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Metabolic Engineering yeast using xylose fermentation for producing ethanol

A technology for engineering bacteria and ethanol production, applied in the biological field, can solve the problems of depletion of reduced coenzyme II, accumulation of reduced coenzyme I, low conversion rate and the like

Inactive Publication Date: 2009-06-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

During xylose fermentation, the xylose reductase derived from Pichia pastoris preferentially utilizes reduced coenzyme II while xylitol dehydrogenase can only specifically utilize oxidized coenzyme I in recombinant S. cerevisiae cells, resulting in the depletion of reduced coenzyme II and the accumulation of reduced coenzyme I
The imbalance of redox coenzymes has caused the low utilization rate of xylose and the accumulation of xylitol. At present, when the engineering bacteria constructed at home and abroad ferment xylose to produce ethanol, the conversion rate of xylose to ethanol is low (Gnansounou et al., Bioresource Technology , 96:985-1002, 2005), this problem has become the key technology of biomass fermentation to produce ethanol

Method used

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  • Metabolic Engineering yeast using xylose fermentation for producing ethanol

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Embodiment 1

[0014] Example 1 Construction of Saccharomyces cerevisiae (Saccharomyces cerevisiae) E33 engineering bacteria

[0015] Saccharomyces cerevisiae W303-1Bα (Pearce, A.K. et al.: Micobiology 147, 391-401., 2001) was used as the starting strain for constructing engineering bacteria. The strains used in the construction process also include Pichia pastoris CBS 6054 (Wahlbom, C.F. et al.: FEMS Yeast Res 3, 319-26., 2003), Candida maltosa (Candida maltosa) XU316 (C.Guo, etc.: J. Appl. Microb. 101, 139-150, 2006) and Escherichia coli (E. coli) JM109 (Invitrogene). Genomes of yeast Pichia CBS 6054, Saccharomyces cerevisiae W303-1Bα, Candida maltosa XU316 and Escherichia coli JM109 were extracted according to standard methods (Ausubel, F.M., et al.: Current protocols in molecular biology. Wiley, Toronto, 1987) DNA. Using Pichia pastoris CBS 6054 genomic DNA as a template, using primers

[0016] Pxdhs (5′-GCAGGATCCATGACTGCTAACCCTTCCTTG-3′) and

[0017] Pxdha (5′-GCACTCGAGTTACTCAGGG CC...

Embodiment 2

[0030] Example two, xylose reductase and transhydrogenase activity of Saccharomyces cerevisiae E33 engineering bacteria

[0031] Recombinant engineering bacteria E33 and starting strain W303-1Bα were inoculated in 5ml of SC medium (0.67%, yeast nitrogen base; 2%, glucose; appropriate amount of essential amino acids (histidine, tryptophan)), cultured (30 ℃, 250rpm shaker culture) overnight. The starting strain W303-1Bα was used as a control. The bacterial cells were collected by centrifugation (5000 rpm, 3 minutes). Cells were lysed by the glass bead method ("Experimental Guide to Yeast Genetics Methods", 87-88), and the protein supernatant was collected by high-speed centrifugation (13000 rpm, 40 minutes). The specific activities of xylose reductase and transhydrogenase in cell extracts were determined according to the assay described by Bruinenberg (Bruinenberg, P.M., et al., J. Gen. Microbiol., 129:953-964, 1983). Only weak coenzyme II-related xylose reductase activity wa...

Embodiment 3

[0032] Embodiment three, Saccharomyces cerevisiae E33 engineered bacteria strain ferments xylose to produce ethanol

[0033] Recombinant engineered bacterial strain E33 is inoculated in 100ml YPD medium (yeast extract 10g l -1 ;Tryptone 20g l -1 ; Glucose 20g l -1 )middle. 30°C, 250rpm shaker culture for 24 hours. The cells were collected by centrifugation (5000 rpm, 5 minutes), and the cells were washed twice with an equal volume of 0.9% physiological saline. Cells were resuspended in 100ml YPX medium (yeast extract 10g l -1 ;Tryptone 20g l -1 ; Xylose 30g l -1 ), cultured on a shaker at 30°C and 100rpm. After 84 hours, the concentration of xylose consumed was 22.2 g l -1 , the concentration of ethanol is 9.0 g l -1 , the yield of xylose to ethanol is 0.405g g -1 , and the conversion rate was 88%.

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Abstract

Xylose fermentation ethanol is one of the key technologies utilizing biomass to produce ethanol. A saccharomyces cerevisiae engineering bacterial strain is set up by a method of metabolic engineering. The bacterial strain not only can ferment the ethanol produced by the xylose, but also can automatically balance oxido-reduction cofactor, so as to be a metabolic engineering microzyme which has little by-product and can effectively use xylose fermentation for producing the ethanol. The conversion rate of xylose fermentation ethanol of bacterial strain reaches 88%, thus laying the foundation for utilizing the biomass to produce fuel ethanol.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a Saccharomyces cerevisiae engineering strain E33 which efficiently utilizes xylose to ferment ethanol and its application. Background technique [0002] With the depletion of petroleum resources, ethanol has replaced part of gasoline as a clean fuel, and the demand is increasing year by year. At present, the raw material for ethanol production is mainly starch from grain, and the cost of raw material accounts for more than half of the entire production cost, which has become a bottleneck for the large-scale promotion of fuel ethanol. Biomass is the most abundant renewable resource on earth. The use of biomass to produce fuel ethanol is a hot spot in international competition. After the carbohydrates in biomass are completely degraded into simple sugars, nearly one-third is xylose. Efficient conversion of xylose to produce ethanol is not only environmentally friendly but also condu...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12P7/06C12R1/865
CPCY02E50/17Y02E50/10
Inventor 江宁郭长缨贺鹏卢大军沈安
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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