CN103627663B - Recombined bacillus subtilis growing by xylose - Google Patents
Recombined bacillus subtilis growing by xylose Download PDFInfo
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- CN103627663B CN103627663B CN201310664891.6A CN201310664891A CN103627663B CN 103627663 B CN103627663 B CN 103627663B CN 201310664891 A CN201310664891 A CN 201310664891A CN 103627663 B CN103627663 B CN 103627663B
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Abstract
The invention discloses recombined bacillus subtilis growing by xylose. An A basic group in the 184th position of an araR gene of the bacillus subtilis is replaced with a G basic group, and a sequence of the araR gene after the A basic group is replaced with the G basic group is as shown in SEQ ID NO.14. Gene mutation favorable for growth of the bacillus subtilis by xylose is discovered by adopting an evolutionary engineering and whole genome sequencing combination method, and point mutation is introduced in wild strains through a genetic engineering technology, so that the recombined bacillus subtilis capable of quickly growing by xylose is obtained.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to three kinds of recombined bacillus subtilis utilizing wood sugar to grow.
Background technology
The renewable biomass resource that lignocellulose extensively exists as nature, has broad application prospects.And wood sugar is the deputy carbohydrate of content in ligno-cellulose hydrolysate, be only second to glucose.Therefore effectively utilizing wood sugar is how one of key factor utilizing lignocellulose efficiently better.But only have minority to have the ability well utilizing wood sugar in microorganism known at present, the numerous essential industry microorganisms comprising subtilis do not have good xylose utilization ability, cannot utilize wood sugar even completely.
The research of wood sugar is utilized all based on traditional genetic engineering means at present about subtilis, by knocking out gene, means such as replacing gene promoter and the corresponding gene of process LAN etc. can make subtilis obtain the ability utilizing wood sugar to grow, but these engineering strains obtained by traditional method are all lower in the ability that consumes containing the energy for growth in the substratum of wood sugar and wood sugar.
And engineering means of evolving are widely used in the aspects such as substrate utilization improvement, being suddenlyd change by the evolution of microorganism self, more directly obtain the bacterial strain with good development proterties.But evolutionary process has uncertainty, while producing along with favourable sudden change, some irrelevant sudden changes also may produce, these sudden changes can cause the genome background of bacterial strain indefinite, affect follow-up genetically engineered operation, and some sudden change even may produce negative effect to some characteristic of bacterial strain, limit bacterial strain application prospect from now on.Therefore by genetic engineering means or evolution engineering method improved strain, all there is obvious weak point.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of recombined bacillus subtilis utilizing wood sugar to grow is provided.
Second object of the present invention is to provide the recombined bacillus subtilis that the second utilizes wood sugar to grow.
3rd object of the present invention is to provide the third recombined bacillus subtilis utilizing wood sugar to grow.
Technical scheme of the present invention is summarized as follows:
A kind of recombined bacillus subtilis (Bacillus subtilis) utilizing wood sugar to grow, araR gene the 184th A base of described bacillus is replaced by G base, and the araR gene order after base replacement is as shown in SEQ ID NO.14.
A kind of recombined bacillus subtilis (Bacillus subtilis) utilizing wood sugar to grow, araR gene the 184th A base of described bacillus is replaced by G base, araR gene order after base replacement is as shown in SEQ ID NO.14, sinR gene the 319th T base of described bacillus is replaced by C base, and the sinR gene order after base replacement is as shown in SEQ ID NO.15.
A kind of recombined bacillus subtilis (Bacillus subtilis) utilizing wood sugar to grow, araR gene the 184th A base of described bacillus is replaced by G base, araR gene order after base replacement is as shown in SEQ ID NO.14, sinR gene the 319th T base of described bacillus is replaced by C base, sinR gene order after base replacement is as shown in SEQ ID NO.15, comP gene the 1121st T base deletion of described bacillus, the comP gene order after base deletion is as shown in SEQ IDNO.16.
Advantage of the present invention: the method that the present invention adopts evolution engineering to be combined with genome sequencing, find the transgenation that Three Favorables subtilis utilizes wood sugar to grow, by genetic engineering technique, wild strain is introduced in point mutation, obtain the recombined bacillus subtilis that wood sugar can be utilized to grow fast.
Accompanying drawing explanation
Fig. 1 is the growth curve of bacterial strain 168AR ferment under aerobic conditions.
Fig. 2 is the growth curve of bacterial strain 168ARSR ferment under aerobic conditions.
Fig. 3 is the growth curve of bacterial strain 168ARSRCP ferment under aerobic conditions.
Embodiment
Below by specific embodiment, the present invention is further illustrated, and the following examples understand the present invention better to enable those skilled in the art to, but do not impose any restrictions the present invention.
The present invention is three kinds of subtilises that wood sugar can be utilized to grow (Bacillus subtilis) called after 168AR, 168ARSR, 168ARSRCP respectively.
Embodiment 1
The subtilis that wood sugar can be utilized to grow is obtained by evolution engineering.
By subtilis 168(is derived from BGSC, BGSC numbering 1A1, conventional gram-positive microorganism physiological Study type strain) continuous passage cultivation in the basic salt culture medium taking wood sugar as carbon source, improves the energy for growth that subtilis utilizes wood sugar.
Concrete operations are as follows: by subtilis 168 first extra add glucose to final concentration be 1g/L be cultivate in the basic salt culture medium of carbon source with wood sugar, culture condition is 37 DEG C, and 220rpm treats thalline OD
600be forwarded to when being about 2 with wood sugar be carbon source basic salt culture medium in cultivate, afterwards at thalli growth to OD
600to about 2 time be cultivation of transferring continuously in the basic salt culture medium of carbon source at wood sugar, transfer after 72 times, being separated to can with specific growth rate 0.45h in the basic salt culture medium taking wood sugar as carbon source
-1the bacterial strain of growth, called after E72.
Taking wood sugar as the formula of the basic salt culture medium of carbon source is: Na
2hPO
46.8g/L, KH
2pO
43.0g/L, NaCl0.5g/L, NH
4cl1.0g/L, MgSO
47H
2o246mg/L, CaCl
216.6mg/L, FeCl
26H
2o13.5mg/L, MnCl
24H
2o1.0mg/L, ZnCl
21.7mg/L, CuCl
22H
2o0.43mg/L, CoCl
26H
2o0.6mg/L, Na
2moO
42H
2o0.6mg/L, tryptophane 50mg/L, wood sugar 9g/L.
Embodiment 2
Nuo Hezhi source, Beijing bioinformation Science and Technology Ltd. subtilis 168 described in embodiment 1 and E72 bacterial strain is entrusted to carry out genome sequencing, by comparison two bacterial strain whole genome sequence, find that araR gene the 184th A base of E72 bacterial strain is replaced by G base, sinR gene the 319th T base is replaced by C base and comP gene 1121 T base deletions.
Embodiment 3
In order to sudden change being introduced wild subtilis 168, need to use the reorganization scheme not staying antibiotic marker, adopt scheme described in reference 1.Upp gene knock-out bacterial strain required in the program can build by reference to mode described in document 1 or other modes, and in the present embodiment, the upp gene knock-out bacterial strain building process of subtilis 168 is as follows:
With pUC18 plasmid (purchased from Thermo company) for carrier, by upp upstream region of gene homology arm, derive from pUB110 plasmid and (come from BGSC, plasmid contained by BGSC numbering 1E6 bacterial strain) kalamycin resistance gene neo and upp downstream of gene homology arm fit together by said sequence, finally obtain the plasmid pUCMupp for upp gene knockout, pUCMupp Plastid transformation is entered in subtilis 168, use kantlex screens, and obtains the starting strain 168 Δ upp of upp gene knockout through PCR checking.
Embodiment 4
The mutant nucleotide sequence of araR gene in the E72 bacterial strain described in embodiment 2 is introduced 168 Δ upp bacterial strains described in embodiment 3.To obtain 168AR strain construction process as follows:
Amplimer Pcu1/Pcu2 is utilized to be derived from BGSC with plasmid pHP13(, plasmid contained by BGSC numbering 1E50 bacterial strain) contain the DNA fragmentation of pBM1ori and chloramphenicol resistance gene cat for template amplification, utilize primer Pcu3/Pcu4 with subtilis 168 genome for template, amplification upp gene and promoter region thereof, above-mentioned 2 DNA fragmentations connected with XhoI/SalI and BamHI/BglII site and passes through bacillus coli DH 5 alpha (purchased from Invitrogen company) clone, obtaining plasmid p9CU.
Utilize amplimer ParaR1/ParaR2 with E72 genome for template, amplification by the DNA fragmentation of G base Substitution containing araR gene the 184th A base, is cloned in plasmid pUC18 (purchased from Thermo company) in SmaI site and obtains plasmid pUCaraR.
Utilize amplimer Pcu3/Pcu5 with plasmid p9CU for template, the DNA fragmentation of amplification containing cat and upp gene, is cloned in plasmid pUCaraR in AfeI site, obtains plasmid pMaraR.
Utilize amplimer ParaR1/ParaR2 to be that template increases with pMaraR, the fragment obtained is transformed into subtilis 168 Δ upp, with the successful positive colony of paraxin screening restructuring, and is verified by PCR, obtains bacterial strain 168 Δ upp Δ araR.
Utilize amplimer ParaR3/ParaR4 with E72 genome for template, amplification is containing araR gene the 184th DNA fragmentation that A base is replaced by G base, the fragment obtained is transformed into subtilis 168 Δ upp Δ araR, with the successful positive colony of 5 FU 5 fluorouracil screening restructuring, and verified by DNA sequencing, obtain bacterial strain 168AR, this bacterial strain is that araR gene the 184th A base of bacillus is replaced by G base, the bacterial strain of araR gene order as shown in SEQ ID NO.14 after base replacement.
168AR bacterial strain is carried out aerobic shake flask fermentation in the basic salt culture medium taking wood sugar as carbon source, and fermented liquid liquid amount is that culture condition is 37 DEG C, 220rpm, and result as shown in Figure 1 containing 50ml fermented liquid in the Erlenmeyer flask of 500ml specification.
The acquisition of 168AR bacterial strain take wood sugar as the ability of carbon source for growth, and under aerobic conditions of flask fermentation, its maximum specific growth rate is 0.28h
-1.Because the sequence of sudden change is open, other people can to build in the present embodiment constructed bacterial strain in other way, and the 168AR bacterial strain that the present embodiment builds is only checking araR gene the 184th A base and is replaced the ability that 168 Δ upp bacterial strains can be made to obtain with wood sugar be carbon source for growth by G base.
Embodiment 5
The 168AR bacterial strain described in embodiment 4 is introduced in the sudden change of sinR gene in the E72 bacterial strain described in embodiment 2, to obtain 168ARSR strain construction process as follows:
Utilize amplimer PsinR1/PsinR2 with E72 strain gene group for template, amplification, containing sinR gene the 319th DNA fragmentation that T base is replaced by C base, is cloned in the plasmid p9CU described in embodiment 4 in SmaI site and obtains plasmid p9CU-sinR.
Plasmid p9CU-sinR is transformed into 168AR bacterial strain, with the successful positive colony of paraxin screening restructuring, when in access LB substratum, 37 DEG C of 220rpm are cultured to OD600=2, get the 5ul bacterium liquid flat board 37 DEG C coated containing 5 FU 5 fluorouracil and cultivate 16 hours screening second time restructuring successfully clone, through PCR and DNA sequencing checking, obtain bacterial strain 168ARSR, this bacterial strain is that araR gene the 184th A base of bacillus is replaced by G base, araR gene order after base replacement is as shown in SEQ IDNO.14, sinR gene the 319th T base of bacillus is replaced by C base, the bacterial strain of sinR gene order as shown in SEQ ID NO.15 after base replacement.
168ARSR bacterial strain is carried out aerobic shake flask fermentation in the basic salt culture medium taking wood sugar as carbon source, and fermented liquid liquid amount is that culture condition is 37 DEG C, 220rpm, and result as shown in Figure 2 containing 50ml fermented liquid in the Erlenmeyer flask of 500ml specification.
168ARSR bacterial strain in the basic salt culture medium taking wood sugar as carbon source aerobic shake flask fermentation specific growth rate comparatively 168AR be improved.Because the sequence of sudden change is open, other people can build bacterial strain constructed in the present embodiment in other way, the 168ARSR bacterial strain that the present embodiment builds is only checking araR gene the 184th A base and is replaced by G base and replaced the ability that 168 Δ upp bacterial strains can be made to obtain with wood sugar be carbon source for growth by C base with sinR gene the 319th T base, and under aerobic conditions of flask fermentation, its maximum specific growth rate is 0.44h
-1, maximum cell concentration is OD600=5.5.
Embodiment 6
The mutant nucleotide sequence of comP gene in the E72 bacterial strain described in embodiment 2 is introduced the 168ARSR bacterial strain described in embodiment 5, to obtain 168ARSRCP strain construction process as follows:
Utilize amplimer PcomP1/PcomP2 with E72 strain gene group for template, the DNA fragmentation of amplification containing the 1121st the T base deletion of comP gene, is cloned in the plasmid p9CU described in embodiment 4 in SmaI site and obtains plasmid p9CU-comP.
Plasmid p9CU-comP is transformed into 168ARSR bacterial strain, with the successful positive colony of paraxin screening restructuring, when in access LB substratum, 37 DEG C of 220rpm are cultured to OD600=2, get the 5ul bacterium liquid flat board 37 DEG C coated containing 5 FU 5 fluorouracil and cultivate 16 hours screening second time restructuring successfully clone, through PCR and DNA sequencing checking, obtain 168ARSRCP bacterial strain, this bacterial strain is that araR gene the 184th A base of bacillus is replaced by G base, araR gene order after base replacement is as shown in SEQ IDNO.14, sinR gene the 319th T base of bacillus is replaced by C base, sinR gene order after base replacement is as shown in SEQ ID NO.15, comP gene the 1121st T base deletion of bacillus, the bacterial strain of comP gene order after base deletion as shown in SEQ ID NO.16.
168ARSRCP bacterial strain is carried out aerobic shake flask fermentation in the basic salt culture medium taking wood sugar as carbon source, and fermented liquid liquid amount is that culture condition is 37 DEG C, 220rpm, and result as shown in Figure 3 containing 50ml fermented liquid in the Erlenmeyer flask of 500ml specification.
168ARSRCP bacterial strain aerobic shake flask fermentation in the basic salt culture medium taking wood sugar as carbon source can obtain the maximum cell concentration higher compared with 168ARSR.Because the sequence of sudden change is open, other people can build bacterial strain constructed in the present embodiment in other way, the 168ARSRCP bacterial strain that the present embodiment builds is only that checking araR gene the 184th A base is replaced by G base, sinR gene the 319th T base is replaced by C base and the 1121st the T base deletion of comP gene can make 168 Δ upp bacterial strains obtain with wood sugar to be the ability of carbon source for growth, and under aerobic conditions of flask fermentation, its specific growth rate is 0.44h
-1, maximum cell concentration is OD600=6.4.
Table 1 strain construction the primer sequence
Embodiment 7
What 168AR, 168ARSR and 168ARSRCP bacterial strain was adjusted to 18g/L at xylose concentration is carry out micro-oxygen shake flask fermentation production acetoin in the basic salt culture medium of carbon source with wood sugar.
Fermented liquid liquid amount is the fermented liquid that 100ml is housed in the Erlenmeyer flask of 250ml specification, culture condition is 37 DEG C, 220rpm, result 168AR bacterial strain exhausted the xylose production of the 18.35g/L acetoin of 4.17g/L in 72 hours, yield is 46% of theoretical yield, 168ARSR bacterial strain run out of the wood sugar of 18.38g/L in 60 hours, produce the acetoin of 4.89g/L, for 54% of theoretical yield, 168ARSRCP bacterial strain run out of the wood sugar of 18.27g/L in 60 hours, produce the acetoin of 5.41g/L, be 61% of theoretical yield, fermenting process is as shown in table 2.
Table 2
As can be seen from the result of micro-oxygen shake flask fermentation, what the subtilis that three strains constructed by the present invention can utilize wood sugar to grow was adjusted to 18g/L at xylose concentration is can effectively accumulate product acetoin in the basic salt culture medium of carbon source with wood sugar, and achieve higher yield, there is good application prospect.
Reference 1:Fabret C, Ehrlich SD, Noirot P.2002.A new mutation delivery systemfor genome-scale approaches in Bacillus subtilis.Mol Microbiol46 (1): 25-36.
Claims (3)
1. the recombined bacillus subtilis utilizing wood sugar to grow, is characterized in that araR gene the 184th A base of described bacillus is replaced by G base, and the araR gene order after base replacement is as shown in SEQ ID NO.14.
2. the recombined bacillus subtilis utilizing wood sugar to grow, it is characterized in that araR gene the 184th A base of described bacillus is replaced by G base, araR gene order after base replacement is as shown in SEQ ID NO.14, sinR gene the 319th T base of described bacillus is replaced by C base, and the sinR gene order after base replacement is as shown in SEQ ID NO.15.
3. the recombined bacillus subtilis utilizing wood sugar to grow, it is characterized in that araR gene the 184th A base of described bacillus is replaced by G base, araR gene order after base replacement is as shown in SEQ ID NO.14, sinR gene the 319th T base of described bacillus is replaced by C base, sinR gene order after base replacement is as shown in SEQ ID NO.15, comP gene the 1121st T base deletion of described bacillus, the comP gene order after base deletion is as shown in SEQ ID NO.16.
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| KR20020087004A (en) * | 2002-09-23 | 2002-11-21 | 주식회사 바이오앤진 | A biological method for producing D-ribose from D-xylose using a Bacillus subtilis mutant |
| WO2006096130A1 (en) * | 2005-03-11 | 2006-09-14 | Forskarpatent I Syd Ab | Arabinose- and xylose-fermenting saccharomyces cerevisiae strains |
| CN101870961A (en) * | 2010-06-11 | 2010-10-27 | 广西民族大学 | Method for extracting xylitol and D-galactitol from xylose mother solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20020087004A (en) * | 2002-09-23 | 2002-11-21 | 주식회사 바이오앤진 | A biological method for producing D-ribose from D-xylose using a Bacillus subtilis mutant |
| WO2006096130A1 (en) * | 2005-03-11 | 2006-09-14 | Forskarpatent I Syd Ab | Arabinose- and xylose-fermenting saccharomyces cerevisiae strains |
| CN101870961A (en) * | 2010-06-11 | 2010-10-27 | 广西民族大学 | Method for extracting xylitol and D-galactitol from xylose mother solution |
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