The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 1805282_ST25.txt. The size of the text file is 50,426 bytes, and the text file was created on Jul. 25, 2018.

FIELD OF THE INVENTION

The present invention relates to the field of gene engineering, and in particular to a D-lactate dehydrogenase, a construction method of abacillusgene engineering strain containing the D-lactate dehydrogenase and use of the engineered strain in production of D-lactic acid.

DESCRIPTION OF THE PRIOR ART

Lactic acid is an important industrial raw material and can be applied to pharmaceutical, food, cosmetic and petrochemical industries, etc. In recent years, as a monomer, lactic acid is also used for synthesizing high-strength biodegradable plastic polylactic acid (PLA). Traditionally, PLA is polymerized by using highly optically-pure L-lactic acid.

Furthermore, by using mixed L-lactic acid and D-lactic acid, the mechanical performance, heat stability and hydrolysis resistance of products using polylactic acid as materials can be obviously improved, thereby greatly exciting the market demands for D-lactic. Although lactic acid can be synthesized by using petroleum as a raw material, the synthesized lactic acid is a mixture of two types of isomerides and is not applicable to production of PLA. Highly optically-pure L-lactic acid and D-lactic acid needed for producing PLA can only be produced through microbial fermentation, and thus many scholars carry out researches on production of D-lactic acid by using a microbial fermentation method.

High-temperature fermentation can minimize the risk of pollution, improve the conversion rate of raw materials and reduce heat supply cost. Up to now, production of optically-pure L-lactic acid at high temperature has been widely studied, which makes a huge contribution to the commercialized production of L-lactic acid. In regard to D-lactic acid, people always eager to realize high-temperature fermentation production of D-lactic acid. However, there are few reports about thermophilic production of D-lactic acid, and the concentration, production speed and rich culture medium components for reported D-lactic acid are not suitable for industrial production. Therefore, it is very necessary to develop a low-cost strong microbial platform to produce D-lactic acid at high temperature.

D-lactate dehydrogenase on which nicotinamide adenine dinucleotide (NADH) depends is a key enzyme for microbial synthesis of D-lactic acid. However, most natural D-lactate dehydrogenases are not stable to heat, and this is one of major bottlenecks for microbial production of D-lactic acid at high temperature. It is reported that, as proved by in-vitro determination of enzyme activity of D-lactate dehydrogenases inLactobacillus plantarum, this enzyme has very high enzyme activity at 42° C., but is fully deactivated after incubation for 3 min at 50° C., while L-lactate dehydrogenases inLactobacillus plantarumafter treatment by using the same process still reserve 94% of enzyme activity. As also shown by other in-vivo experiments, D-lactate dehydrogenases are easily deactivated under a high-temperature condition. On the whole, the low activity or extremely poor heat stability of the D-lactate dehydrogenases reported above prevents the production of D-lactic acid under a high-temperature condition by using the D-lactate dehydrogenases. In order to realize high-efficiency production of D-lactic acid at high temperature, it is an important task to find a D-lactate dehydrogenase having high heat stability.

Heat-resistantBacillus licheniformisATCC 14580 is a facultative anaerobic, Gram-positive endospore-type bacterium. For a microbial fermentation platform, it has a lot of potential advantages as follows: 1) it can use various pentoses and hexoses; 2) it has a fast cell growth speed and thus can shorten the fermentation period; 3) it can be subjected to genetic operations; and 4) it is a “Generally Recognized as Safe” strain generally accepted by the Food and Drug Administration. These advantages show that strain ATCC 14580 can be used as an ideal platform strain. Up to now, this strain has already been proved to be usable for high-temperature fermentation production of 2,3-butanediol.

Therefore, one skilled in the art devotes himself to developing an engineered strain for D-lactic acid with a high yield at high temperature and preparation and use thereof, by using heat-resistantBacillus licheniformisATCC 14580 as an original strain.

SUMMARY OF THE INVENTION

With regard to the defects that most natural D-lactate dehydrogenases in the prior art are poor in heat stability, the cost of the existing thermophilic production of D-lactic acid is high and the concentration and production speed of D-lactic acid are not suitable for industrial production, the present invention provides an engineered strain for producing D-lactic acid with a high yield at high temperature, and preparation method and use of the strain. The present invention discloses a D-lactate dehydrogenase which has unique properties and is extracted fromThermodesulfatator indicus, and this enzyme has extremely good thermophily and heat stability. After performing codon optimization for the enzyme via colibacillus, by using the optimized enzyme and the gene engineering construction method, a fermentation product of the reconstructedBacillus licheniformiscan be redirected to optically-pure D-lactic acid with a high yield from naturally produced 2,3-butanediol.

In one aspect, the present invention provides a D-lactate dehydrogenase. In one specific implementation mode, the D-lactate dehydrogenase has one of the following amino acid sequences:

1) an amino acid sequence expressed by SEQ ID No. 1;

2) an amino acid sequence derivatively produced from the amino acid sequence expressed by SEQ ID No. 1 through substitution, deletion, insertion or addition of one or more amino acid residues, and a protein produced by the amino acid sequence having D-lactate dehydrogenase activity;

3) an amino acid sequence produced from the amino acid sequence expressed by SEQ ID No. 1 through conservative replacement; and

4) an amino acid sequence having at least 80% of homology with the amino acid sequence expressed by SEQ ID No. 1.

Further, the substitution, deletion, insertion or addition of the amino acid residues occurs outside a functional domain comprising enzyme catalytic sites, ligand binding sites and NAD binding sites of the D-lactate dehydrogenase.

Further, according to relative positions, key amino acids at the enzyme catalytic sites are Arg at position 229, Glu at position 258 and His at position 290; key amino acids at the ligand binding sites are Ser, Ala and Gly positions 75-77, Tyr at position 99, Arg at position 229, His at position 290 and Phe at position 293; and key amino acids at the NAD binding sites are Tyr at position 99, Ile at position 104, Gly at position 150, Gly, Lys, Ile and Gly at positions 152-155, Tyr, Asp and Pro at positions 172-174, His, Cys, Pro and Leu at positions 199-202, Asn at position 206, Met at position 209, Thr, Ala and Arg at positions 227-229, Asp and Val at positions 253 and 254, His at position 290, and Ala and Phe at positions 292 and 293.

In another aspect, the present invention provides a nucleotide sequence. In one specific implementation mode, the nucleotide sequence codes the D-lactate dehydrogenase according to claim1.

Further, the nucleotide sequence is one of the following nucleotide sequences:

1) a nucleotide sequence expressed by SEQ ID No. 2;

2) a nucleotide sequence expressed by SEQ ID No. 3;

3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3; and

4) a nucleotide sequence hybridized with a complementary chain of the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3 under a high-stringency condition.

In another aspect, the present invention provides use of the D-lactate dehydrogenase as described above or the nucleotide sequence as described above in production of D-lactic acid.

In another aspect, the present invention provides a gene engineering strain. In one specific implementation mode, the gene engineering strain has the D-lactate dehydrogenase as described above or a D-lactate dehydrogenase coded by the nucleotide sequence as described above.

Further, in the gene engineering strain, a pathway for synthesizing L-lactic acid by pyruvic acid is blocked and a pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked.

Preferably, the pathway for synthesizing L-lactic acid by pyruvic acid is blocked by deactivating or deleting an L-lactate dehydrogenase gene; and the pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked by deactivating or deleting one or both of an acetolactate synthase gene and an acetolactate decarboxylase gene.

Preferably, in the gene engineering strain, one or more of a pathway for synthesizing formic acid by pyruvic acid, a pathway for synthesizing acetic acid by pyruvic acid, and a pathway for synthesizing ethanol by pyruvic acid are blocked.

Preferably, the pathway for synthesizing formic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate formate-lyase gene and a pyruvate formate-lyase activating enzyme gene; the pathway for synthesizing acetic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an acetokinase gene; and the pathway for synthesizing ethanol by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an ethanol dehydrogenase gene.

Preferably, an original D-lactate dehydrogenase gene in the gene engineering strain is deactivated or deleted.

Further, an original strain of the gene engineering strain isthermophilus.

Further, an original strain of the gene engineering strain isbacillus.

Preferably, thebacillusis selected from a group consisting ofBacillus licheniformis, Bacillus coagulans, B. subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus circulansandAneurinibacillus aneurinilyticus.

Preferably, a restrictive modification system in the original strain is deactivated or knocked out.

Preferably, the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 ofBacillus licheniformisATCC 14580.

Further, a promoter of a coding gene of the above D-lactate dehydrogenase is reconstructed into a constitutive promoter.

Preferably, the constitutive promoter is a promoter Palsof α-acetolactate synthase ofBacillus licheniformisATCC 14580, a Pcpromoter in plasmid pMMPc, a P43promoter inB. subtilisor a promoter Pldhof L-lactate dehydrogenase ofBacillus licheniformisATCC 14580.

More preferably, the constitutive promoter is a promoter Palsof α-acetolactate synthase ofBacillus licheniformisATCC 14580.

Further, the gene engineering strain isBacillus licheniformisBN11, the preservation number of which is CCTCC NO: M2016026 and which was preserved in China Center for Type Culture Collection on Jan. 8, 2016.Bacillus licheniformisBN11 was deposited on Jan. 8, 2016 under the Budapest Treaty with the China Center for Type Culture Collection at Wuhan University, Wuhan 430072, P.R. China, under accession CCTCC M 2016026.

In another aspect, the present invention provides a method for preparing the gene engineering strain as described above. In one specific implementation mode, the method comprises: deactivating or deleting an L-lactate dehydrogenase gene in an original strain; and introducing the nucleotide sequence of the D-lactate dehydrogenase as described above.

Further, the method further comprises: deactivating or deleting an original D-lactate dehydrogenase gene in the original strain; and blocking a pathway for synthesizing 2,3-butanediol by pyruvic acid.

Further, the method further comprises: reconstructing a promoter of the introduced nucleotide sequence into a constitutive promoter.

Further, the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 ofBacillus licheniformisATCC 14580; and α-acetolactate decarboxylase (alsD) and acetolactate synthase (alsS) genes are replaced with the nucleotide sequence as described above.

In still another aspect, the present invention further provides use of the above gene engineering strain, in particular use in production of D-lactic acid.

In one specific implementation mode, a carbon source adopted in the production is one or more selected from a group consisting of glucose, xylose, maltose, lactose and sucrose.

Preferably, low-price culture mediums containing peanut meal or dried corn steep liquor powder are adopted for fermentation in the production.

Preferably, fermentation temperature for the production is 45° C.-55° C.

Preferably, pH of the fermentation culture mediums in the production is 6.0-8.0.

Preferably, a fermentation process in the production is a continuous fermentation or fed-batch process.

In another aspect, the present invention provides a D-lactate dehydrogenase. In one specific implementation mode, the D-lactate dehydrogenase has one of the following amino acid sequences:

1) an amino acid sequence expressed by SEQ ID No. 1;

2) an amino acid sequence of a derivative protein produced from the amino acid sequence expressed by SEQ ID No. 1 through substitution, deletion or insertion of one or more amino acid residues, the derivative protein having D-lactate dehydrogenase activity; and

3) an amino acid sequence having at least about 80% of homology with the amino acid sequence expressed by SEQ ID No. 1.

The protein comes fromT. indicus. The above protein may be artificially synthesized and may also be obtained by firstly synthesizing a coding gene thereof and then performing biological expression.

The present invention further provides nucleic acid molecules coding the above D-lactate dehydrogenase, and these nucleic acid molecules may be DNA such as cDNA, genome DNA or recombinant DNA; and may also be RNA such as mRNA, hnRNA or tRNA.

Further, the present invention provides a nucleotide sequence coding the above D-lactate dehydrogenase. The nucleotide sequence is one of the following nucleotide sequences:

1) a nucleotide sequence expressed by SEQ ID No. 2;

2) a nucleotide sequence expressed by SEQ ID No. 3; and

3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3.

In another aspect, the present invention provides use of the above D-lactate dehydrogenase or the above nucleotide sequence in production of D-lactic acid.

In another aspect, the present invention provides a gene engineering strain, characterized in that the gene engineering strain has the D-lactate dehydrogenase according to claim1or a D-lactate dehydrogenase coded by the nucleotide sequence according to claim2.

Further, in the above gene engineering strain, the L-lactate dehydrogenase gene is deactivated or deleted, and acetolactate synthase and/or acetolactate decarboxylase genes are deactivated or deleted.

Further, the above gene engineering strain further has one or more genes which are deactivated or deleted, and the one or more genes are selected from pyruvate formate-lyase gene, pyruvate formate-lyase activating enzyme gene, pyruvate dehydrogenase gene, acetokinase and ethanol dehydrogenase gene.

Further, an original strain of the above gene engineering strain isbacillus. Further, thebacillusare selected from a group consisting ofBacillus licheniformis, Bacillus coagulans, B. subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus circulansandAneurinibacillus aneurinilyticus. Preferably, the original strain isBacillus licheniformis. Preferably, the original strain isBacillus licheniformisATCC 14580. Preferably, the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 ofBacillus licheniformisATCC 14580.

Further, a promoter of the above D-lactate dehydrogenase gene is a promoter Palsof α-acetolactate synthase ofBacillus licheniformisATCC 14580, a Pcpromoter in plasmid pMMPc, a P43promoter inB. subtilisor a promoter Pldhof L-lactate dehydrogenase ofBacillus licheniformisATCC 14580. Preferably, the promoter is directly connected with an original codon “ATG” of ldhTigene.

Further, the above gene engineering strain isBacillus licheniformisBN11, the preservation number of which is CCTCC NO: M2016026 and which was preserved in China Center for Type Culture Collection on Jan. 8, 2016.

In still another aspect, the present invention provides a method for preparing the above gene engineering strain, comprising the following steps:

1) knocking out an L-lactate dehydrogenase gene in an original strain;

2) introducing the above nucleotide sequence coding the D-lactate dehydrogenase;

3) deactivating or deleting an acetolactate synthase gene and/or acetolactate decarboxylase gene; and

4) while or after performing step 3), adding a promoter sequence in the front of the nucleotide sequence introduced in step 2).

Specifically, by using a mutant strain MW3 ofBacillus licheniformisATCC 14580 with the L-lactate dehydrogenase gene which is knocked out as a host, and replacing α-acetolactate decarboxylase (alsD) and acetolactate synthase (alsS) with the above D-lactate dehydrogenase, a heat-resistant engineeringbacillusstrain CCTCC NO: M2016026 capable of producing D-lactic acid through fermentation at temperature of 30° C.-55° C. by using glucose is obtained.

In yet another aspect, the present invention further provides use of the above gene engineering strain, in particular use in production of D-lactic acid. Further, the strain is used for producing D-lactic acid by using glucose, xylose, maltose, lactose, sucrose or a combination thereof as a carbon source. Further, low-cost culture mediums containing peanut meal and dried corn steep liquor powder are used for fermentation. Further, D-lactic acid is produced through fermentation under an environment condition between an anaerobic environment condition and a microaerobic environment condition with pH of 6.0-8.0 and temperature of 45° C.-55° C. Further, the fermentation process is a continuous fermentation or fed-batch process.

Further, firstly seed culture is performed to the above gene engineering strain to obtain seed culture solution, and then fermentation culture is performed in fermentation culture mediums using one of glucose, xylose, maltose, lactose and sucrose as a carbon source and using yeast powder, peptone, peanut meal and dried corn steep liquor as a nitrogen source to obtain D-lactic acid. Specifically, the process comprises the following steps:

1) slant culture: inoculating an engineeredbacillusstrain into a solid slant culture medium containing 20 g/L agar and culturing for 24-48 h at 45-55° C.;

2) seed culture: inoculating the engineeredbacillusafter slant culture into a seed culture medium under an aseptic condition, statically culturing for 24-36 h at 45-55° C. to obtain seed culture solution, wherein a neutralizing agent is added to control pH of fermentation liquid; and

3) fermentation culture: inoculating the seed culture solution into a fermentation culture medium in an inoculation amount according to a volume ratio of 5-20%, and culturing for 48-90 h at 45° C.-55° C., wherein the temperature is preferably 50° C.

Preferably, each liter of the seed culture medium in step 2) contains 60-120 g of glucose or 40-70 g of xylose, 8-12 g of yeast powder, 3-8 g of peptone, 50 g of calcium carbonate and balance of water, and preferably contains 90 g of glucose, 10 g of yeast powder, 5 g of peptone, 50 g of calcium carbonate and balance of water; and pH of the seed culture medium is 6.0. Sterilization is performed for 15 min at 115° C. The neutralizing agent comprises one or more of NaOH, NH4OH and Ca(OH)2.

Preferably, the fermentation culture medium in step 3) comprises the following components and contents thereof: 40-180 g/L carbon source and 5-20 g/L nitrogen source. Preferably, the fermentation process in step 3) is a fed-batch process, and the fed-batch process refers to that, when the total content of reducing sugars in the fermentation liquid is lower than 20 g/L, the carbon source is added to enable the total content of the reducing sugars to be maintained at 30-70 g/L or reach 50-70 g/L. Preferably, pH of the fermentation culture medium is 6.0-8.0.

The reconstructed gene engineering strain provided by the present invention can produce, by fermentation, high-concentration and highly optically-pure D-lactic acid at higher fermentation temperature by using low-cost raw materials, which can improve the production efficiency while the cost is reduced, so that the production cost is reduced and the method is suitable for application and dissemination in industrial production. The highest yield of D-lactic acid can reach 226 g/L with an optical purity of 99.9%, the highest sugar-acid conversion rate can reach 93.6% and the fermentation production capacity is 3.2 g/[L·h]. Therefore, by using the inventive method to produce D-lactic acid, the cost can be reduced, the operation flow can be simplified and the method has a wide industrial application prospect.

The concept, specific steps and produced technical effects of the present invention will be further described below with reference to the drawings, so as to fully understand the purposes, features and effects of the present invention.

