Butyl butyrate synthesis strain as well as construction method and application thereof

Abstract

The invention discloses a butyl butyrate synthesis strain as well as a construction method and application thereof, the butyl butyrate synthesis strain takes clostridium tyrobutyricum as host bacteria, and overexpresses genes of aldol dehydrogenase AdhE2 and lipase LipA; the aldol dehydrogenase AdhE2 and the lipase LipA are derived from clostridium acetobutylicum. The butyl butyrate synthesis strain provided by the invention can realize de novo synthesis of butyl butyrate with high efficiency and high selectivity without exogenous addition of precursors and lipase, and provides an adaptive gene element basis for subsequent modification.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation engineering, in particular to a butyl butyrate synthesis strain and a construction method and application thereof. Background Art

[0002] Butyrate is an important short-chain fatty acid ester widely used in food, cosmetics, solvents, and biofuels. It can be used as a food additive to enhance flavor, a fuel additive, or even as a substitute for aviation fuel. Traditional methods for its synthesis are harsh and environmentally unfriendly. Microbial synthesis of butyl butyrate has garnered attention in recent years as a new, more environmentally friendly, cost-effective, and efficient pathway. Currently, microbial synthesis of butyl butyrate can be achieved through de novo synthesis (without the need for exogenous addition of precursors or esterification enzymes) or through the use of exogenous precursors or enzymes. The latter is relatively easy to implement and has already yielded good yields, but the production cost is relatively high.

[0003] At present, some progress has been made in the de novo synthesis of butyl butyrate by microorganisms. For example, the applicant's previous application CN114317623A discloses a method for synthesizing butyl butyrate based on a lipase-producing Escherichia coli surface-displaying strain. This method uses a mixed culture of Escherichia coli and some butyric acid-producing Clostridium to synthesize esters including butyl butyrate. Different strains provide the precursors required for synthesis, and the lipase expressed in the mixed bacterial system then undergoes an esterification reaction to obtain the final product, butyl butyrate. However, this scheme has the limitation that the ratio of mixed bacteria inoculation must be strictly controlled, and the mixed bacterial system is relatively unstable. CN114395575A discloses a recombinant strain of Clostridium tyrobutyricum for producing butyl butyrate. By overexpressing the aldol dehydrogenase AdhE2 from Clostridium acetobutylicum ATCC824 and the alcohol acyltransferase VAAT from strawberry in Clostridium tyrobutyricum ATCC 25755, butyl butyrate can be synthesized using glucose and mannitol as raw materials. However, microbial synthesis of butyl butyrate still faces many challenges, such as the difficulty in subsequent modification of plant-derived esterases and low selectivity for butyl butyrate. Summary of the Invention

[0004] The first object of the present invention is to provide a butyl butyrate synthesis strain, which can efficiently synthesize butyl butyrate from scratch without the need for additional butanol or lipase catalysis and has high selectivity.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A butyl butyrate-producing strain, which uses Clostridium tyrobutyricum as a host bacterium and overexpresses genes for alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA; Wherein, the alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA are derived from Clostridium acetobutylicum.

[0006] In some embodiments of the present invention, the genes of the alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA are overexpressed using the endogenous promoter of the host bacteria.

[0007] In some embodiments of the present invention, the host bacterium is Clostridium tyrobutyricum L319, whose deposit number is GDMCC No: 62289; the endogenous promoter Pth1 of Clostridium tyrobutyricum L319 is used to overexpress the genes of alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA.

[0008] In some embodiments of the present invention, the alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA are derived from Clostridium acetobutylicum ATCC824.

[0009] The second object of the present invention is to provide a method for constructing the above-mentioned strain, which comprises: The promoter, the genes of alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA were connected and constructed into a vector plasmid to obtain a recombinant plasmid; Using Escherichia coli as a donor bacterium, the recombinant plasmid is introduced into the host bacterium by conjugation transfer.

[0010] In some embodiments of the present invention, the promoter, the genes of alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA are connected by Gibson assembly.

[0011] In some embodiments of the present invention, the vector plasmid is pMTL82151.

[0012] The third object of the present invention is to provide the use of the above-mentioned strain in the synthesis of butyl butyrate.

[0013] In some embodiments of the present invention, the butyl butyrate-synthesizing strain is inoculated into a fermentation medium to synthesize butyl butyrate de novo.

[0014] In some embodiments of the present invention, the fermentation medium is TGY medium.

[0015] Clostridium tyrobutyricum is not a model strain and has poor expression capacity for exogenous genes. In addition, many common lipases or lipase-like enzymes are derived from fungi such as yeast and Candida, as well as from plants. These lipases are incompatible with expression in prokaryotes. However, the present invention found that co-expressing the alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA from Clostridium acetobutylicum in Clostridium tyrobutyricum has little effect on its growth and can achieve efficient and highly selective de novo synthesis of butyl butyrate without the need for exogenous addition of precursors or lipases, providing a foundation of adaptive genetic elements for subsequent modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1It is the recombinant synthesis pathway of butyl butyrate by Clostridium tyrobutyricum.

[0017] Figure 2 Comparison of the growth performance of the recombinant strains Ct(pMTL82151-Pthl-adhE2-lipA) and Ct(pMTL82151-Pthl-adhE2).

[0018] Figure 3 Comparison of the fermentation performance of the recombinant strains Ct(pMTL82151-Pthl-adhE2-lipA) and Ct(pMTL82151-Pthl-adhE2).

[0019] Figure 4 The ester fermentation yield and composition of the recombinant strain Ct (pMTL82151-Pthl-adhE2-lipA) are shown. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] The host bacteria used in the examples is Clostridium tyrobutyricum L319, which has been disclosed in the applicant's prior application CN115305219A, with a deposit number of GDMCC No: 62289.

[0022] The culture medium components used in the examples are as follows: LB medium: NaCl 10 g / L, yeast powder 5 g / L, tryptone 10 g / L; Fermentation medium (TGY medium): 30 g / L peptone, 50 g / L glucose, 10 g / L yeast powder, 40 g / L calcium carbonate.

[0023] Example 1 Construction of engineered strain Ct (pMTL82151-Pthl-adhE2-lipA) (1) Construction of recombinant plasmid pMTL82151-Pthl-adhE2-lipA: Using the vector plasmid pMTL82151 as a template, the primers V82-F and V82-R shown in Table 1 were used to amplify the linearized vector V82151 according to the system and procedures in Tables 2 and 3. Using the genome of Clostridium tyrobutyricum L319 as a template, the primers thl-F and thl-R were used to amplify the thl promoter fragment (P thl , the nucleotide sequence is shown in SEQ ID NO: 1), and using Clostridium acetobutylicum ATCC834 as a template, the fragment AdhE2 ( adhE2The lipase fragment (lipA, nucleotide sequence shown in SEQ ID NO:2) was amplified using primers LP-F and LP-R. This fragment was ligated into the vector pMTL82151 between the restriction sites SacI and XbaI via Gibson assembly. The fragment was then transformed into competent E. coli CA434 cells using the heat shock method, plated onto LB medium plates containing 30 μg / mL of lipase, and incubated at 37°C for 12 hours. Colonies growing on the plate were subsequently verified using primers JP-F and JP-R. Amplified colonies that matched the expected band were then transferred to liquid LB medium. When cultured to stationary phase (OD = 3), the plasmid was extracted and sent to Anhui General Biotechnology (Chuzhou) Co., Ltd. for Sanger sequencing for further verification.

[0024] Table 1 Primers for constructing recombinant plasmid pMTL82151-Pthl-adhE2-lipA

[0025] Table 2 PCR system

[0026] Table 3 PCR reaction procedure

[0027] (2) Conjugation transformation of recombinant plasmid: The E. coli CA434 with the correct recombinant plasmid verified above was activated in liquid LB medium, and then transferred to fresh LB medium at a 1% inoculation volume. When the culture reached OD = 1-2, 5 mL of bacteria were collected and centrifuged (5000 rpm, 2 min), and then resuspended and washed with an equal amount of 10 mM 1xPBS buffer. After centrifugation again, the bacterial sludge was retained for later use. Then, 2.5 mL of E. coli was mixed with 0.4 mL of Clostridium tyrobutyricum (OD = 2-3) statically cultured in TGY culture concentrate, resuspended and spread on non-resistant TGY plates, and cultured anaerobically for 24 h. Then, the colonies on the plates were washed with 0.5-0.6 mL of fresh TGY liquid medium and transferred to a plate containing (15 μg / mL thiamphenicol and 250 The culture medium was plated on TGY plates containing 1 μg / mL cycloserine for 2-5 days until a single colony appeared. PCR verification was performed using primers JP-F and JP-R, and the engineered Clostridium tyrobutyricum Ct (pMTL82151-Pthl-adhE2-lipA) was finally obtained.

[0028] Example 2 Construction of engineered strain Ct (pMTL82151-Pthl-adhE2) The plasmid was constructed using the same method as in Example 1, using the following primers: Table 4 Primers for constructing recombinant plasmid pMTL82151-Pthl-adhE2

[0029] The engineered Clostridium tyrobutyricum Ct (pMTL82151-Pthl-adhE2) was obtained by the same conjugation method as in Example 1.

[0030] Example 3 Fermentation test of engineered strain Ct (pMTL82151-Pthl-adhE2) and engineered strain Ct (pMTL82151-Pthl-adhE2-lipA) (1) After activating the engineered strain using TGY medium for 12 h, the activated culture medium was transferred to a serum bottle containing 50 mL of fresh TGY at an inoculum volume of 5% (v / v). After static culture at 37°C for 12 h, hexadecane with a volume of 1 / 2 of the culture medium was added. After 60 h, the upper layer sample was taken for gas chromatography detection, and the lower layer was used for high-performance liquid chromatography detection.

[0031] (2) The results are as follows Figure 2 As shown, compared with the control strain Ct (pMTL82151-Pthl-adhE2), the engineered strain Ct (pMTL82151-Pthl-adhE2-lipA) had little effect on its growth after the introduction of Clostridium-derived lipase. Only after butyl butyrate accumulated during the stable period did the bacteria rupture, resulting in a relatively faster decrease in the OD value during the final fermentation period. At the same time, the yield composition in the fermentation broth was detected by liquid phase ( Figure 3 ) shows that the overall composition of acetic acid, butyric acid, and butanol has not changed. However, due to the introduction of lipase, the production of the three has decreased relatively. This is evidence that lipase uses acetic acid, butyric acid, and butanol as catalytic precursors to convert them into esters. Among them, acetic acid decreased by 13.9%, butyric acid decreased by 23.1%, and butanol decreased by 7.5%. Finally, the production of esters in the organic phase (n-hexadecane) was determined, as shown in Figure 2. Figure 4 As shown, the yield of butyl butyrate was 1.57 g / L and the yield of butyl acetate was 0.03 g / L, of which butyl butyrate accounted for 98.3%.

[0032] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A butyl butyrate-synthesizing strain, characterized in that: The strain uses Clostridium tyrobutyricum as a host bacterium and overexpresses the genes of alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA; Wherein, the alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA are derived from Clostridium acetobutylicum.

2. The strain according to claim 1, characterized in that The endogenous promoter of the host bacteria is used to overexpress the genes of the alcohol-aldehyde dehydrogenase AdhE2 and the lipase LipA.

3. The strain according to claim 1, characterized in that The host bacterium is Clostridium tyrobutyricum L319, and its preservation number is GDMCC No: 62289. The endogenous promoter Pth1 of Clostridium tyrobutyricum L319 is used to overexpress the genes of the alcohol-aldehyde dehydrogenase AdhE2 and the lipase LipA.

4. The strain according to claim 1, characterized in that The aldol dehydrogenase AdhE2 and lipase LipA are derived from Clostridium acetobutylicum ATCC824.

5. The method for constructing the strain according to any one of claims 1 to 4, characterized in that: The construction method comprises: The promoter, the genes of alcohol-aldehyde dehydrogenase AdhE2 and lipase LipA were connected and constructed into a vector plasmid to obtain a recombinant plasmid; Using Escherichia coli as a donor bacterium, the recombinant plasmid is introduced into the host bacterium by conjugation transfer.

6. The method according to claim 5, characterized in that The promoter, aldol dehydrogenase AdhE2 and lipase LipA genes were connected by Gibson assembly.

7. The method according to claim 5, characterized in that The vector plasmid is pMTL82151.

8. Use of the strain according to any one of claims 1 to 4 in the synthesis of butyl butyrate.

9. The use according to claim 8, characterized in that The butyl butyrate-synthesizing strain is inoculated into a fermentation medium to synthesize butyl butyrate de novo.

10. The use according to claim 8 or 9, characterized in that: The fermentation medium is TGY medium.

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