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Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome.

ABSTRACT: Arabinoxylan is a heteropolymeric chain of a ?-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from Clostridium thermocellum contained one or three cohesin modules and was tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a ?-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated d-xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed.

SUBMITTER: Sun J

PROVIDER: S-EPMC3346407 | biostudies-other | 2012 Jun

REPOSITORIES: biostudies-other

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Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome.

Sun Jie J Wen Fei F Si Tong T Xu Jian-He JH Zhao Huimin H

Applied and environmental microbiology 20120323 11

Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/C ...[more]

PMID: 22447594

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Project description:Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, inefficient conversion of glycerol to ethanol by natural Saccharomyces cerevisiae limits its use in these processes. In this study, we have developed an efficient glycerol-converting yeast strain by genetically modifying the oxidation of cytosolic NAD (NADH) by an O2-dependent dynamic shuttle and abolishing both glycerol phosphorylation and biosynthesis in S. cerevisiae strain D452-2, as well as by vigorous expression of whole genes in the dihydroxyacetone (DHA) pathway (Candida utilis glycerol facilitator, Ogataea polymorpha glycerol dehydrogenase, endogenous dihydroxyacetone kinase, and triosephosphate isomerase). The engineered strain showed conversion efficiencies (CE) up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with a production rate of >1 g liter-1 h-1 when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain converted a mixture of glycerol and glucose into bioethanol (>86 g/liter) with 92.8% CE. To the best of our knowledge, this is the highest reported titer of bioethanol produced from glycerol and glucose. Notably, we developed a glycerol-utilizing transformant from a parent strain which cannot utilize glycerol as a sole carbon source. The developed strain converted glycerol to ethanol with a productivity of 0.44 g liter-1 h-1 on minimal medium under semiaerobic conditions. Our findings will promote the utilization of glycerol in eco-friendly biorefineries and integrate bioethanol and plant oil industries. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Coconversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, low conversion efficiency from glycerol and sugars limits the industrial application of this process. Therefore, the generation of an efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Hence, metabolic flux control in yeast grown on glycerol will lead to the generation of cell factories that produce chemicals, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.

Project description:BackgroundMobile Genetic Elements (MGEs) are selfish DNA integrated in the genomes. Their detection is mainly based on consensus-like searches by scanning the investigated genome against the sequence of an already identified MGE. Mobilomics aims at discovering all the MGEs in a genome and understanding their dynamic behavior: The data for this kind of investigation can be provided by comparative genomics of closely related organisms. The amount of data thus involved requires a strong computational effort, which should be alleviated.ResultsOur approach proposes to exploit the high similarity among homologous chromosomes of different strains of the same species, following a progressive comparative genomics philosophy. We introduce a software tool based on our new fast algorithm, called regender, which is able to identify the conserved regions between chromosomes. Our case study is represented by a unique recently available dataset of 39 different strains of S.cerevisiae, which regender is able to compare in few minutes. By exploring the non-conserved regions, where MGEs are mainly retrotransposons called Tys, and marking the candidate Tys based on their length, we are able to locate a priori and automatically all the already known Tys and map all the putative Tys in all the strains. The remaining putative mobile elements (PMEs) emerging from this intra-specific comparison are sharp markers of inter-specific evolution: indeed, many events of non-conservation among different yeast strains correspond to PMEs. A clustering based on the presence/absence of the candidate Tys in the strains suggests an evolutionary interconnection that is very similar to classic phylogenetic trees based on SNPs analysis, even though it is computed without using phylogenetic information.ConclusionsThe case study indicates that the proposed methodology brings two major advantages: (a) it does not require any template sequence for the wanted MGEs and (b) it can be applied to infer MGEs also for low coverage genomes with unresolved bases, where traditional approaches are largely ineffective.

Dataset Information

Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome.

Publications

Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome.

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