CN109355239B

CN109355239B - Clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors and preparation method and application thereof

Info

Publication number: CN109355239B
Application number: CN201811173004.4A
Authority: CN
Inventors: 王菊芳; 傅宏鑫; 廖正平
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-10-09
Filing date: 2018-10-09
Publication date: 2021-01-19
Anticipated expiration: 2038-10-09
Also published as: CN109355239A

Abstract

The invention discloses a clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors, and a preparation method and application thereof, and belongs to the technical field of biology. The strain is Clostridium acetobutylicum (Clostridium acetobutylicum)824(proABC), is preserved in China general microbiological culture collection center in 2018 and 08 and month 06, and has a preservation number: CGMCC NO. 16223. The strain has strong tolerance to various cellulose hydrolysate inhibitors (formic acid, ferulic acid, syringaldehyde and coumaric acid), can directly utilize cellulose biomass hydrolysates such as non-detoxified soybean straws, rice straws and corn straws to carry out acetone-butanol-ethanol (ABE) fermentation, and the butanol yield of the strain is respectively 2 times, 3.4 times and 4.4 times of that of wild bacteria. The invention provides an effective strain and a method for efficiently utilizing cellulose biomass to produce butanol.

Description

Clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors, and a preparation method and application thereof.

Background

In recent years, with the depletion of fossil energy and the worsening of environmental problems, the development of new environmentally friendly renewable energy has become a research hotspot for researchers in various countries. The cellulose biomass which has the characteristics of reproducibility, wide distribution, large reserve, low price and the like is used as the raw material to produce the biological butanol, so that the method has a wide prospect.

Traditional strain for butanol production-acetobutylClostridium alcolohol has a wide substrate utilization ability, and can directly utilize starch, glucose, xylose, fructose and arabinose to perform ABE fermentation (Jones DT, Woods DR (1986) Acetone-butanol fermentation review. Microbiol Rev 50(4): 484-524). In the cellulosic biomass hydrolysate, glucose and xylose are the main components (Suo Y, Fu H, Ren M, Yang X, Liao Z, Wang J (2018) butyl acid production from lignocellulosic biological biomass by engineered Clostridium butyricum overexpression protein group. BioResourr technique 250: 691-. Thus, conducting ABE fermentation with cellulosic biomass will greatly reduce the input of feedstock costs. However, during the pretreatment of cellulosic biomass (usually carried out under high temperature and high pressure conditions using dilute acids or bases), various byproducts are produced which have toxic effects on microbial cells, such as furans, weak acids, phenols, and the like (R) ((R))

LJ, Alriksson B, Nilvebrandt N-O (2013) Bioconversion of lignocellulose: inhibitors and detoxication.Biotechnol Biofuels 6(1): 16). Wherein Formic and ferulic acids, syringaldehyde and coumaric acids are typical representatives of weak acids and phenols, these inhibitors have a significant inhibitory effect on the growth, sugar consumption rate and Butanol production of Clostridium acetobutylicum (Wang S, Zhang Y, Dong H, Mao S, Zhu Y, Wang R, Luan G, Li Y (2011) microorganism of Acid Crash of Acetone-Butanol-Ethanol Fermentation by Clostridium acetobutylicum. appl Environ Microbiol77(5): 1674. sup. 1680; Ezeji T, Blastek HP (2008) Fermentation of dried bacteria' and solvents (DDGS) hydrosaltes to solutions and values-added products 5242. Biotechnology). The natural clostridium acetobutylicum has low tolerance to cellulose hydrolysate inhibitors, so that the growth of thalli is severely inhibited when the non-detoxified cellulose hydrolysate is used as a substrate for producing the butanol by fermentation, the sugar consumption rate is low, the yield of the butanol is low, the fermentation efficiency is influenced, and the industrial production of the cellulose butanol is seriously hindered. The traditional detoxification treatment can effectively remove most inhibitors,but the fermentation process is complicated, the operation cost is increased, and the market competitiveness of the cellulose butanol is not improved. Therefore, the improvement of the tolerance of the clostridium acetobutylicum strain to the cellulose hydrolysate inhibitor is a key measure for improving the fermentation efficiency of the cellulose butanol, and has important significance for reducing the fermentation cost and improving the market competitiveness of the cellulose butanol.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the clostridium acetobutylicum which is resistant to various cellulose hydrolysate inhibitors.

The invention also aims to provide a preparation method of the clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors.

It is a further object of the present invention to provide the use of clostridium acetobutylicum that is tolerant to multiple cellulose hydrolysate inhibitors as described above.

The strain has higher tolerance to formic acid, ferulic acid, syringaldehyde and coumaric acid, can directly utilize non-detoxified lignocellulose hydrolysate to perform butanol fermentation, and has important significance for reducing the cost of cellulose butanol fermentation and improving the market competitiveness of butanol.

The purpose of the invention is realized by the following technical scheme:

the invention provides a strain of Clostridium acetobutylicum with tolerance to various cellulose hydrolysate inhibitors, which is named as Clostridium acetobutylicum 824(proABC) and has the preservation information as follows: the preservation unit: china general microbiological culture Collection center (CGMCC), the preservation date is 2018, 08 and 06 days, and the preservation address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC NO. 16223.

In some embodiments of the present invention, the cellulose hydrolysate inhibitor is preferably at least one of formic acid, ferulic acid, syringaldehyde and coumaric acid.

The invention also provides a preparation method of the clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors, namely, the genes proA, proB and proC related to proline synthesis are overexpressed in the clostridium acetobutylicum by constructing a recombinant expression vector. The method comprises the following steps:

(1) PCR amplifying target genes, including promoter and proline synthesis related genes proA, proB and proC;

(2) connecting a target gene with a vector;

(3) the ligation product was transformed into E.coli DH5 α;

(4) the recombinant plasmid is transformed into escherichia coli TOP10(pAN2) and is subjected to methylation treatment;

(5) preparing acetone butanol clostridium electrotransformation competent cells;

(6) and (4) performing electric conversion to obtain the clostridium acetobutylicum recombinant transformant.

The Clostridium acetobutylicum is preferably Clostridium acetobutylicum (Clostridium acetobutylicum) ATCC 824.

The promoter is clostridium acetobutylicum thiolase promoter Pthl.

The proline synthesis related gene has the following sequence (1) or (2):

(1) the nucleotide sequences of proA, proB and proC related to proline synthesis pathway in clostridium acetobutylicum are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3;

(2) a sequence having at least 70% homology with the nucleotide sequence of the proline synthesis pathway-related genes proA, proB and proC in Clostridium acetobutylicum.

In some embodiments of the invention, the proline synthesis-related gene is preferably proA, proB and proC.

Proline, as a cell-compatible substance, has various cellular functions such as stabilization of protein and cell membrane structures, reduction of DNA lysis temperature, and elimination of Reactive Oxygen Species (ROS) in cells, and the like, and thus can improve the tolerance of cells to osmotic pressure, oxidative pressure, ethanol, and the like (Appl Microbiol Biotechnol,2008, 81(2), 211). Therefore, the invention improves the synthesis capacity of the proline in cells by over-expressing the proline synthesis related gene, thereby improving the tolerance performance to the cellulose hydrolysate inhibitor. Most of the current studies on solvent production clostridium are directed at improving the tolerance of one cellulose hydrolysate inhibitor, while in the invention, the tolerance of the clostridium acetobutylicum to various cellulose hydrolysate inhibitors is remarkably improved, including formic acid, ferulic acid, syringaldehyde and coumaric acid.

The invention also provides application of the clostridium acetobutylicum in producing butanol by fermenting cellulose biomass hydrolysate. Preferably, the application in the production of butanol by using non-detoxified cellulose biomass hydrolysate through fermentation.

In some embodiments of the invention, the cellulosic biomass is an inexpensive agricultural waste. Preferably, the cellulose biomass is at least one of soybean straw, rice straw and corn straw.

The clostridium acetobutylicum can directly utilize non-detoxified soybean straw hydrolysate, rice straw hydrolysate and corn straw hydrolysate to perform ABE fermentation to obtain higher butanol yield, so that detoxification treatment of cellulose biomass hydrolysate is not needed, the fermentation process is simplified, the cost investment is reduced, and the market competitiveness of the biological butanol is improved.

Compared with the prior art, the invention has the following advantages and effects:

the clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors has strong tolerance to various cellulose hydrolysate inhibitors (formic acid, ferulic acid, syringaldehyde and coumaric acid), can directly utilize non-detoxified cellulose biomass hydrolysates such as soybean straws, rice straws and corn straws to perform acetone-butanol-ethanol (ABE) fermentation, and has butanol yield respectively 2 times, 3.4 times and 4.4 times of that of wild bacteria. The invention provides an effective strain and a method for efficiently utilizing cellulose biomass to produce butanol.

Drawings

FIG. 1 is a map (A) of a recombinant vector overexpressing a proline synthesis pathway-associated gene and the transcription levels (B) of the genes proA, proB and proC after transformation of C.acetobutylicum with the recombinant vector;

FIG. 2 is the intracellular proline content (A) of wild type ATCC824 and engineered 824(proABC) and intracellular ROS levels (B) under stress with different inhibitors;

FIG. 3 is a graph of the growth of wild type ATCC824 and engineered 824(proABC) bacteria on RCM plates containing different inhibitors;

FIG. 4 is a graph of the growth of wild ATCC824 and engineered 824(proABC) bacteria in RCM liquid media with different inhibitors; wherein, (A)1.5g/L formic acid; (B)1.5g/L ferulic acid; (C)2.5g/L syringaldehyde; (D)1.0g/L coumaric acid;

FIG. 5 shows butanol fermentations with wild type ATCC824 and engineered 824(proABC) bacteria with mixed sugars (40G/L glucose +20G/L xylose) (A and B), concentrated soybean straw hydrolysate (C and D), concentrated rice straw hydrolysate (E and F), and concentrated corn straw hydrolysate (G and H) as substrates.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are all commercially available reagents and materials unless otherwise specified.

Example 1: construction and transformation of co-expression vector of proline synthesis pathway related genes proA, proB and proC and RT-PCR detection of gene transcription level

(1) Recombinant plasmid construction and transformation

The specific construction process of the recombinant vector pMTL-Pthl-proABC is as follows:

the promoter Pthl was amplified using the genome of Clostridium acetobutylicum ATCC824 (standard strain) as a template with primers Pthl-F and Pthl-R. The proAB gene is amplified with the proAB-F and proAB-R primers, the proC gene (containing its own promoter) is amplified with the proC-F and proC-R primers, and the proABC gene is amplified by PCR with the proAB and proC primers as templates and the proAB-F and proC-R primers as overlapping primers.

Pthl-F：5′-CCGGAATTCTCGACTTTTTAACAAAATATATTGAT-3'; (EcoRI restriction sites underlined)

Pthl-R：5′-CGCGGATCCTCTAACTAACCTCCTAAATTTTGATAC-3'; (underlined BamH I restriction sites)

proAB-F：5′-AGGAGGTTAGTTAGAGGATCCAGGAGGAAAACATTATGAATACAAGAGAAAAATACT-3'; (underlined BamH I restriction sites)

proAB-R：5′-ATTATGAATACAAGAGAATTATTTTCTTATTTGCCCATCTCC-3′；

proC-F：5′-GGGCAAATAAGAAAATAATTCTCTTGTATTCATAATGTTTTGCTACT-3′；

proC-R：5′-ACGACGGCCAGTGCCAAGCTTCTATTTTGATGACATTAGCTTTGATT-3'. (HindIII restriction sites are underlined)

The PCR amplification system is as follows:

reaction components	Dosage of
		PrimeSTAR HS(Premix)	25μL
Upstream primer	1.5μL
		Downstream primer	1.5μL
DNA template	2μL
		ddH₂O	Up to 50μL

The PCR reaction program is: at 98 ℃ for 5 min; 30 cycles of 98 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 4 min; 72 ℃ for 10 min;

plasmid pMTL82151 (professor Yangtze university of Ohio State, USA, also available from Biovector NTCC collection) and promoter Pthl were first cut with EcoRI and BamHI in a double enzyme system:

reaction components	Dosage of
		Plasmids or Pthl	2μg
EcoRⅠ	1μL
		BamHⅠ	1μL

	10×FastDigest buffer	2μL
ddH₂O			Up to 20μL

The PCR reaction program is: react for 2h at 37 ℃.

The cleavage product was purified and recovered and pMTL82151 and promoter Pthl were ligated. The connecting system is as follows:

reaction components	Dosage of
		pMTL82151 double enzyme digestion product	50ng
Pthl double enzyme digestion product	22ng
		T4DNA Ligase	0.5μL
10×T4DNA Ligase Buffer	1μL
		ddH₂O	Up to 10μL

The reaction conditions are as follows: ligation was performed overnight at 16 ℃ to give an intermediate vector designated pMTL-Pthl.

Subsequently, plasmid pMTL-Pthl was double digested with BamHI and HindIII as follows:

reaction components	Dosage of
		Plasmid pMTL-Pthl	2μg
HindⅢ	1μL
		BamHⅠ	1μL

	10×FastDigest buffer	2μL
ddH₂O			Up to 20μL

The reaction conditions are as follows: at 37 ℃ for 2 h.

The cleavage products were recovered and pMTL-Pthl and the gene proABC were inserted behind the promoter Pthl using the One-Step recombinant ligase Clon express II One Step Cloning Kit (Nanjing Novodazen Biotech Ltd.). The connecting system is as follows:

reaction components	Dosage of
		pMTL-Pthl enzyme digestion product	50ng
proABC PCR product	150ng
		Exnase II	2μL
5×CE IIBuffer	4μL
		ddH₂O	Up to 20μL

The reaction conditions are as follows: 30min at 37 ℃.

The recombinant vector pMTL-Pthl-proABC is obtained, and the plasmid map is shown in figure 1A. After each ligation, E.coli DH 5. alpha. competent cells were transformed with the ligation product, and cultured overnight to obtain transformants. Single clones were picked and primed with pMTL-F: 5'-TGAAGTACATCACCGACGAGCAAG-3' and primer pMTL-R: 5'-TGCTGCAAGGCGATTAAGTTGGGT-3' for colony PCR verification. Positive clones were verified by sequencing.

The recombinant vector pMTL-Pthl proABC electrotransformation of Clostridium acetobutylicum ATCC824 has the following specific steps:

before electrotransformation, the correctly sequenced recombinant expression plasmid pMTL-Pthl-proABC was transformed into E.coli TOP10(pAN2) competent cells (Tiangen Biochemical technology Co., Ltd.) for methylation. The methylated plasmid pMTL-Pthl-proABC was extracted for electrotransformation. The specific process of electrotransformation is as follows: activating and culturing Clostridium acetobutylicum ATCC824 overnight, transferring the activated seed solution to fresh RCM culture medium, and culturing to OD₆₀₀About 1.0. The cells were pre-cooled on ice for 15min, then centrifuged at 8000rpm at 4 ℃ to collect the cells, resuspended in ice-pre-cooled electrotransfer buffer EPB (270mM sucrose and 5mM sodium dihydrogen phosphate, pH7.4), centrifuged at 8000rpm at 4 ℃ to collect the cells, and the above washing step was repeated once. And (3) resuspending the thalli by using an appropriate amount of ice-precooled electrotransformation buffer solution EPB to obtain the electrotransformation competent cells. About 10. mu.g of methylated plasmid pMTL-Pthl-proABC and 600. mu.L of competent cells were mixed well and added into an ice-precooled electric shock cup (diameter 0.4cm), and the body and bottom of the electric shock cup were wiped dry with a piece of mirror wiping paper and then placed into an electric converter for electric conversion. The electrotransfer conditions were: u is 2500V, t is approximately equal to 4ms, and 3 times of continuous electric shock. After electric shock, the electrotransformation mixed solution is quickly added into a fresh nonreactive RCM liquid culture medium and is placed in an anaerobic incubator at 37 ℃ for incubationCulturing for 4h, coating a proper amount of bacterial liquid on a thiamphenicol resistant plate, and placing the plate in an anaerobic incubator at 37 ℃ for culturing for 36h to obtain the Clostridium acetobutylicum recombinant transformant named Clostridium acetobutylicum 824 (proABC).

The deposit information of Clostridium acetobutylicum (Clostridium acetobutylicum)824 (proABC): the preservation unit: china general microbiological culture Collection center (CGMCC), the preservation date is 2018, 08 and 06 days, and the preservation address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC NO. 16223.

(2) RT-PCR analysis of the transcription of the genes proA, proB and proC in wild-type ATCC824 and engineered 824(proABC)

Wild ATCC824 and engineered 824(proABC) were grown in RCM broth overnight at 37 ℃ anaerobically, inoculated at 5% (v/v) to RCM containing 20g/L glucose at 37 ℃ anaerobically, and sampled after 12 hours. Centrifuging at 8000rpm for 10min, discarding supernatant, and precipitating for RNA extraction. RNA was extracted using an RNAprep pure Cell/Bacteria Kit (Tiangen Biochemical technology Co., Ltd.). Extracting RNA with RNase Free dH₂O is dissolved and reverse transcription is carried out. The reverse transcription step is as follows:

1) removing genome DNA, and reacting:

reaction components	Dosage of
		5×gDNA Eraser Buffer	2.0μL
gDNA Eraser	1.0μL
		Total RNA	1.0μg
RNase Free dH₂O	Up to 10.0μL

The reaction conditions are as follows: at 42 ℃ for 2min, immediately on ice.

2) Reverse transcription, the reaction system is as follows:

reaction components	Dosage of
		Reaction solution of step 1)	10.0μL
PrimeScript RT Enzyme MixⅠ	1.0μL
		RT primer Mix	1.0μL
5×PrimeScript Buffer 2	4.0μL
		RNase Free dH₂O	Up to 20.0μL

The reaction conditions are as follows: 15min at 37 ℃ and 5s at 85 ℃.

Subsequently, the cDNA obtained by the above reverse transcription was diluted 10-fold and used as a template for RT-PCR analysis. The RT-PCR reaction system is as follows:

reaction components	Dosage of
		SYBR premix Ex(2×)(Tli RNaseH Plus),Bulk	10.0μL
Forward Primer(10μM)	0.8μL
		Reverse Primer(10μM)	0.8μL
DNA template (< 100ng)	2.0μL
		ddH₂O	Up to 20.0μL

The reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃; 30s at 60 ℃; 40 cycles.

The primers used in the RT-PCR reaction were as follows:

the experimental results are shown in fig. 1B, and the transcription levels of the genes proA, proB, and proC in the engineered bacterium 824(proABC) are significantly higher than those of the wild-type bacterium ATCC824, which are 11.34 times, 16.58 times, and 8.61 times, respectively, that of the wild-type bacterium. The genes proA, proB and proC are shown to be efficiently expressed in the engineering bacteria 824 (proABC).

Example 2: overexpression of proline synthesis related genes proA, proB and proC enhances synthesis of intracellular proline of strain and improves scavenging capacity of strain to intracellular ROS

(1) Determination of intracellular proline content

Wild type ATCC824 and engineered 824(proABC) were grown anaerobically in RCM broth overnight at 37 ℃. The activated inoculum was transferred to RCM containing 20g/L glucose at 37 ℃ for anaerobic culture at 5% (v/v), and samples were taken after 8 hours. The sample was centrifuged at 8000rpm for 10min, the cells were collected and washed 3 times with SMM buffer. The cells were resuspended in 1mL of 3% 5-sulfosalicylic acid and extracted at room temperature for 12 h. Centrifuging to remove precipitate, and mixing 1mL of supernatant extractive solution with 0.5mL of acidic ninhydrin solution and 0.5mL of glacial acetic acid. The mixture was put in a water bath at 100 ℃ for 1 hour and immediately placed on ice to terminate the reaction. 2mL of xylene and the reaction mixture are shaken and mixed evenly and placed at room temperature for 20 min. And (3) after the mixed solution is layered, taking the upper layer liquid, and measuring the light absorption value at 520nm in an ultraviolet spectrophotometer.

The result is shown in fig. 2A, the intracellular proline content of the engineering bacteria 824(proABC) is significantly higher than that of the wild bacteria ATCC824, and is increased by 38.2% compared with the wild bacteria. Indicating that the synthesis of intracellular proline is remarkably enhanced by over-expressing proline synthesis related genes proA, proB and proC.

(2) Intracellular ROS (reactive oxygen species) level determination

Intracellular ROS levels were determined using the ROS Assay Kit. Firstly, wild bacteria ATCC824 and engineering bacteria 824(proABC) are cultured in RCM liquid culture medium overnight, activated bacteria liquid is inoculated in 25mL of fresh RCM liquid culture medium containing 0.1% (v/v) DCFH-DA (2',7' -dichlorofluorosis yellow diacetate) according to the inoculation amount of 5% (v/v), and after the bacteria body grows to OD₆₀₀When the concentration is about 1.0, the broth is divided into 5 parts, 4 parts are added with 1.0g/L formic acid (formic acid), 1.0g/L ferulic acid (ferulic acid), 2.0g/L syringaldehyde (syringaldehyde) and 1.0g/L coumaric acid (p-coumaric acid), and the rest part is not added with inhibitor (withoutinhibitor) as a control. Subsequently, the cells were incubated anaerobically at 37 ℃ for 1 hour while the cells were mixed by inverting the cells every 5 minutes. Then, the thalli is collected centrifugally and washed for 3 times by using a fresh RCM liquid culture medium, the thalli is resuspended by using 5mL of the fresh RCM liquid culture medium, 200 mu L of bacterial liquid is taken to be positioned in a multifunctional microplate reader SpectraMax M5 to measure the fluorescence value, wherein the excitation wavelength is 488nm, and the emission wavelength is 525 nm.

The results are shown in fig. 2B, where the intracellular ROS levels of the engineered bacterium 824(proABC) were significantly lower than the wild-type bacterium ATCC824 under the stress of the cellulose hydrolysate inhibitor. It is shown that the ROS scavenging capacity of the thallus is enhanced along with the enhancement of the synthesis of the intracellular proline.

Example 3: co-expression of proline synthesis pathway related genes proA, proB and proC to enhance tolerance of strain to cellulose hydrolysate inhibitor

(1) Preparation of culture Medium

RCM solid medium: 10g/L of peptone, 3g/L of yeast extract, 10g/L of beef powder, 5g/L of glucose, 1g/L of soluble starch, 5g/L of sodium chloride, 3g/L of sodium acetate, 0.5g/L of cysteine hydrochloride and 15g/L of agar powder.

RCM liquid medium: 10g/L of peptone, 3g/L of yeast extract, 10g/L of beef powder, 20g/L of glucose, 1g/L of soluble starch, 5g/L of sodium chloride, 3g/L of sodium acetate and 0.5g/L of cysteine hydrochloride.

(2) Tolerance test

Activating and culturing Clostridium acetobutylicum ATCC824 and engineering bacteria 824(proABC) overnight, inoculating into fresh RCM liquid culture medium at an inoculum size of 5% (v/v), and allowing the bacteria to grow to OD₆₀₀About 1.0, sample and dilute the sample gradient to 10⁰、10^-1、10^-2、10^-3And 10^-4. mu.L of the diluted sample was pipetted with a pipette and dropped evenly onto RCM plates containing different inhibitors (1.0g/L formic acid, 1.0g/L ferulic acid, 2.0g/L syringaldehyde and 1.0g/L coumaric acid). After air drying, the mixture is placed in an anaerobic incubator at 37 ℃ for culturing for 36h, and the growth condition of the thalli is observed.

Activating and culturing Clostridium acetobutylicum ATCC824 and engineering bacteria 824(proABC) overnight, inoculating into fresh RCM liquid culture medium at an inoculum size of 5% (v/v), and allowing the bacteria to grow toOD₆₀₀About 1.0, inoculating 10% (v/v) of the strain into RCM liquid medium containing different inhibitors (1.5g/L formic acid, 1.5g/L ferulic acid, 2.5g/L syringaldehyde and 1.0g/L coumaric acid) and culturing, and sampling every 4h to determine OD₆₀₀。

The experimental results are shown in fig. 3 and 4, and the growth of the engineered bacteria 824(proABC) on RCM plates or in RCM liquid medium containing different inhibitors is significantly better than that of the wild bacteria ATCC 824. It is demonstrated that as proline synthesis is enhanced, the tolerance of clostridium acetobutylicum to cellulose hydrolysate inhibitors is also enhanced.

Example 4: production of butanol by fermentation of cellulose biomass hydrolysate by using wild bacteria ATCC824 and engineering bacteria 824(proABC)

(1) Fermentation medium:

preparation of P2 culture medium: 80g/L glucose, 1g/L yeast extract, and acetic acid buffer (ammonium acetate 2.2g/L, KH)₂PO₄ 0.5g/L，K₂HPO₄0.5g/L), mineral solution (MgSO)₄·7H₂O 0.2g/L，MnSO₄·H₂O 0.01g/L，Fe SO₄·7H₂O0.01 g/L, NaCl 0.01g/L), vitamin solution (rho-para-aminobenzoic acid 1mg/L, thiamine 1mg/L, biotin 0.01 mg/L).

Preparing cellulose hydrolysate: respectively crushing soybean straws, rice straws and corn straws to the diameter of about 200 mu m by a crusher, and drying the crushed materials according to the solid-liquid ratio of 1: 10(w/v) the above cellulosic biomass powder was mixed with 0.02M H₂SO₄Mixing, and pretreating at 121 deg.C for 30 min. After cooling to room temperature, the pH was adjusted to 5.0 with NaOH. Adding cellulase according to the volume ratio of 1.8%

CTec2(Novozymes) and was hydrolyzed at 150rpm at 50 ℃ for 72 h. Centrifuging at 8000rpm, removing precipitate, concentrating the supernatant until the total reducing sugar concentration is about 60g/L, and respectively recording as non-detoxified soybean straw hydrolysate concentrated solution, rice straw hydrolysate concentrated solution and corn straw hydrolysate concentrated solution.

(2) Clostridium acetobutylicumATCC824 and engineering bacteria 824(proABC) are activated and cultured overnight, inoculated into fresh RCM liquid culture medium according to the inoculation amount of 5% (v/v), and the bacteria grow to OD₆₀₀When the inoculation amount is about 1.0 percent, the strain is inoculated into P2 culture medium (marked as Mixed sugar) containing 40g/L glucose and 20g/L xylose, and non-detoxified soybean straw hydrolysate concentrated solution (marked as SSH), rice straw hydrolysate concentrated solution (marked as RSH) and corn straw hydrolysate concentrated solution (marked as CSH) according to the inoculation amount of 10 percent (v/v) for fermentation. Samples were taken periodically to determine residual sugar concentration as well as product concentration.

As shown in FIG. 5, when the fermentation was carried out using a mixed sugar of 40g/L glucose and 20g/L xylose as a substrate, the fermentation performance of ATCC824 and engineering bacteria 824(proABC) was similar, while when the fermentation was carried out using a concentrated solution of soybean straw hydrolysate, a concentrated solution of rice straw hydrolysate and a concentrated solution of corn straw hydrolysate as substrates, the sugar uptake efficiency and solvent production of ATCC824, which is the original strain, were severely inhibited, while the engineering bacteria 824(proABC) were only slightly inhibited. The final yield of Butanol (Butanol) of the engineering bacteria 824(proABC) when fermentation is carried out by taking non-detoxified soybean straw hydrolysate concentrated solution, rice straw hydrolysate concentrated solution and corn straw hydrolysate concentrated solution as substrates is respectively 8.0g/L, 7.80g/L and 6.30g/L, which are obviously higher than that of wild bacteria ATCC824 by 4.0g/L, 2.30g/L and 1.42 g/L. The engineering bacteria 824(proABC) remarkably improves the performance of fermentation by utilizing cellulose hydrolysate by enhancing the synthesis of intracellular proline, so that normal fermentation can be carried out without detoxification treatment, the fermentation process is simplified, the cost input is reduced, and the market competitiveness of the biological butanol is improved.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of southern China's science

<120> clostridium acetobutylicum capable of tolerating various cellulose hydrolysate inhibitors and preparation method and application thereof

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1257

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proA nucleotide sequence

<400> 1

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ctttcttacg cagacactaa caccaaaaat aaggccctta tagaaatgag taaggctctt 120

ttagaaaata aggactatat attatctcaa aataaaatag acattgaaaa tgcagaaaaa 180

attggtactt ctaaagcact tatagataga cttactttga acgataaaag aattactgac 240

atggcagaag cacttataaa aacctcctct cttcaagatc caattggaga agtaataaaa 300

atgtggaaaa cacctgatga gcttcaaatt ggtcaaatga gagtgccact tggagtcatt 360

ggaataatct atgaggcaag acctaatgtt actgtagatg ctgctgctct atgcataaaa 420

tccggtaact cagtaatact cagaggtgga aaagaagcta taaattctaa cactgctatt 480

gcaaaaataa taaaaaatgc tgtagtaact gctggtcttc cagatggttc aattgagttt 540

atagatatca ccgatagaga aaccgttaat gtaatgatga gattaaatgg tcttatagat 600

gtattaattc caagaggtgg agcagggctc ataaaaagtg ttgttgagaa ctcttccgta 660

cctgtaatag aaacaggaac cggtaattgt catgtatatg tagataaata tgctgatttt 720

gataaggctg aaagaataat aataaacgct aagcttcagc gtccagcagt atgcaatgcc 780

atggaaagtc ttttagttca caaggatgta gctcatgaat ttcttcctag aatttcgagt 840

aagcttaagg agctaaaggt tcaaataaga ggctgcgctg ctactcagaa aattgtaaaa 900

gatatagttc ctgctactga tgaggatttc ggaaaagaat ttttggactt aatattatcc 960

gtaaaggttg ttgattcact tgaagaggct attgatcata tatttaaata tagtacaaag 1020

cactctgaag caataataac cgaaaactat actaacgctc aaagattttt gaaggaagtt 1080

gatgctgctg ctgtatatgt aaatgcttca acacgattca cggatggcga acaatttgga 1140

tttggcggcg aaataggtat aagcactcaa aaattgcatg caagaggtcc tatggggtta 1200

gaacagctta ctacaacaaa atatgtaatt tatggagatg ggcaaataag aaaataa 1257

<210> 2

<211> 804

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proB nucleotide sequence

<400> 2

atgaatacaa gagaaaaata cttgagtaat gtaaatagac ttgtcataaa agtaggaagt 60

tcaactttaa cacatccaag tggacttctt aacttttata aaatagaaca tattgtaaga 120

caaattgcag acttacataa tcaaggcatc aaggttatac tagtttcttc tggtgctata 180

ggtgctggaa ttgggaagct cagactaaaa gagagaccaa aaaccattcc agaaaagcag 240

gcagcagctg ctgtaggtca aggtgtttta atgcatactt acgaaaagct ttttgctgaa 300

tatggtcaaa ttgtagggca aatactaata acacgagaag acttatctag taagaaaaga 360

gttgtaaatg ttcaaaacac cttttcagca cttcttgatc atggaataat ccctattgta 420

aatgaaaatg atgctacagt cgttgaggaa ataaaattcg gagataatga taccttatct 480

gcaagagttg caagcttaat aaaagcagat ttacttattt tactctctga tatagatggt 540

ttgtacgatt caaaccctgc tgtaaacaaa aatgctgttt taatagatac agttaacgaa 600

gttaatgaag aagttaaagc atcagctggt ggcgctggtt caaagctcgg cactggtgga 660

atggcaacaa aaatcagagc agcagaaata gctactgaaa atggaatatc gatggttata 720

gcaaatggtg aaaagcaaga ggccataaga aatattttga attttgagaa tgagggaact 780

ctctttatac ctaaaaacaa ataa 804

<210> 3

<211> 1031

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proC nucleotide sequence

<400> 3

ttctcttgta ttcataatgt tttgctacta aaatattgta taaatatttt gtgaatttat 60

aataataata tttaagattg attattcaat attttaagta acgcagcctc ctttataaat 120

tattagctga taaaaagatt ttattgtata ataaaaaata tacatttatt cataagatta 180

tatcataaaa aatcgtttgt tttcaaggag tgaagaagat ggatagcaaa gttggattta 240

ttggctgcgg gaatatggca caggcaataa taaagggaat ggttaaggca aaggtagtac 300

caaatgaaaa tataattgta agtaatcctt ctaatgaaaa gctagagaag attagtaaag 360

aatgcggagt tttaactaca aatgacaaca agcaggttgc attaaaggca gatattattg 420

tattatctgt taaacctaat aaatacgaaa aggttataag tgaaatcaag gatttagtac 480

aacaaagtgt tataatagtt gcaatagcag cgggggtttc aatagagaaa acaagaatta 540

tgtttaaaaa tagcaatata agagtagtaa gggctatgcc aaatactcca gcgcttgtag 600

gagaggctat gacagctata tcaccatgtg aggaaataga caaaggtgaa ctaaaaaatg 660

taacggagat atttgagtca tttggaaaat gtgaagttgt agatgaagct ttaatgaatg 720

cagtaacagg ggtaagtggt tcatcacctg cttatgtgta tatgtttgta gaagctatgg 780

cagatgctgc tgttttgaac ggaatgacta gggaaaaagc atataagttt gcagctcaat 840

cagtacttgg agcagctaaa atgattcttg aaacaggaga acatcctggt aaacttaagg 900

atgatgtatg ttcaccttct ggaactacca ttgaagcagt atacgcactt gaaaaaagtg 960

gatttagagc ttcggtaatt gcagcagttg atgcatgcat aaaaaaatca aagctaatgt 1020

catcaaaata g 1031

<210> 4

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Pthl-F

<400> 4

ccggaattct cgacttttta acaaaatata ttgat 35

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Pthl-R

<400> 5

cgcggatcct ctaactaacc tcctaaattt tgatac 36

<210> 6

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proAB-F

<400> 6

aggaggttag ttagaggatc caggaggaaa acattatgaa tacaagagaa aaatact 57

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proAB-R

<400> 7

attatgaata caagagaatt attttcttat ttgcccatct cc 42

<210> 8

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proC-F

<400> 8

gggcaaataa gaaaataatt ctcttgtatt cataatgttt tgctact 47

<210> 9

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proC-R

<400> 9

acgacggcca gtgccaagct tctattttga tgacattagc tttgatt 47

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pMTL-F

<400> 10

tgaagtacat caccgacgag caag 24

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pMTL-R

<400> 11

tgctgcaagg cgattaagtt gggt 24

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proA-F

<400> 12

gaggtggagc agggctcata 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proA-R

<400> 13

ttgcatactg ctggacgctg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proB-F

<400> 14

aggtgctgga attgggaagc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proB-R

<400> 15

acaccttgac ctacagcagc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proC-F’

<400> 16

tccagcgctt gtaggagagg 20

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> proC-R’

<400> 17

gcaggtgatg aaccacttac cc 22

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CAC2679-F

<400> 18

gacattactt caaacgaacc tg 22

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CAC2679-R

<400> 19

cccttagccc atttattcct 20

Claims

1. A strain of clostridium acetobutylicum tolerant to various cellulose hydrolysate inhibitors is characterized in that: the microbial strain preservation and management center is named as Clostridium acetobutylicum 824(proABC) and is preserved in China general microbiological culture collection center of microbiological research institute of China academy of sciences, No. 3, West Lu 1 institute of China, North Cheng, Tokyo, Chaoyang, in 2018, 08 and 06 days, and the preservation number is as follows: CGMCC NO. 16223;

the cellulose hydrolysate inhibitor is at least one of formic acid, ferulic acid, syringaldehyde and coumaric acid.

2. The method of making a clostridium acetobutylicum strain tolerant to multiple cellulose hydrolysate inhibitors as claimed in claim 1, wherein:

the proline synthesis related genes proA, proB and proC are overexpressed in clostridium acetobutylicum by constructing a recombinant expression vector.

3. The method of claim 2, wherein: the method comprises the following steps:

(2) connecting a target gene with a vector;

(3) the ligation product was transformed into E.coli DH5 α;

4. The production method according to claim 3, characterized in that:

the Clostridium acetobutylicum is Clostridium acetobutylicum (Clostridium acetobutylicum) ATCC 824.

5. The production method according to claim 3, characterized in that:

the promoter is clostridium acetobutylicum thiolase promoter Pthl.

6. The production method according to claim 3, characterized in that:

the nucleotide sequences of proline synthesis related genes proA, proB and proC are respectively shown in SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO. 3.

7. Use of the clostridium acetobutylicum of claim 1 that is tolerant to multiple cellulose hydrolysate inhibitors for the fermentative production of butanol using cellulose biomass hydrolysates.

8. Use according to claim 7, characterized in that:

the clostridium acetobutylicum tolerant to various cellulose hydrolysate inhibitors is applied to the production of butanol by fermentation of non-detoxified cellulose biomass hydrolysate.

9. Use according to claim 7 or 8, characterized in that:

the cellulose biomass is agricultural waste.