102 results about "Zymomonas mobilis" patented technology

According to the invention, through amplifying a strong promoter in Ensifer adhaerens and carrying out bioinformatics analysis and functional verification, a strong promoter is obtained, and the strong promoter can be widely used for gene expression, genetic manipulation and strain improvement in Sinorhizobium meliloti, Zymomonas mobilis, Caulobacter crescentus, Pseudomonas denitrificans, Agrobacterium tumefaciens, Brucella abortus, Pseudomonas fluorescens, Rhizobium leguminosarum, Ensifer adhaerens and other alpha-proteobacteria and has a nucleotide sequence of SEQ ID NO: 1. The invention also relates to a plasmid vector containing the strong promoter, a method for constructing a genetic engineering strain by using the strong promoter, and an application of the corresponding strain and ahost cell in starting expression of a target gene.

The invention discloses a method for co-producing a plurality of saccharic acids from cellulose fuel ethanol. The method comprises the following steps: performing dilute acid hydrolysis pretreatment or dilute acid steam explosion pretreatment on plant fiber raw materials to obtain a hemicellulose pre-hydrolysate and cellulose-enriched solid material through solid-liquid separation, wherein an enzyme hydrolysate is obtained by neutralizing and performing cellulose enzyme hydrolysis on the solid material, and fuel ethanol is produced by anaerobically fermenting the enzyme hydrolysate through saccharomyces cerevisiae or zymomonas mobilis; and a plurality of saccharic acids are produced by whole-cell co-catalyzing through gluconobacter oxydans or pseudomonas fragi under an aerobiotic condition after the hemicellulose pre-hydrolysate is concentrated and neutralized. The plurality of saccharic acids can be co-produced from fuel ethanol prepared from the plant fiber raw materials, the inhibiting effect of hemicellulose pre-hydrolysate sugar fermentation can be effectively overcome, the utilization efficiency of various carbohydrate substances in the plant fiber raw material can be obviously improved, the pollution load in industrial wastewater is effectively reduced, thereby improving the economic benefit of bio-refinery of the plant fiber raw materials. The utilization rate of total carbohydrate in the raw materials is over 80%, and the carbohydrate conversion rate is more than 90%.

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

The invention relates to an ethanol fermentation engineering bacteria capable of reducing a fermentation byproduct. The ethanol fermentation engineering bacteria is characterized in that a pyruvate formate-lyase gene and a lactate dehydrogenase gene of Escherichia coli are inactivated; and a Zymomonas mobilis gene is transferred into the Escherichia coli. When ethanol is produced by fermenting hexose and pentose with the engineering bacteria, lactic acid and formic acid are not produced and the byproduct is reduced, so that the ethanol productivity is improved.

The invention belongs to the technical field of genetic engineering, and particularly relates to a multi-gene locus simultaneous editing method based on an endogenous CRISPR-Cas system of zymomonas mobilis and application thereof. The method mainly comprises the following steps of: constructing a plasmid containing an artificial CRISPR expression unit; selecting a plurality of target editing target loci, and designing a gRNA sequence; connecting a plurality of gRNA sequences in series, and constructing the gRNA sequences on the plasmid containing the artificial CRISPR expression unit; constructing a donor DNA sequence on a vector to obtain an edited plasmid; and transforming the edited plasmid into competent cells for editing. The method constructs a gene editing platform by utilizing theendogenous CRISPR-Cas system of zymomonas mobilis, breaks the limitation of low efficiency of exogenous CRISPR-Cas9 genome editing in the strain, realizes rapid and efficient knockout of multiple genes in the strain, and promotes the development of metabolic engineering, systemic biology and synthetic biology.

The invention relates to pure-culture mixed fermented mildewed bean dregs and a preparation method thereof. The method comprises the following steps: (1) fresh wet bean dregs are washed and squeezed to reduce water content to 70-80 %; (2) the squeezed bean dregs are boiled for 15-30 min at normal pressure; (3) the boiled bean dregs are cooled to room temperature, 0.1-10ml Actinomucor elegans spore suspension with a concentration of 104-106 cfu/ml and 0.2-20ml Zymomonas mobilis suspension with a concentration of 106-108 cfu/ml are vaccinated in the bean dregs, after uniform stir, the inoculated bean dregs are pressed into spherical or pie-shaped bean dregs; (4) the spherical or pie-shaped bean dregs are fermented in an artificial climate case for 3-6 d at 18-30 DEG C, with 90-95% relative humidity, so as to obtain the mildewed bean dregs. The invention has the advantages of reasonable technology, simple operation, reasonable utilization of bean dregs, reducing pollution and improving the nutrition value of bean dregs.

The invention belongs to the technical field of gene editing, and particularly relates to a Zymomonas mobilis genome editing method based on a CRISPR-Cas12a system and application of the method. The method for editing a Zymomonas mobilis genome is established on the basis of the CRISPR-Cas12a system with Zymomonas mobilis (ZM4) as the modulus strain, the oriented editing of the genome is realized,a gene editing tool is provided for developing the rational design of an allogenic metabolic pathway and cell factory for producing biomass fuel and biological materials in the strain, and the development in metabolic engineering and related research fields is promoted. The method is technically characterized by including the steps of establishing an inducible expression Cas12a recombinant strainand an editing plasmid containing an artificial CRISPA expression unit, designing guiding RNA, connecting the guiding RNA to the editing plasmid after annealing the primer sequence of the guiding RNA, and shifting a target plasmid into a competent cell for expression editing.

The invention provides a method for producing ethyl alcohol through crop stalks by use of recombinant zymomonas mobilis. According to the method, the crop stalks are used as a base material, aspergillus niger and trichoderma are mixed and fermented to produce a crude enzyme solution containing cellulase, the crop stalks are processed through the crude enzyme solution to enable cellulose in the crop stalks to be decomposed to produce reducing sugar, and finally recombinant zymomonas mobilis is used for fermenting the enzymatically-decomposed reducing sugar to produce ethyl alcohol. In the method, fermentation products of aspergillus niger and trichoderma are mixed according to the ratio of 1: 1 to obtain the cellulase crude enzyme solution, so that the product feedback inhibition is effectively controlled, and the cellulase crude enzyme with higher activity and yield is obtained. As zymomonas mobilis is inoculated in the enzymatically-decomposed fermentation liquor containing reducing sugar, the ethyl alcohol production efficiency through the crop stalks by use of zymomonas mobilis is greatly improved, and defects that the use range of a zymomonas mobilis substrate is narrow, and the crop stalks cannot be effectively utilized are overcome. According to the method, the conversion rate of ethyl alcohol through the crop stalks is 5%, that is, 100 g of the crop stalks can produce 5 g of ethyl alcohol.

The invention relates to a colon bacillus alcohol fermentation engineering bacterium resisting alcohol. A colon bacillus mutant strain highly resisting the alcohol is obtained through a plurality of generations of directional screening; and an alcohol dehydrogenase gene II and a pyruvate decarboxylase gene of zymomonas mobilis are transferred into the mutant strain to obtain the novel colon bacillus alcohol fermentation engineering bacterium. When the engineering bacterium uses pentose and hexose as substrates to ferment for producing the alcohol, the engineering bacterium can still maintain higher fermentation rate under high-concentration alcohol, and has higher transformation rate of the alcohol.

The invention belongs to the technical field of genetic engineering, particularly relates to a genome editing method based on a zymomonas mobilis endogenous CRISPR-Cas system and application thereof and aims at taking Z.mobilis 4 as a type strain and utilizing the I-F type CRISPR-Cas system coded by its genome to establish a genome editing platform. A powerful tool is provided for basic research and application research in the strain and similar cells, and the development of metabolic engineering, system biology and synthetic biology is promoted. According to the technical scheme, the genome editing method is characterized by comprising the steps of constructing a plasmid containing an artificial CRISPR expression unit; selecting a guide RNA sequence aiming at a target site; constructing aguide RNA primer sequence on the plasmid containing the artificial CRISPR expression unit; constructing a donor DNA sequence on a vector to obtain an editing plasmid; transforming the editing plasmidinto competent cells for editing.

Xylose-utilizing, ethanol producing strains of Zymomonas mobilis with improved performance in medium comprising biomass hydrolysate were isolated using an adaptation process. Independently isolated strains were found to have independent mutations in the same coding region. Mutation in this coding may be engineered to confer the improved phenotype.

The invention discloses an FLP-containing pBBR1MCS-2 recombinant plasmid and a method for modifying a zymomonas mobilis genome DNA. The plasmid is constructed by the step of: inserting a segment Pgap-FLP into pBBR1MCS-2 through C1aI and XbaI sites to obtain pBBR1MCS-2-Pgap-FLP, wherein the Pgap-FLP is a nucleotide sequence shown in SEQ ID NO:1 in a sequence table. The plasmid can be conveniently transferred in an electroporation manner to enter zymomonas mobilis, and can be conveniently removed. By combining with an FRT-FLP site specificity recombinant system from saccharomyces cerevisiae, the invention provides a rapid, efficient, convenient and stable method for modifying zymomonas mobilis genome DNA. The invention provides a method for modifying DNAs at multiple sites of the zymomonas mobilis genome by circularly utilizing a selective marker gene.

The invention provides zymomonas mobilis resistant to high-concentration acetic acid and high-concentration furfural and application thereof. The accession number of the zymomonas mobilis is CMGCC No.10946. The zymomonas mobilis can quick grow in a culture solution containing 7.0 g/L of acetic acid and 3.0 g/L of furfural, and alcoholic fermentation can be performed quickly. Therefore, the zymomonas mobilis can directly perform fermentation on cellulose enzymatic hydrolysates on the conditions of high-concentration acetic acid and high-concentration furfural, and fuel alcohol is obtained through preparation.

Zymomonas mobilis mutant strains tolerant to high-concentration acetic acid and applications thereof are disclosed. The taxonomic name of one of the strains is zymomonas mobilis AQ8-1, and the accession number of the zymomonas mobilis AQ8-1 is GDMCC60258. The taxonomic name of the other strain is zymomonas mobilis AQ8-9, and the accession number of the zymomonas mobilis AQ8-9 is GDMCC60259. Mutation screening includes 1) performing room-temperature plasma induced mutation; 2) performing renewing culture by utilizing a medium; and 3) repeatedly screening under a high acetic acid concentration to obtain the strains. The strains can rapidly grow in a culture liquid containing 5.0-8.0 g/L of acetic acid, and can perform alcoholic fermentation. The invention also provides an alcoholic fermentation method.

The invention discloses a method for producing fuel ethanol by using regrouping zymomonas mobilis to ferment xylose. The method comprises the following steps of: A), inoculating the regrouping zymomonas mobilis into 100mL of T liquid nutrient solution (containing 10g/L of yeast extract, 2g/L of KH2PO4, 1g/L of MgSO4.7H2O1, 1g/L of (NH4)2SO4 and 20g/L of xylose) according to the inoculum size of 1 percent (v/v), wherein the nutrient solution contains 30 to 50 mu g.mL-1 of kanamycin or ampicillin, standing at the temperature of 20 to 30 DEG C to culture until the OD600 is equal to 1.0, centrifuging the nutrient solution for 5 to 10 minutes under the condition of 8,000 to 1,000rpm, and obtaining the supernatant; and B), accurately measuring 20ml of fermentation liquor, adding the fermentation liquor into a flask with a round bottom, after adding 20ml of water into the fermentation liquor, placing the fermentation liquor in a distilling apparatus, collecting 20ml of distillate, and measuring the concentration of ethanol by gas chromatography.

The invention relates to a method for synthesizing biodiesel with microorganisms in vivo, which is realized in escherichia coli. The method comprises the following steps of: respectively cloning an acinetobacter wax fat synthetase gene atfA, an escherichia coli acyl coenzyme A synthetase gene fadD, a zymomonas mobilis pyruvate decarboxylase gene pdc and an alcohol dehydrogenase gene adhB gene on a pET expression vector in series to obtain a plasmid pXT11; transferring the plasmid pXT11 into escherichia coli containing a plasmid pMSD8 and a plasmid pMSD15 to obtain an escherichia coli stain XT31; and generating biodiesel by the escherichia coli stain XT31. The method realizes the microbial transformation from sugar to the biodiesel, lays the foundation for biologically preparing the biodiesel from lignocelluloses, and radically solves the problem of insufficient raw material supply in biodiesel preparation. A step of esterifying methanol is not needed in the technology, thereby the cost is greatly reduced and the problems that glycerol as a side product is formed, and the like are avoided.

An object of the present invention is to provide a method for producing ethanol from polysaccharide alginate contained in a large amounts in brown algae, using the alginate assimilation capacity of the Sphingomonas sp. strain A1 and the strong ethanol production capacity of bacteria such as Zymomonas mobilis. Specifically, a method for producing ethanol using alginate as a raw material comprises causing genes encoding proteins and enzymes involved in Sphingomonas sp. strain A1-derived alginate assimilation and genes encoding enzymes involved in ethanol production to co-exist in a single microorganism and then culturing the microorganism in a medium containing alginate.

The invention discloses a construction method of high-conversion-ratio zymomonas mobilis. Meanwhile, the invention further relates to the high-conversion-ratio zymomonas mobilis. Thirdly, the invention also relates to application of the high-conversion-ratio zymomonas mobilis to a gene engineering field. The construction method comprises the following steps: combining reference documents and a database to predicate related genes of a zymomonas mobilis ZM4 whole-genome limitation-modification system; designing gene knockout primers for the predicated two genes related to the limitation-modification system respectively; taking ZM4 and ZM401 as starting strains, and knocking out the related genes through a plasmid pEX18Tc, so as to finally obtain ZM4m and ZM401m which are used as knockout strains of the related genes of the limitation-modification system. According to the construction method disclosed by the invention, the conversion efficiency of the obtained strains is remarkably improved, and the quantity of obtained transformants is great; the strains have a high positive rate, and the growth performance and the fermentation performance of the strains are the same as those of wildtype strains. The double knockout strains are used as the starting strains, so that molecular modification of the zymomonas mobilis and related mechanism researches are facilitated.

The invention discloses a series of expression vectors pSUZM1a, pSUZM1b, pSUZM1c, pSUZM2a, pSUZM2b, pSUZM2c, pSUZM3a, pSUZM3b and pSUZM3c, and a construction method thereof. The expression vectors are annular vectors, and respectively contain replication origin from Escherichia coli plasmid on plasmid pUC18, and a kanamycin screening marking gene, wherein pSUZM1(a, b, c) respectively contain replication origin oriC from Zymomonas mobilis chromosome, pSUZM2(a, b, c) respectively contain replication origin from Zymomonas mobilis endogenous plasmid pZZM401, and the pSUZM3(a, b, c) respectively contain replication origin from Zymomonas mobilis endogenous plasmid pZZM402. The pSUZM1a, pSUZM2a and pSUZM3a respectively contain promoter Ppdc from Zymomonas mobilis; pSUZM1b, pSUZM2b and pSUZM3b respectively contain promoter Para from vector pKD46; and pSUZM1c, pSUZM2c and pSUZM3c respectively contain tac promoter from vector pGEX. The method realizes the efficient expression of exogenous gene in Zymomonas mobilis, and has a very good application prospect.

The invention provides ethanol-tolerant zymomonas mobilis. The ethanol-tolerant monad contains a mutated and transformed rpoD gene; point mutation of the RpoD protein encoded by the gene is Q57L, G426C and I448N; the ethanol-tolerant zymomonas mobilis can tolerate ethanol of which the initial concentration at least is 9%; the classification of the ethanol-tolerant zymomonas mobilis is named zymomonas mobilis (ZM4-mropD); the ethanol-tolerant zymomonas mobilis is preserved at the China General Microbiological Culture Collection Center; the preservation number is CGMCC NO.10617; and the preservation date is March 12, 2015. The invention further provides a method for preparing a bacterial strain. The method comprises the following steps: preparation of a mutant rpoD gene, construction of recombinant plasmid pBmrpoD, plasmid transformation, and screening by different gradients of ethanol. The bacterial strain can tolerate ethanol of which the initial concentration at least is 9%; and the preparation method is easy to operate. The invention further discloses an application of the bacterial strain in production of ethanol.