| The food crops were used as primary raw material for traditional ethanol fermentation. In order to save the cost, using lignocellulose as renewable biomass resources and fully transforming the components into fuel ethanol has important economic and social significance. Lignocellulose mainly consists of cellulose, hemicellulose and lignin and the xylose is mainly monosaccharides component of hemicellulose. However, the traditional Saccharomyces cerevisiae used for ethanol fermentation can use glucose well, but cannot use xylose and arabinose. Pentose effective utilization is the necessary foundation for industrialization of biomass resources to ethanol. Therefore, the focus of the study is introduction of the pentose metabolic pathway in Saccharomyces cerevisiae for co-fermentation of hexose and pentose. In this work, the efficient integrated/replicative plasmid, the optimal promoter and optimal pentose pathway genes were used as strategy to unfold experiments, and the pentose metabolic pathways were introduced into Saccharomyces cerevisiae for constructing xylose and arabinose utilization engineering strain.This is about the study of the autonomously replicating sequence(ARS). Four different ARSs(ARS304, ARS315, ARS735, and ARS1512) were introduced into Saccharomyces cerevisiae integrated vector PNTS2 K. It can be concluded that all of the four ARSs were able to work with ARS315 having the highest replicate ability in S. cerevisiae after electroporation. Consequently, ARS315 can bepotentially used for high expression and stable recombinant strain construction and metabolic engineering of S. cerevisiae.This is research on optimal promoter. Expression plasmids were constructed using three constitutive promoters(TEF1p, PGK1 p, TDH3p) for xylose isomerase gene expression, and three Saccharomyces cerevisiae expression plasmid(pJFE11, pPY1, pTP2) preserved in our laboratory were used for the skeleton. It can be identified promoter PGK1 p is more suitable for out host expressing xylose isomerase and producing ethanol through the comparasion of transformants growth, xylose consumption rate and ethanol productivity. It is a good foundation for stability xylose isomerase expression in industrial Saccharomyces cerevisiea.This is the study on expression of arabinose metabolic pathway. Three genes araA, araB, araD of Lactobacillus plantarum encoding arabinose consumption pathway were expressed in Saccharomyces cerevisiae through different ways. After the comparasion of three co-expression method: expression by multiply-plasmids with multiply-promoters; expression by one plasmid with multiply-promoters; expression by one plasmids with one promoter. We found that plant the genes araA, araB, araD of Lactobacillus plantarum encoding arabinose consumption pathway were functional well under the single promoter TEF1. And this expression frame can be easily integrated into yeast chromosome. Therefore, this expression method is a good foundation for the later integration expression.The construction of engineering strain of Saccharomyces cerevisiae for xylose, arabinose utilization. Using Saccharomyces cerevisiae Juk36 a as host, and using integrated/ replicative plasmid pNTS2 KA as skeleton, a mix expression cassettes containing xylose isomerase(XI) cassettes and arabinose pathway cassettes are assembled and integrated in the genome of Juk36 a strain. The best functional strain 36aS1 was selected for xylose and arabinose utilization. The strain 36aS1 exhibited a maximum specific growth rate of 0.32h-1 and 0.22h-1 in synthetic medium with D-xylose or L-arabinose as sole carbon source. For the strain 36aS1, in glucose and xylose co-fermentation, it can completely consumed 180 g/L sugars to produce up to 75 g/L ethanol and almost no xylitol and acetic acid by-products produced. The ethanol yield was 0.42, which is 82% of the theoretical value. This breakthrough build a technology platform for the construction of engineering strains which can ferment total sugar group in lignocellulose hydrolysate. |
PDF Full Text Request