) EG relative activity with CMC as the sole substrate. (C) BGL relative activity assay with pNPG as the sole substrate. (D) Total specific cellulase activity assay with filter paper as the sole carbon substrate. The samples collected from KY3, KR5 and KR7 cultures were estimated using the same protein concentration for enzyme reaction at 40°C. One unit of FPA is defined as one mmol reducing sugar releasing from filter paper in one minute. *: P < 0.05 (significant), **: P < 0.01; ***: P < 0.001; N.S., non-significant.

Figure 6

The cellulosic ethanol production assays. KY3, KR5, and KR7 were inoculated with OD of 20 cells, and a 10% avicel was added into 10 ml YP medium as the solo carbon source. The semi-anaerobic batch cultures were performed at 40°C with 250 rpm shaking.