Saccharomyces cerevisiae engineering bacterium capable of utilizing whey efficiently to produce ethyl alcohol and construction method of saccharomyces cerevisiae engineering bacterium

A technology of Saccharomyces cerevisiae and construction method, which is applied in the field of bioengineering, can solve problems such as releasing the regulation of galactose metabolism, and achieve the effect of wide application prospects

Active Publication Date: 2015-12-30
HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] At present, there is no report at home and abroad that overexpresses

Method used

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  • Saccharomyces cerevisiae engineering bacterium capable of utilizing whey efficiently to produce ethyl alcohol and construction method of saccharomyces cerevisiae engineering bacterium
  • Saccharomyces cerevisiae engineering bacterium capable of utilizing whey efficiently to produce ethyl alcohol and construction method of saccharomyces cerevisiae engineering bacterium
  • Saccharomyces cerevisiae engineering bacterium capable of utilizing whey efficiently to produce ethyl alcohol and construction method of saccharomyces cerevisiae engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of Saccharomyces cerevisiae Genetically Engineered Bacteria to Remove Galactose Metabolism Regulation

[0034] (1) Construction of genetic engineering strains

[0035] 1) Using the genome of Saccharomyces cerevisiae AY5 (SaccharomycescerevisiaeCGMCCNo2.1364) as a template, using G6A-U and G6A-D as primers, PCR was used to amplify the fragment GAL6A with a length of 646bp upstream of the full sequence of the GAL6 gene, and similarly use G6B- U and GB6-D were used as primers to amplify the 665bp fragment GAL6B downstream of the full sequence of the GAL6 gene (see Table 1 for primer sequences and restriction sites), and then connect GAL6A and GAL6B to the pUC19 plasmid to obtain the plasmid pUC- G6AB;

[0036] 2) Using the Yep-C plasmid as a template and using Cup-U, Cup-D1, and Cup-D2 as primers, a 1410bp copper resistance gene CUP1 was amplified and connected to the plasmid pUC-G6AB to obtain the plasmid pUC-G6AB-CUP1 (see the build process fig...

Embodiment 2

[0050] Example 2: Research on Fermentation Performance of Engineering Bacteria Using Whey to Produce Fuel Ethanol

[0051] Inoculate AY5MG and its parent AY-510B24M into 20mL glucose culture medium, and culture overnight at 30°C for 12 hours; after centrifugation and washing, transfer all the bacterial liquid to 200mL whey medium, and culture and ferment at 30°C. Whey medium (g / L): whey powder 120 (lactose content is 53.1g / L), (NH 4 ) 2 SO 4 5. MgSO 4 ·7H 2 O1, make up to 1L with water. During the fermentation period, the samples were shaken every 24 hours, and the weight loss was recorded; after the fermentation, the culture was stopped and weighed;

[0052] Table 2 Fermentation performance of Saccharomyces cerevisiae receptor strain and engineered strain in whey

[0053]

[0054] Note: The data shown are the average of the results of three parallel experiments.

Embodiment 3

[0055] Example 3: Study on glucose repression of whey decomposition products of whey-utilizing Saccharomyces cerevisiae engineering bacteria and starting strains

[0056] The engineering bacteria and the recipient bacteria were respectively inserted into 5mL YEPD culture medium, and cultured overnight at 30°C for 12h; all the bacterial liquids were transferred to 20mL galactose culture medium, and cultured at 30°C for 24h. Prepare simulated whey breakdown product medium: glucose 3g, galactose 3g, (NH 4 ) 2 SO 4 0.5g, MgSO 4 ·7H 2 O0.1g, yeast powder 0.2g, peptone 0.1g, KH 2 PO 4 0.3g, distilled water 100mL. Inoculate according to 10% inoculum amount, and culture statically at 30°C. Shake samples at regular intervals during fermentation, measure different sugar concentrations, and see the results Figure 5 . Lactose in whey is first decomposed into glucose and galactose in the engineered strain of Saccharomyces cerevisiae, and then enters their respective metabolic pat...

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Abstract

Disclosed is a saccharomyces cerevisiae engineering bacterium capable of utilizing whey efficiently to produce ethyl alcohol. A strong promoter PGK1 is selected to express a lactose lytic enzyme gene LAC4 and a lactose permease gene LAC12 respectively, MIG1, NTH1 and GAL6 genes are knocked out, and semi-lactose metabolism regulation of saccharomyces cerevisiae is eliminated during relief of glucose repression to obtain the high-durability saccharomyces cerevisiae engineering bacterium AY5MG capable of utilizing the whey efficiently to produce the ethyl alcohol, wherein the collection number is CGMCC No.11223. The bacterium can grow and ferment to generate the ethyl alcohol in a culture medium with the whey concentration of 120g/L (the lactose content being 53.1g/L) without influences on fermenting property, the fermentation cycle is 54 hours, utilization rate of lactose in the whey is 98.7%, and the yield of the absolute ethyl alcohol to the lactose is 49.7% (equivalent to 92.3% of theoretical yield); glucose repression on semi-lactose is relieved, and accordingly, glucose and the semi-lactose are utilized at the same time; normal fermentation can be realized when the content of the ethyl alcohol is 19%(v/v) or the fermentation temperature is 39 DEG C.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the breeding of industrial microorganisms, in particular to a strain of Saccharomyces cerevisiae engineering bacteria that not only alleviates the regulation of galactose metabolism but also improves stress resistance and efficiently utilizes whey and its construction method. Background technique [0002] Whey refers to the extremely thin liquid left after the flocs are separated when making cheese or casein. It is a by-product of industrial production of cheese and casein. Every 1 ton of cheese produced produces 9t of whey, which contains 55% of milk nutrition, but because whey has high BOD and COD, it causes a great burden on the environment. At present, the global whey production is about 1.6 billion tons, and only 50% of it is processed for food, feed, etc. The remaining about 800 million tons are not effectively used and are excreted into nature, which not only causes e...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/90C12N15/04C12R1/865
CPCY02E50/10
Inventor 邹静李军康维民崔蕊静朱凤妹张建才刘素稳
Owner HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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