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

Abstract

BackgroundMany 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.ResultsTo 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.ConclusionSeven 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.

Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824

“…To date, most studies have focused on the production of ethanol (for an overview of developments in various organisms, see recent reviews by Olson et al [2] and la Grange et al [3]). Recent examples of engineering for ethanol production via CBP via the native method include the production of 38.1 g/l ethanol from 92.1 g/l Avicel via a co-culture of modified Clostridium thermocellum and Thermoanaerobacterium saccharolyticum [4]; the ‘recombinant’ approach has recently generated strains of Saccharomyces cerevisiae [5] and Kluyveromyces marxianus [6] able to ferment hot water treated rice straw and Avicel respectively, although final ethanol titres were still low.…”

1928 - Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain

“…gRNA integrated in Cas9-carrying yeast strain by PGASO. The six gRNA cassettes were together integrated into the selected Cas9-carrying strains using a modified PGASO technique 15,16 . We retained their own SNR52 promoters and SUP4 terminators in these gRNA cassettes.…”

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

“…These six multiple genes were assembled into the genome of the host using PGASO technology. Experimental results indicated that the developed strain KR7 contains five recombinantly expressed heterologous cellulase genes and that strain could transform crystalline cellulose into ethanol (Chang et al, 2013).…”