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Enhancement of microbial ethanol production

A technology of microorganisms, thermophilic microorganisms, applied in the field of microorganisms

Inactive Publication Date: 2009-06-03
BIOCONVERSION TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The present invention seeks to solve the problem of high yield ethanol production from biomass

Method used

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  • Enhancement of microbial ethanol production
  • Enhancement of microbial ethanol production
  • Enhancement of microbial ethanol production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1. Construction of synthetic formate dehydrogenase gene ( figure 2 )

[0077] Amino acid sequence of Pseudomonas 101 formate dehydrogenase (NCBI protein database accession number P33160 - SEQ ID NO: 3) back translated into DNA sequence by codon optimization for Geobacillus thermoglucosidase . The promoter and rho-independent terminator regions from the Bacillus strain were added upstream and downstream of the translated sequence ( figure 2 ). The new sequence has less than 40% similarity to the known fdh gene sequence (37% identity to the known fdhl gene). Xba1 sites were designed on both sides of this construct to facilitate cloning into a suitable vector.

[0078] Using the method of Gao et al. (see Xinxin Gao, Peggy Yo, Andrew Keith, Timothy J.Ragan and Thomas K.Harris (2003). Nucleic Acids Research, 31(22), e143) to synthesize the required sequence, and clone it into pCR - Unique Xba1 location in Blunt. The resulting vector pCR-F1 ( Figure 4 )...

Embodiment 2

[0080] Example 2. Insertion of the fdh gene into multiple (IS) sites

[0081] This strategy works for some strains, such as the Bacillus stearothermophilus LLD-R strain, which contain insertion sequences (IS) that frequently recombine at multiple insertion sites. It is expected that a vector carrying the fdh gene and this IS sequence will be stably integrated at one or more of these positions.

[0082] Construction of plasmid pUB-ISF1 ( Figure 5 )

[0083] First, using Bacillus stearothermophilus LLD-15 as a template, use forward primer (AGTACTGAAATCCGGATTTGATGGCG-SEQ ID NO: 6) and reverse primer (AGTACTGCTAAATTTCCAAGTAGC-SEQ ID NO: 7) to PCR amplify the known LLD-R strain Insertion sequence (SEQ ID NO: 5 and image 3 ). Sca1 restriction sites were introduced at both ends of this sequence. The PCR product was first cloned into plasmid pCR-TOPO2.1 and the resulting plasmid pCR-IS was introduced into E. coli DH5α cells and used to isolate the IS region by Sca1 restrict...

Embodiment 3

[0088] Example 3. Construction of ldh-deleted strains.

[0089] The first step involves cloning the Bacillus kanamycin resistance marker (kan) and the gene cassette for the ldh gene of the Bacillus stearothermophilus LLD-R strain into plasmid pUC18, which replicates only in Gram-negative microorganisms .

[0090] Construction of Bacillus cloning vector plasmid pUCK ( Image 6 ).

[0091] The kanamycin resistance gene (kan) was cloned into the unique Zra1 site in plasmid pUC18, which is outside of any coding region and reporter gene (lacZ) in the plasmid. To clone the kan gene, use the plasmid pUB110 as a template, and use the following primers to amplify a 1.13kb fragment containing the kanamycin resistance gene by PCR:

[0092] kan-Bs Z-F (ACACAGACGTCGGCGATTTGATTCATAC-SEQ ID NO: 10) and

[0093] kan-Bs Z-R (CGCCATGACGTCCATGATAATTACTAATACTAGG-SEQ ID NO: 11).

[0094] The Zra1 sites were introduced into both ends of the kan gene by primers. The PCR product was then di...

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Abstract

A thermophilic microorganism lacks lactate dehydrogenase activity and preferably contains an active pyruvate formate lyase pathway. The thermophilic microorganism contains a gene encoding an NAD-linked formate dehydrogenase. The gene encoding an NAD-linked formate dehydrogenase is preferably a codon optimised version of the gene encoding a thermostable NAD-linked formate dehydrogenase. DNA constructs allow stable expression of the gene encoding an NAD-linked formate dehydrogenase in the thermophilic microorganism. The DNA constructs are based upon use of an insertion sequence to achieve stable expression or recombination to insert the gene encoding an NAD-linked formate dehydrogenase into the lactate dehydrogenase gene, thus achieving gene knockout and new functionality in a single step. The microorganisms are useful in fermentation of sugars to produce ethanol.

Description

technical field [0001] The present invention relates to fermentation processes and microorganisms used therein, and in particular to the enhancement of microbial ethanol production. More specifically, the present invention relates to the enhancement of ethanol production by thermophilic bacteria such as Bacillus sp. using mixed sugars derived from biomass hydrolysis. In particular, the present invention designs a novel ethanol production pathway by cloning a gene encoding an NAD-linked formate dehydrogenase into a microorganism that encodes a pyruvate-formate lyase complex but lacks Functional gene for lactate dehydrogenase activity. Background technique [0002] Currently, bioethanol is produced from glucose, maltose or sucrose derived from cereal starches, sugar cane or sugar beets, all of which have food value. Cellulose and hemicellulose form a major part of agricultural by-products and could, in principle, be a major source of low-cost, renewable bioethanol. However,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/06C12N1/21C12N15/53C12N15/90C12N9/02
CPCY02E50/17C12P7/065C12N9/0008Y02E50/10
Inventor 穆罕默德·贾韦德纳姆达尔·巴格哈伊-亚兹迪
Owner BIOCONVERSION TECH INC
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