CN101781634B - Recombinant zymomonas mobilis capable of producing ethanol by using xylose and fermentation method thereof - Google Patents

Abstract

The invention discloses a recombinant Zymomonas mobilis capable of producing ethanol from xylose and a fermentation method thereof, and a recombinant Zymomonas mobilis GX1 capable of producing ethanol from xylose, which is preserved in China Microorganisms The General Microorganism Center of the Preservation Management Committee, the deposit number is CGMCC 3438. For the first time in the present invention, the E. coli rrnB regulatory element is fused with xylose metabolism-related genes to construct a plasmid with an artificial fusion operon, which can be used by electrotransformation and domestication A recombinant Zymomonas mobilis strain that produces ethanol from xylose, which can efficiently express foreign genes, can efficiently utilize glucose and xylose to co-ferment alcohol at 30-34°C, and can use corn cobs containing xylose and glucose to hydrolyze Liquid alcohol. The bacterial strain of the invention is of great significance for effectively utilizing waste lignocellulose to produce clean energy and solving the current energy crisis.

Description

Recombinant Zymomonas mobilis capable of producing ethanol from xylose and fermentation method

technical field

The invention belongs to the field of bioengineering, and relates to a recombinant Zymomonas mobilis capable of producing ethanol from xylose and a fermentation method.

Background technique

With the depletion of fossil energy and the gradual emergence of the greenhouse effect, clean alternative energy has become a global research hotspot, and the research and development of fuel ethanol produced by fermentation of lignocellulose as raw material has attracted more attention. Renewable lignocellulose resources are extremely abundant in nature, and one of its main components is hemicellulose. 85% to 90% of hemicellulose is xylose (Lachke 2002), so the use of xylose to ferment ethanol is of great significance to the effective utilization of lignocellulose and to solve the current energy crisis.

However, high-efficiency ethanol-producing bacteria in nature can only use six-carbon sugars such as glucose to produce ethanol, and cannot use xylose; while a few strains that can use xylose to produce alcohol have low ethanol-producing ability, which limits the use of xylose in nature. Effective use of cellulose. Zymomonas mobilis has the advantages of high alcohol production, less biomass formation, no oxygen demand, high ethanol tolerance, and high sugar concentration tolerance, but its substrate range is narrow, and only glucose and fructose can be used. and six-carbon sugars such as sucrose (Rogers 2007). To construct a recombinant Zymomonas mobilis that can utilize xylose to produce alcohol, so that it can effectively utilize various components of lignocellulose, is of great significance to the production of clean and alternative energy.

Zymomonas mobilis utilizes the ED (Entner-Doudoroff) pathway to achieve high-efficiency alcohol production, but lacks a complete pentose phosphate pathway, and needs to introduce xylose isomerase, xylulokinase, transaldolase and transketone Enzymes can constitute a complete metabolic pathway, convert xylose into an intermediate product of the ED pathway, and enable Zymomonas mobilis to ferment xylose to produce ethanol.

To express exogenous xylose isomerase, xylulokinase, transaldolase and transketolase genes in Zymomonas mobilis, the properties of expression vector and exogenous gene, promoter and SD sequence, host Bacterial control system, etc. When using genetic engineering to improve the metabolic pathway of host bacteria or using low-copy plasmids as expression vectors to express target genes, gene expression can be regulated by changing the mRNA stability of the target gene. The increase in mRNA stability will increase the abundance of mRNA. lead to a proportional increase in protein expression (Smolke 2000; Pfleger 2006). Improving mRNA stability can be achieved by changing the regulatory elements of the target gene, such as changing the secondary structure of the untranslated region, ribosome binding sequence and other regulatory elements (Carrier 1997). Transcription terminators play an important role in the high expression of foreign genes in bacteria.

Escherichia coli rrnB operator T1T2 is a powerful transcription terminator, which can effectively prevent the read-through phenomenon in transcription and ensure the stability of expression plasmids, thereby increasing the expression level of foreign genes in prokaryotic cells. rrnB of Escherichia coli can increase The expression level of exogenous genes in Escherichia coli and Bacillus subtilis (Rong 2005; Wang 1992); however, there is no report on the use of rrnB and xylose metabolism-related genes as operons in Zymomonas mobilis . J. Sambrook, D.W. Russell, Molecular Cloning Experiment Guide (Volume 1) (Third Edition), translated by Huang Peitang, Beijing: Science Press, 2002

Carrier TA and Keasling JD. Controlling Messenger RNA Stability in Bacteria: Strategies for engineering Gene Expression. Biotechnol. Prog. 1997, 13: 699-708.

Lachke A. Biofuel from D-xylose-the second most abundant sugar. Resource, 2002, 50:50-58.

Pfleger BF, Pitera DJ, Smolke CD, Keaslin JD. Combinatorial engineering of intergenic regions inoperons tunes expression of multiple genes. Nature Biotechnology. 2006, 24: 1027-1032.

Rogers PL, Jeon YJ, Lee KJ, et al. Zymomonas mobilis for Fuel Ethanol and Higher Value Products. Adv Biochemical Eng Biotechnology, 2007, 108: 263-288.

Smolke CD, Carrier TA, Keasling JD. Coordinated Differential Expression of Two Genes through Directed mRNA Cleavage and Stabilization by

Wang, LF, Doi RH. Heterologous gene expression in Bacillus subtilis. Biotechnology, 1992, 22: 63-104.

Rong Jingjing, Diao Zhenyu, Zhou Guohua. Research progress of Escherichia coli expression system [J]. Pharmaceutical Biotechnology, 2005, 12(006): 416-420.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, to provide a kind of recombinant Zymomonas mobilis GX1 (Zymomonas mobilis) CGMCC 3438 that can utilize xylose to produce ethanol.

The second object of the present invention is to provide a method for obtaining recombinant Zymomonas mobilis capable of utilizing xylose to produce ethanol.

The third object of the present invention is to provide a fermentation method of recombinant Zymomonas mobilis capable of producing ethanol from xylose.

A recombinant Zymomonas mobilis GX1 (Zymomonas mobilis) that can use xylose to produce ethanol was preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on November 11, 2009, address: Beichen West Road 1, Chaoyang District, Beijing No. 3 Courtyard, preservation number CGMCC 3438.

A method for obtaining a recombinant Zymomonas mobilis that can utilize xylose to produce ethanol comprises the steps:

(1) Construction of the shuttle expression vector pZ capable of replicating in Escherichia coli and Zymomonas mobilis: first extract the plasmid in Z. mobilis (ATCC NO.10988) by SDS alkaline lysis method (Sam Brook, 2002), Electrophoresis after digesting the plasmid with AvaI, reclaiming a 2.7kb plasmid linear fragment, and obtaining the Zymomonas mobilis plasmid pZM2 after ligation; extracting the E. coli plasmid pACYC, combining the E. coli plasmid pACYC and the Zymomonas mobilis Bacterial plasmid pZM2 was digested with AvaI, and the expression vector pZ was constructed after ligation;

(2) Construction of the shuttle vector pXylose expressing xylose metabolism genes:

Extract Escherichia coli K12 genomic DNA and Zymomonas mobilis (ATCC NO.31821) genomic DNA;

Using Zymomonas mobilis (ATCC NO.31821) genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.1 and SEQ ID NO.2 as PCR primers to amplify the promoter Pgap of Zymomonas mobilis;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as PCR primers, amplifying the genes encoding xylose isomerase XylA and xylokinase XylB and the untranslated regions Sequence, the amplified product was digested with Kpn I and Xba I and ligated into the pUC19 plasmid, then digested with Nsi I and Sma I, blunt the sticky end and ligated to obtain the vector pXX;

Using pXX as a template, using the sequences described in the sequence table SEQ ID NO.13 and SEQ ID NO.4 as PCR primers to re-amplify XylA-XylB, and connecting the Pgap and XylA-XylB into an operon by overlapping extension PCR, After being digested with EcoR I, it is connected in the vector pZ to obtain the plasmid pZ-XX;

Using Zymomonas mobilis (ATCC NO.31821) genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.5 and SEQ ID NO.6 as PCR primers to amplify the promoter of the eno gene;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.7 and SEQ ID NO.8 in the sequence table as PCR primers to amplify the Tal gene encoding transaldolase, and form Peno-Tal by overlapping extension PCR. The fusion gene fragment was constructed into the TA vector pGMT to obtain the plasmid pTal;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.9 and SEQ ID NO.10 as PCR primers to amplify the transketolase Tkt gene;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.11 and SEQ ID NO.12 as PCR primers to amplify rrnB, obtain the fusion DNA fragment of Tkt-rrnB by overlap extension PCR; use Xba I and SphI double-cut plasmid pTal and DNA fragment Tkt-rrnB were ligated to obtain plasmid pTT; the pTT and plasmid pZ-XX were digested with NcoI and ligated to obtain the xylose isomerase gene, an exogenous gene required for xylose metabolism , the plasmid pXylose of xylulokinase gene, transketolase gene, transaldolase gene and gene expression regulatory element Escherichia coli terminator rrnB, Zymomonas mobilis gap and eno promoter;

(3) Transformation of plasmid pXylose to Zymomonas mobilis: transforming plasmid pXylose into Zymomonas mobilis CP4 (CICC 10232) to obtain a recombinant strain;

(4) Inoculate the recombinant strain into 5ml xylose-containing RM liquid medium with an inoculum size of 2-10% by volume, and transfer to another new medium containing xylose after culturing at 30°C until the OD600 is 1.0. Continue to cultivate in the RM liquid medium of xylose, the number of times of described transfer is 8-12 times, obtains a kind of recombinant Zymomonas mobilis bacterial strain GX1 (Zymomonas mobilis) CGMCC 3438 that can utilize xylose to produce ethanol; RM liquid medium containing xylose is 20g/L xylose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, and the balance is water.

The transformation of the step (3) plasmid pXylose to Zymomonas mobilis is as follows: inoculating the Zymomonas mobilis CP4 (CICC10232) culture into 50ml RM liquid culture medium with an inoculum size of 0.5-1.5% by volume culture at 30°C until OD ₆₀₀ = 0.5; centrifuge at 6000rpm for 5 minutes, collect the cells, wash 2-4 times with 10% glycerol aqueous solution in ice bath, and use 2ml of 10% Suspend and mix the glycerol aqueous solution to obtain competent cells; take 40 μl competent cells, add 1 μg plasmid pXylose to mix, transfer to a sterile pre-cooled 1mm electric shock cup, 1800V electric shock, quickly add 450ul RM liquid medium, and mix well Transfer to a 1.5ml sterile centrifuge tube, and culture at 30°C for 12-17 hours; spread the culture on RM solid medium screening plate containing 20μg/ml tetracycline, and place it upside down in an incubator at 30°C for 2-3 hours. day, select the transformant; after the colony is identified by PCR, select the transformant to measure the enzyme activity of exogenous protein xylose isomerase, xylulokinase, transketolase or transaldolase, and obtain a kind of ethanol that can use xylose to produce ethanol The recombinant Zymomonas mobilis strain, the RM liquid medium is: 20g/L glucose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, the balance is water, and the RM solid medium is: 20g/L glucose, 10g/L yeast extract, 20g/L agar powder and KH ₂ PO ₄ 2g/L, the balance is water.

A method for fermenting a recombinant Zymomonas mobilis strain that can utilize xylose to produce ethanol, comprising the steps of:

The bacterial strain GX1 (Zymomonas mobilis) CGMCC3438 of the recombinant Zymomonas mobilis that can utilize xylose to produce ethanol is cultivated in the liquid RM medium containing xylose to the bacterial cell concentration OD600=1.0-1.5, and the bacterial cell is concentrated by centrifugation, and inoculated into In the RM liquid medium containing xylose and glucose, the concentration of the initial bacterium is controlled at OD600=0.1-0.2, and the above bacteria are fermented at 30°C-34°C for 48-96 hours respectively; the RM containing xylose and glucose The liquid medium is 25g/L xylose, 25g/L glucose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, and the balance is water.

Innovation point of the present invention:

The present invention fuses Escherichia coli rrnB regulatory element with xylose metabolism-related genes for the first time, constructs a plasmid with an artificial fusion operon, and obtains a recombinant Zymomonas mobilis strain that can use xylose to produce ethanol by means of electrotransformation and domestication , it can efficiently express foreign genes and utilize xylose to produce alcohol. The natural Zymomonas mobilis can only ferment six-carbon sugars such as glucose, fructose and sucrose, but a recombinant Zymomonas mobilis that can use xylose to produce ethanol in the present invention can be fermented under the condition of 30-34°C Co-fermentation of glucose and xylose can be used efficiently to produce alcohol, and corncob hydrolyzate containing xylose and glucose can be used to produce alcohol. There are a lot of waste lignocellulose in nature, one of its main components is hemicellulose. 85%～90% in the hemicellulose is xylose (Lachke 2002), so the bacterial strain of the present invention is of great significance to the effective utilization of waste lignocellulose to produce clean energy and solve the current energy crisis.

Description of drawings

Figure 1 is the construction process of the expression vector PZ

Figure 2 shows the construction process of Zymomonas mobilis and Escherichia coli shuttle vectors.

Figure 3 is the growth curve of the recombinant strain in 2% xylose.

Figure 4 is a growth curve of a recombinant Zymomonas mobilis strain capable of producing ethanol from xylose in 2% xylose.

Fig. 5 A recombinant Zymomonas mobilis strain capable of producing ethanol from xylose co-ferments glucose and xylose to produce alcohol under various temperature conditions. A is the consumption of glucose and ethanol production under various temperature conditions; B is the growth curve of fermentation strains under various temperature conditions and the consumption of xylose; C is the production of by-product glycerol under various temperature conditions ; D is the formation of by-product acetic acid under various temperature conditions; E is the formation of by-product xylitol under various temperature conditions.

Fig. 6 is the situation that a kind of recombinant Zymomonas mobilis bacterial strain that can utilize xylose to produce ethanol is fermented in the fermenter tank of NBS company, in the RM culture medium of glucose and xylose of 25g/L of 30 degree pH5.5 Conditions of fermentation, cell concentration, sugar concentration, ethanol production, etc.

Figure 7 shows the fermentation of a recombinant Zymomonas mobilis strain capable of producing ethanol from xylose in corncob hydrolyzate at 30°C and pH 5.5.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

The construction of the shuttle expression vector pZ that embodiment 1 can replicate in E. coli and Zymomonas mobilis: first extract the plasmid of Z.mobilis (ATCC10988) with SDS alkaline lysis method (Sam Brook, 2002), use Ava I enzyme After cutting the plasmid, electrophoresis was performed to recover a 2.7kb plasmid linear fragment, and after ligation, the pZM2 plasmid of Zymomonas mobilis was obtained. Escherichia coli plasmid pACYC was extracted by SDS alkaline lysis method. Based on Escherichia coli plasmid pACYC and Zymomonas mobilis plasmid pZM2, it was digested with AvaI and ligated into plasmid pZ. pZ contains the replication origins of Escherichia coli and Zymomonas mobilis (Figure 1), that is, it can replicate in the above two host bacteria.

Example 2 Construction of the shuttle vector pXylose expressing xylose metabolism genes

Genomic DNA of Escherichia coli K12 and Zymomonas mobilis (ATCC NO.31821) were extracted with the bacterial genome extraction kit of Tiangen Company;

Using the genome of Zymomonas mobilis (ATCC NO.31821) as a template, using the sequences described in the sequence table SEQ ID NO.1 and SEQ ID NO.2 as PCR primers, amplifying the promoter Pgap of Zymomonas mobilis;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as PCR primers, amplifying the genes encoding xylose isomerase XylA and xylokinase XylB and the untranslated regions , the amplified product was digested with Kpn I and Xba I and ligated into the pUC19 plasmid, then digested with Nsi I and Sma I, blunt the sticky ends and ligated to obtain the vector pXX;

Using pXX as a template, using the sequences described in the sequence table SEQ ID NO.13 and SEQ ID NO.4 as PCR primers to re-amplify XylA-XylB, and connecting the Pgap and XylA-XylB into an operon by overlapping extension PCR, After being digested with EcoR I, it was connected to the vector pZ constructed in Example 1 to obtain plasmid pZ-XX;

Using Zymomonas mobilis (ATCC NO.31821) genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.5 and SEQ ID NO.6 as PCR primers to amplify the promoter of the eno gene;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.7 and SEQ ID NO.8 in the sequence table as PCR primers to amplify the Tal gene encoding transaldolase, and form Peno-Tal by overlapping extension PCR. The fusion gene fragment was constructed into the TA vector pGMT to obtain the plasmid pTal;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.9 and SEQ ID NO.10 as PCR primers to amplify the transketolase Tkt gene;

Using Escherichia coli K12 genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.11 and SEQ ID NO.12 as PCR primers to amplify rrnB, obtain the fusion DNA fragment of Tkt-rrnB by overlap extension PCR; use XbaI and SphI The double-cut plasmid pTal and the DNA fragment Tkt-rrnB are connected to obtain the plasmid pTT; the pTT and the plasmid pZ-XX are digested with NcoI and then connected to obtain the xylose isomerase gene, an exogenous gene required for xylose metabolism, Plasmid pXylose of xylulokinase gene, transketolase gene, transaldolase gene and gene expression regulatory elements Escherichia coli terminator rrnB, Zymomonas mobilis gap and eno promoters (see Figure 2). Plasmid pXylose contains xylose isomerase, xylokinase, transaldolase and transketolase genes that can encode xylose metabolism. These four enzymes can form new metabolic pathways with existing components in Zymomonas mobilis Efficiently converts xylose into ethanol.

SEQ ID NO.1: 5'ctcagaattctgtcgatgccgagttggactt3';

SEQ ID NO.2: 5'aataggcttgcatgtttaattctcctaactta3';

SEQ ID NO.3: 5'ctcggtaccatgcaagcctattttgacca3';

SEQ ID NO.4: 5'gtttctagagaattctgcctctcgtgtcagcgtg 3';

SEQ ID NO.13: 5'gagaataaacatgcaagcctattttgacca3';

SEQ ID NO.5: 5'cccatggctcgagatctccagttactcaata3';

SEQ ID NO.6: 5'tgtccgtcatatcgaaacctttcttaaaat3';

SEQ ID NO.7 5'aggtttcgatatgacggacaaattgacctc3'

SEQ ID NO.8 5'cgtctagattacagcagatcgccgatca3'

SEQ ID NO.9: 5'cgtctagacgatctggagtcaaaatgtcc3';

SEQ ID NO.10: 5'cgccaaaacagttacagcagttcttttgctttc3';

SEQ ID NO.11: 5'aactgctgtaactgttttggcggatgagaga3';

SEQ ID NO.12: 5'tcaatgcatgccatggaagagtttgtagaaacgcaa3'.

The acquisition of embodiment 3 recombinant strains

Plasmid pXylose was extracted with the large extraction plasmid kit of Promega Company. Inoculate the culture of Zymomonas mobilis CP4 (CICC10232) into 50ml RM liquid medium at 0.5% or 1.5% inoculum size, and culture it statically at 30°C until _OD600 =0.5; centrifuge at 6000rpm for 5min, collect the thalli, and use Wash 2 or 4 times with 10% glycerol aqueous solution in ice bath, suspend and mix with 2ml 10% glycerol aqueous solution to obtain competent cells; take 40 μl competent cells, add 1 μg Plasmid pXylose was mixed, transferred to a sterile pre-cooled 1mm electric shock cup, 1800V electric shock, quickly added 450ul RM body culture medium, mixed evenly, transferred to a 1.5ml sterile centrifuge tube, and stood at 30°C for 14 hours (optional 12 hours or 17 hours), the culture was coated with RM solid medium screening plate containing 20 μg/ml tetracycline, placed upside down in the incubator at 30°C for 2 to 3 days, and the transformants were selected; after the colonies were identified by PCR, three The enzyme activities of the exogenous protein xylose isomerase measured by the transformants were 0.0471, 0.0552 and 0.0676 U/mg respectively, that is, the recombinant strains were successfully obtained. (The enzyme activity of xylulokinase, transketolase or transaldolase can also be determined), RM liquid medium is: 20g/L glucose, 10g/L yeast extract and 2g/L KH ₂ PO ₄ , the balance is Water, RM solid medium: 20g/L glucose, 10g/L yeast extract, 20g/L agar powder and 2g/L KH ₂ PO ₄ , the balance is water.

Embodiment 4 Recombinant strain utilizes xylose growth observation

Cultivate the recombinant strain statically in RM medium containing 20g/L glucose at 30°C until the _OD600 is about 1.2. After centrifugation, resuspend the bacteria in RM medium without any sugar components and wash once , inoculate the concentrated bacteria into RM medium containing 20g/L xylose, the initial OD ₆₀₀ after inoculation is 0.08, and place it in a 30°C incubator for static culture. From 0 to 96 hours, the OD600 of the bacteria did not change significantly from 0.08 to 0.088; from 120 to 192 hours, the OD600 increased from 1.161 to 1.359, and at 312 hours, the maximum bacterial concentration _OD600 was around 1.48 (Figure 3).

Embodiment 5 Recombinant bacterial strain adapts to the domestication culture of xylose fermentation alcohol production

In order to improve the ability of the recombinant strain to utilize xylose, the recombinant strain is inoculated into 5ml of xylose-containing RM liquid medium with an inoculum size of 5% (or 2% or 10%) by volume, and cultivated at 30°C. After OD600 is 1.0, it is transferred to another new RM liquid medium containing xylose to continue culturing. The number of times of said transfer is 10 times (8 times or 12 times can also be selected), and a kind of xylose can be obtained. A recombinant Zymomonas mobilis strain that produces ethanol from sugar; the RM liquid medium containing xylose is 20g/L xylose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, and the balance is water. The growth ability of the bacteria to adapt to xylose is enhanced. The initial culture concentration of the strain is OD600=0.114. After 34 hours, the OD600 can reach 1.775, and the maximum bacterial concentration _OD600 is 1.915 (Fig. 4). Subsequent fermentation use.

Example 6 Optimization of the fermentation temperature for the co-fermentation of glucose and xylose by recombinant Zymomonas mobilis capable of producing ethanol from xylose

In the RM liquid medium containing xylose and glucose, the recombinant Zymomonas mobilis that can use xylose to produce ethanol was fermented under five temperature gradient conditions of 26°C, 30°C, 34°C, 37°C and 40°C respectively , through EChrom98HPLC to determine the concentration of recombinant Zymomonas mobilis cells that can use xylose to produce ethanol in the fermentation broth, the consumption of sugar, the generation of ethanol, the content of by-products such as xylitol, and optimize the temperature of alcohol production by fermentation .

The strains were fermented under the above-mentioned temperature conditions. The higher the fermentation temperature, the faster the glucose consumption, and the glucose was fully utilized within 24 hours ( FIG. 5A ). The consumption of xylose varies significantly with the fermentation temperature: when fermented at 30°C and 34°C, the utilization of xylose is the best: when fermented for 96 hours, the utilization of xylose reaches 93.4% and 95.4% When fermented at 40°C, the utilization rate of xylose decreased; at 26°C, the utilization rate of xylose was the lowest (Figure 5B), that is, too high or too low temperature was not conducive to the utilization of xylose.

With the consumption of glucose and xylose, ethanol is gradually produced. At 30°C and 34°C, the ethanol production was the highest, reaching 23.0g/L and 23.3g/L, and the final cell concentration was also the highest; while at 37°C and 40°C, the ethanol production decreased and the cell concentration decreased (Fig. 5A,B). However, at 26°C, the bacterial cell concentration was the lowest, and ethanol was only 17.54 g/L (Fig. 5B). Fermented at 30°C, 34°C, 37°C, and 40°C, the ethanol production basically reached the maximum at 48 hours, and there was no significant increase in ethanol production at 72 hours and 96 hours; better effect.

The higher the fermentation temperature, the more by-product glycerol was produced (Fig. 5C). When fermented at 34°C and 37°C, the maximum acetic acid production was higher, 0.53g/L and 0.52g/L, respectively, followed by acetic acid production at 40°C and 30°C, and the lowest at 26°C (Figure 5D). Xylitol was 1.25g/L and 1.06g/L when fermented at 30°C and 34°C, respectively, which was higher than about 0.8g/L produced by fermentation at other temperature conditions (Fig. 5E).

In summary, fermentation at 30°C and 34°C for 48-96 hours is the optimum temperature for fermentation, with high utilization rate of xylose and ethanol production, and less by-products.

The RM liquid medium containing xylose and glucose is 25g/L xylose, 25g/L glucose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, and the balance is water.

Example 7 Co-fermentation of glucose and xylose by recombinant Zymomonas mobilis capable of producing ethanol from xylose to produce ethanol

Take the recombinant Zymomonas mobilis that can use xylose to produce ethanol and inoculate it into 5ml of RM xylose medium, culture at 30°C for 21h, transfer the above 5ml bacterial liquid to 300ml of RM xylose medium, and culture at 30°C for 36h Afterwards, the thalli were collected, resuspended and inoculated into a 3-L BIOFLO 3000 fermenter (New Brunswick Scientific, Edison, NJ) for fermentation. The culture medium in the fermenter was RM liquid medium containing xylose and glucose. The initial OD ₆₀₀ = 0.2 and the working volume was 1.5 L. According to Example 6, fermentation was selected under the conditions of 30° C., pH value 5.5 and rotation speed 150 rpm. Regular sampling and analysis of biomass, sugar, ethanol and by-product concentrations.

Glucose can be completely consumed in 20 hours, and the concentration of xylose is reduced to 0.45g/L in 24 hours, and the utilization rate is 98.2%. At this time, the concentration of ethanol is also close to the highest value, reaching 22.4g/L. When fermented for 32 hours, the highest yield of ethanol was 22.54g/L, and the yield of ethanol was 87.8%. The bacterial cell concentration reached the maximum OD ₆₀₀ of 2.0 in 16 hours, and the by-products xylitol, acetic acid and glycerol were produced during the fermentation process, which were 0.9g/L, 0.8g/L and 1.5g/L respectively ( Figure 6).

Example 8 Fermentation of recombinant Zymomonas mobilis capable of producing ethanol from xylose in corncob hydrolyzate

The recombinant Zymomonas mobilis which can produce ethanol from xylose was inoculated into corn cob cellulose hydrolyzate containing 35.6g/L glucose and 12.9g/L xylose, and fermented at 30°C. Samples were taken to determine the concentration of by-products such as sugar, ethanol and xylitol, glycerol and acetic acid. Glucose was basically consumed within 24 hours of fermentation, leaving only 0.3g/L; the utilization rate of xylose was 84.8%, remaining 1.96g/L, and the concentration of ethanol finally produced was 20.26g/L (Figure 7). The ethanol yield calculated by consuming sugar) reached 87.6% of the theoretical value, and the ethanol yield calculated based on the total sugar was 83.0% of the theoretical value, that is, the recombinant bacteria had a strong alcohol-producing ability in the corncob hydrolyzate. There are fermentation by-products xylitol, acetic acid, etc. in the hydrolyzate itself, and the amount of by-products xylitol and acetic acid produced in the fermentation is basically the same as the amount produced by fermentation in the medium with the same sugar concentration. The recombinant bacterial strain of the invention can preferably use the hydrolyzed solution of cellulose as a raw material for fermentation, and lays a foundation for efficiently utilizing various components of lignocellulose to produce ethanol.

Claims (4)

1. A recombinant Zymomonas mobilis (Zymomonas mobilis) GX1 that can utilize xylose to produce ethanol, its preservation number is CGMCC 3438. 2. obtain a kind of method that can utilize xylose to produce the recombinant Zymomonas mobilis of ethanol of claim 1, it is characterized in that comprising the steps: (1) Construction of the shuttle expression vector pZ capable of replicating in Escherichia coli and Zymomonas mobilis: first, extract the plasmid of Zymomonas mobilis Z. Electrophoresis after digesting the plasmid with Ava I, reclaim the plasmid linear fragment of 2.7kb, and obtain the Zymomonas mobilis plasmid pZM2 after connection; extract the E. The bacterium plasmid pZM2 was digested with AvaI, and the expression vector pZ was constructed after ligation; (2) Construction of the shuttle vector pXylose expressing xylose metabolism genes: Extract the genomic DNA of Escherichia coli K12 and Zymomonas mobilis, the preservation number is: ATCC NO.31821 genomic DNA; Using Zymomonas mobilis, the preservation number is ATCC NO.31821 genomic DNA as a template, using the sequences described in SEQ ID NO.1 and SEQ ID NO.2 in the sequence table as PCR primers to amplify the promoter of Zymomonas mobilis Pgap; Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as PCR primers, amplifying the genes encoding xylose isomerase XylA and xylokinase XylB and the untranslated regions The amplified product was digested with Kpn I and Xba I and ligated into the pUC19 plasmid, and then digested with Nsi I and Sma I, and the sticky ends were blunted and ligated to obtain the vector pXX; Using pXX as a template, using the sequences described in the sequence table SEQ ID NO.13 and SEQ ID NO.4 as PCR primers to re-amplify XylA-XylB, and connecting the Pgap and XylA-XylB into an operon by overlapping extension PCR, After being digested with EcoR I, it is connected in the vector pZ to obtain the plasmid pZ-XX; Using Zymomonas mobilis, the preservation number is ATCC NO.31821 genomic DNA as a template, and using the sequences described in SEQ ID NO.5 and SEQ ID NO.6 in the sequence table as PCR primers to amplify the promoter of the eno gene; Using Escherichia coli K12 genomic DNA as a template, using the sequences described in SEQ ID NO.7 and SEQ ID NO.8 in the sequence table as PCR primers to amplify the Tal gene encoding transaldolase, and form Peno-Tal by overlapping extension PCR. The fusion gene fragment was constructed into the TA vector pGMT to obtain the plasmid pTal; Using Escherichia coli K12 genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.9 and SEQ ID NO.10 as PCR primers to amplify the transketolase Tkt gene; Using Escherichia coli K12 genomic DNA as a template, using the sequences described in the sequence table SEQ ID NO.11 and SEQ ID NO.12 as PCR primers to amplify rrnB, obtain the fusion DNA fragment of Tkt-rrnB by overlap extension PCR; use Xba I and The Sph I double-cut plasmid pTal and the DNA fragment Tkt-rrnB are ligated to obtain the plasmid pTT; the pTT and the plasmid pZ-XX are digested with Nco I and ligated to obtain the exogenous xylose isomerization gene required for xylose metabolism. Plasmid pXylose of enzyme gene, xylulokinase gene, transketolase gene, transaldolase gene and gene expression regulatory elements Escherichia coli terminator rrnB, Zymomonas mobilis gap and eno promoter; (3) Transformation of the plasmid pXylose into Zymomonas mobilis: transforming the plasmid pXylose into Zymomonas mobilis CP4 with a preservation number of CICC 10232 to obtain a recombinant strain; (4) Inoculate the recombinant bacterial strain into 5ml xylose-containing RM liquid medium with an inoculum size of 2-10% by volume, and transfer to another new strain after cultivating at 30°C until the OD600 is _1.0 Continue culturing in the RM liquid medium containing xylose, the number of times of the transfer is 8-12 times, and obtain a recombinant Zymomonas mobilis that can utilize xylose to produce ethanol; the RM liquid culture containing xylose The base is 20g/L xylose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, and the balance is water. 3. the method for obtaining a kind of recombinant Zymomonas mobilis that can utilize xylose to produce ethanol according to claim 2 is characterized in that the conversion of the step (3) plasmid pXylose to Zymomonas mobilis is: Zymomonas mobilis CP4, the preservation number is: CICC 10232 culture is inoculated into 50ml RM liquid medium at an inoculum volume of 0.5% to 1.5% by volume, cultured statically at 30°C until OD ₆₀₀ =0.5; centrifuged at 6000rpm 5min, collect the bacteria, wash 2-4 times with 10% glycerol aqueous solution in ice bath, suspend and mix with 2ml 10% glycerol aqueous solution by volume, and obtain competent cells; take 40 μl For competent cells, add 1 μg plasmid pXylose to mix, transfer to a sterile pre-cooled 1mm electric shock cup, electric shock at 1800V, quickly add 450ul RM liquid medium, mix well, transfer to a 1.5ml sterile centrifuge tube, and let stand at 30°C Cultivate for 12 to 17 hours; spread the culture on RM solid medium screening plate containing 20 μg/ml tetracycline, place it upside down in an incubator at 30°C for 2 to 3 days, and select transformants; after colonies are identified by PCR, select transformants Measure the enzyme activity of exogenous protein xylose isomerase, xylulokinase, transketolase or transaldolase to obtain a recombinant Zymomonas mobilis bacterial strain that can utilize xylose to produce ethanol, and the RM liquid culture The base is: 20g/L glucose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, and the balance is water. The RM solid medium is: 20g/L glucose, 10g/L yeast extract, 20g/L L agar powder and KH ₂ PO ₄ 2g/L, the balance is water. 4. the fermentation method of a kind of recombinant Zymomonas mobilis that can utilize xylose to produce ethanol according to claim 1, it is characterized in that comprising the steps: Cultivate the strain of recombinant Zymomonas mobilis that can use xylose to produce ethanol in liquid RM medium containing xylose to a cell concentration of OD ₆₀₀ = 1.0-1.5, concentrate the cells by centrifugation, and inoculate into xylose- and glucose-containing In the RM liquid culture medium, the concentration of the initial bacteria is controlled at OD ₆₀₀ =0.1～0.2, and the above bacteria are fermented at 30°C～34°C for 48～96 hours respectively; the RM liquid medium containing xylose and glucose is 25g/L xylose, 25g/L glucose, 10g/L yeast extract and KH ₂ PO ₄ 2g/L, the balance is water.

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