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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus

Definitions

• the present invention relates to genetic modification for industrial applications of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic.
• Lactic acid and its salts are commercially viable products useful in various fields including medicine, biodegradable polymers and food processing.
• Moderately thermophilic Bacillus species that are facultative anaerobic and homolactic are ideal organisms for the industrial manufacture of lactic acid. They are capable of growing at temperatures between 30-65 0 C and allow anaerobic industrial fermentation at temperatures above 50 0 C. This high temperature has several advantages when fermenting on industrial scale: less risk of infections and thus higher enantiomeric purity, faster reactions etcetera.
• the homolactic nature allows the production of lactic acid from hydrocarbon sources (including hexose and pentose sugars; See WO 04/063382) without the formation of more than 15 wt% side products such as formic acid and acetic acid.
• the facultative anaerobic nature of the bacilli allows fermentation under anaerobic conditions, or at least under a low partial pressure of oxygen, which for Industrial scale is desirable because it allows for relatively inexpensive equipment and processing.
• the nutrient requirements of these bacteria are less demanding than those of lactic acid bacteria such as Lactobacillus species, which also allows for relatively inexpensive Industrial processes.
• R-lactate-producing bacteria are either mesophilic (e.g. Bacillus laevolacticus) or have demanding nutrient requirements (e.g. Lactobacillus delbrueckii), which makes the manufacture of R-lactate much more expensive than that of S-lactate.
• one object of the present invention is to avail of a moderately thermophilic Bacillus strain which is facultative anaerobic and produces R-lactic acid by homolactic fermentation.
• Another object of the invention is to avail of a method to produce genetically engineered moderately thermophilic bacilli which are facultative anaerobic and homolactic.
• the genus Bacillus comprises over 200 different species (See Sneath, P. H. A., 1986: Endospore-forming Gram-positive rods and cocci, in Bergey's manual of systematic bacteriology. VoI 2. Sneath, P.H.A., Mair, N. S., Sharpe, M. E., Holt, J. G.
• Transformation of Bacillus spp. An Examination of the transformation of Bacillus protoplasts by plasmids pUB110 and pHV33, Current Microbiology, VoI 13 (1986), pp 191-195, the protoplast transformation in various bacilli was described. However the transformation in B. coagulans was reported as unsuccessful
• Electroporation is widely used for bacteria but requires species-specific (or even strain- specific) optimisation of the growth medium and electroporation buffer. Successful electroporation of Bacillus species often requires in vivo or in vitro methylation of plasmid DNA to prevent its restriction after transformation.
• WO 02/29030 discloses introduction of in vivo methylated plasmid in cells of a thermophilic Bacillus strain TN by electroporation. The plasmid used is based on the thermosensitive plasmid pUB110. However, we found that this plasmid did not yield transformed cells when used to transform moderately thermophilic Bacillus species that are facultative anaerobic and homolactic.
• the present invention shows the genetic engineering of moderately-thermophilic facultative-anaerobic homolactic Bacillus species by transformation.
• the bacilli can also be genetically engineered for production of industrial compounds, including other organic acids than lactic acid, alcohol, enzymes, amino acids, and vitamins.
• the moderately thermophilic Bacillus species is homolactic because this ensures that the newly introduced functionality will result in high yield production with a small amount of side products. Furthermore, the use of homolactic Bacillus species enables that only a few modifications have to be applied in order to obtain an industrially applicable micro-organism.
• Moderately thermophilic Bacillus species are defined as bacteria that are capable of growing at temperatures between 30-65 0 C.
• moderately thermophilic facultative- anaerobic homolactic species are Bacillus coagulans and Bacillus smithii.
• Homolactic Bacilli can naturally produce S-lactate by homolactic fermentation. Which specific strains can produce lactic acid by homolactic fermentation can easily be determined by the person skilled in the art.
• the present invention also encompasses strains derived from moderately thermophilic facultative-anaerobic Bacillus species, wherein the homolactic phenotype is modified.
• a strain or derivative is chosen which is sporulation deficient.
• Plasmids tested were plL253 (See Simon, D., and A. Chopin, 1988: Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70:559-566), pMV158 (See Burdett, V., 1980: Identification of tetracycline resistant R-plasmids in Streptococcus aqalactiae (group B), Antimicrob. Agents Chemother. 18: 753-766.), pHP13 (See Haima, P., S. Bron, G.
• Venema 1987: The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. MoI. Gen. Genet. 209:335-342.
• pUB1 10 See Keggins, K.M., P. S. Lovett, E. J. Duvall, 1978: Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110, Proc. Natl. Acad. Sci. USA 75:1423-1427
• pAMSIOO See Kiewiet, R., J. Kok, J. F. M. L. Seegers, G. Venema, S.
• pWCFS105 See Van Kranenburg, R., N. Golic, R. Bongers, RJ. Leer, W. M. de Vos, R.J. Siezen, M. Kleerebezem, 2005: Functional anvalysis of three plasmids from Lactobacillus plantarum, Appl. Environ. Microbiol. 71 : 1223-1230
• pNZ280 See Platteeuw, C, F. Michiels, H. Joos, J. Seurinck, and W. M. de Vos, 1995:
• the present invention discloses a method for genetic modification of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic by means of genetic engineering.
• the method comprises the steps of: introducing DNA cloned in a thermosensitive plasmid system containing a pSH71 replicon or a homologue thereof into cells of a moderately thermophilic Bacillus species that is facultative anaerobic and homolactic; culturing the cells on a selective medium at a permissive temperature for plasmid replication to select transformed cells capable of growing on said selective medium at said permissive temperature; culturing said transformed cells on a selective medium at a non-permissive temperature for plasmid replication to select transformed cells capable of growing on said selective medium at said non-permissive temperature.
• the culturing of the cells at the permissive temperature on a selective medium allows for selection of transformants, i.e. cells that have taken up the transforming DNA.
• transformants i.e. cells that have taken up the transforming DNA.
• transformed colonies are isolated prior to culturing the transformed cells at a non-permissive temperature, to allow checking the integrity of the transforming DNA. Cells of one or a few individual colonies are then cultured at a non-permissive temperature, to allow the selection of integrants.
• thermosensitive plasmid system containing a pSH71 replicon (GenBank Accession Number A09338) or a homologue thereof.
• a pSH71 replicon is a replicon providing a thermosensitive replication functionality.
• a thermosensitive replication functionality provides replication of a plasmid containing the replicon at a permissive temperature and lack of replication of said plasmid at a non- permissive temperature.
• This thermosensitive replication functionality of the pSH71 replicon is provided by an origin of replication and a replication protein (RepA) that is encoded by the replicon.
• RepA replication protein
• a "pSH71 replicon or a homologue thereof is defined as a DNA comprising a DNA sequence encoding a polypeptide having a thermosensitive replication functionality (the RepA protein) and having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is 80% identical to SEQ ID NO:1 , preferably 90% identical, more preferably 95, 96%, 97%, 98%, 99% identical.
• the pSH71 replicon or a homologue thereof further comprises an origin of replication where the RepA protein as defined above is capable to act upon.
• An example of a homologue of the pSH71 replicon is pWV01 (GenBank Accession Number X56954).
• the pSH71 replicon or a homologue thereof may further comprise a regulatory protein (RepC).
• the degree of identity between two amino acid sequences refers to the percentage of amino acids that are identical between the two sequences.
• the degree of identity is determined using the BLAST algorithm, which is described in Altschul, et al., J. MoI. Biol. 215: 403-410 (1990).
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• an integration plasmid with a conditional replicon is desirable.
• a plasmid can be introduced under permissive conditions after which the growth conditions can be changed (e.g. by a shift to a non-permissive temperature) making the plasmid non-replicating and allowing for selection for chromosomal integration events caused by homologous or nonhomologous recombination.
• a thermosensitive replicon can be used such as pSH71 present in pNZ124, or plasmids essentially identical (homologous) thereto or a derivative thereof having retained the plasmid's thermosensitive properties.
• Permissive temperatures for this replicon in moderately thermophilic Bacillus species that are facultative anaerobic and homolactic are preferably between 37 and 50 0 C.
• Non- permissive temperatures are preferably above 50 0 C.
• Permissive and non-permissive temperatures may not only depend on the replicon, but also on the host Bacillus species. The latter is a known phenomenom from the prior art, exemplified by pG+host plasmids for which 37°C is a permissive temperature in Lactobacillus delbrueckii but a non-permissive temperature for Lactococcus lactis (See US 5919678 ).
• Conditional cloning vectors can also be obtained as thermosensitive derivatives of pNW33n or other plasmids able to replicate in moderately thermophilic Bacillus species that are facultative anaerobic and homolactic.
• thermosensitive plasmid system containing a pSH71 replicon or a homologue thereof is introduced into cells of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic by:
• Transformation of these Bacillus species by electroporation can be achieved by a high- voltage discharge through a suspension containing a moderately thermophilic Bacillus species that is facultative anaerobic and homolactic and a suitable transforming DNA comprising the desired functionality and/or DNA sequences homologous to genomic sequences of the specific Bacilli.
• Transformation of these Bacillus species by conjugation can be achieved by contacting (a population of) the moderately thermophilic Bacillus species that is facultative anaerobic and homolactic with (population of) a donor cell containing self-transmissible or mobilizable plasmid having the desired functionality.
• Self-transmissible plasmids encode all the functions they need to move among cells, and sometimes they also aid in the transfer of chromosomal DNA and mobilizable plasmids.
• Mobilizable plasmids do not encode all of the proteins required for transfer and consequently need these functions to be provided by the donor genome (chromosome or plasmid-encoded). Mobilizable plasmids at least contain an origin of transfer (or/T) region.
• Any donor cell which can be co-cultured with the moderately thermophilic Bacillus to be modified is in principle suitable to serve as a donor cell.
• suitable donor cells are those of Bacillus species, including B. alkalophilus, B. amyloliquefaciens, B. brevis, B. cereus, B. circulans, B. coagulans, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. smithii, B. subtilis, B. thermoamylovorans, B. thuringiensis, Geobacillus stearothermophilus, E. coli, Enterococcus faecalis, Lactobacillus species, including L. acidophilus, L.
• amylophilus L. amylovorus, L. casei, L. coryniformis, L. crispatus, L. curvatus, L. delbrueckii, L gasseri, L. helveticus, L. johnsonii, L. plantarum, L. reuteri, L. rhamnosus, L. sakei, L. sanfriscensis, 'Streptococcus species, including S. agalactiae, S. mutans, S. oralis, S. pneumoniae, S. salivarius, S. sobrinus, and S. thermophilus.
• Suitable self-transmissible plasmids include pRK24, pLS20, pAM ⁇ i , or a plasmid essentially identical thereto or a derivative hereof having retained the plasmid's self- transmissible capability.
• Suitable mobilizable plasmids include pAT18, pAT28, pJS28, or a plasmid essentially identical thereto or a derivative hereof having retained the plasmid's mobilizing capability.
• the inventors further managed to develop a natural transformation protocol for these Bacillus species. This required determining the right medium composition for growth, starvation, and transformation, the right timing for developing and harvesting competent cells, and the right transformation procedure. While natural transformation is not known to be wide-spread among Bacillus species the inventors discovered that moderately thermophilic bacilli that are facultative anaerobic and homolactic can be made naturally competent.
• the source of the transforming DNA may influence the transformation outcome.
• the source of the DNA to be transformed isolated from either Lactococcus lactis MG 1363, Escherichia coli DH5 ⁇ , E. coli JM 109, or E. coli JM 1 10) did not affect transformation efficiencies in the present Bacillus species, indicating that the methylation state of the DNA is not important. This is in contrast to other Bacillus species (see e.g. WO 02/29030).
• the transforming DNA for introduction into Bacillus is isolated from Lactococcus lactis More preferably, cloning steps to constuct the transforming DNA are also performed in Lactococcus lactis This, because cloning in E coli often appeared to result in deletions and/or rearrangements in the cloned DNA
• thermosensitive plasmid methods for transformation by preferably induction of natural competence, transformation of electrocompetent cells, and conjugation are provided, the use of a a thermosensitive plasmid is demonstrated, and application of these elements for the production of genetically engineered moderately thermophilic Bacillus species that are facultative anaerobic and homolactic, for instance for the production of R-lactate, is demonstrated
• the transforming DNA contains a pSH71 replicon or a homologue thereof and a DNA of interest capable of providing a desired functionality to the Bacillus cells
• Chromosomal modification is the preferred modification of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic, since chromosomal modification will ensure a stable distribution of said functionality over the progeny cells
• Introduction of a desired functionality in the chromosome can be done with non-homologous as well as with homologous recombination Homologous recombination is preferred, as it opens the opportunity to introduce, to remove or to simultaneously introduce and remove a functionality
• a functionality may be a gene encoding a desired polypeptide to be produced by the cell and/or a gene encoding a polypeptide involved in production of a primary or secondary metabolite by the cell and/or a DNA sequence enabling deletion of a DNA sequence from the chromosome of the cell
• Genes encoding polypeptides are provided with regulatory sequences functional in the cell, for instance a promoter sequence
• the regulatory sequences may be sequences that are natively associated with the coding sequence, or may be heterologous thereto
• a gene encoding a polypeptide may be fused to any regulatory or promoter sequence functioning in the Bacillus species of choice Suitable promoter sequences include promoters obtainable from the Bacillus species of choice, hybrid promoters derived from different native Bacillus promoters and artificial promoters.
• a preferred promoter is the promoter of a Bacillus gene to be inactivated by homologous recombination. Especially preferred is the IdhL promoter from Bacillus coagulans. or the promoter of the amylase gene as disclosed in US 5171673.
• a selection marker is part of the transforming DNA.
• the selection marker may be present on the same DNA fragment or plasmid as the functionality of interest or on a separate DNA fragment or plasmid.
• a preferred selection marker is the cat gene coding for chloramphenicol acetyltransferase from pMH3.
• Desired functionalities that can be introduced are R-lactic acid production as described below, and other functionalities that provide the production of compounds that are metabolizable from pyruvate.
• these compounds are pyruvate, acetolactate, diacetyl, acetoin, 2,3-butanediol, 1 ,2-propanediol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2-oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate.
• the transforming DNA further contains a DNA sequence that is homologous to a genomic target sequence of the specific Bacillus to be engineered.
• a DNA sequence that is homologous to a genomic target sequence of the specific Bacillus to be engineered The skilled person will understand that no 100% identity is required to obtain homologous recombination. A percentage identity of about 90% will also suffice.
• the DNA sequence of interest to be inserted in the chromosome by homologous recombination is flanked by homologous sequences with a sufficient length to enable homologous recombination. Such a length may be at least about 200 bp, for instance between about 200 and about 1500 bp, preferably between about 200 and about 1000 bp.
• the present invention is directed to the modification by means of genetic engineering of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic wherein a desired functionality is introduced, preferably by homologous recombination.
• the present invention is also directed to the modification by means of genetic engineering of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic wherein an undesired functionality is removed by homologous recombination.
• the invention is further directed to the modification by means of genetic engineering of moderately thermophilic Bacillus species that are facultative anaerobic and homolactic wherein a desired functionality is introduced in the chromosome and at the same time an undesired functionality is removed by homologous recombination
• the modification by means of genetic engineered includes introduction of a functionality in, removal of a functionality from or simultaneous introduction of a functionality in and removal of a functionality from the bacterial chromosome
• the modification by means of genetic engineered occurs by homologous recombination
• the genetically engineered derivative of a moderately thermophilic Bacillus species that is facultative anaerobic and homolactic is a strain wherein the conversion of pyruvate to lactate is blocked and pyruvate accumulates, or wherein additional modifications are applied to redirect pyruvate towards production of other products including acetolactate, diacetyl, acetoin, 2,3-butaned ⁇ ol, 1 ,2-propaned ⁇ ol, acetate, formate, acetaldehyde, ethanol, L-alanine, oxaloacetate, S-malate, succinate, fumarate, 2- oxoglutarate, oxalosuccinate, isocitrate, citrate, glyoxylate
• the genetically engineered derivative of a moderately thermophilic Bacillus species that is facultative anaerobic and homolactic is a strain wherein the IdhL gene coding for S-lactate dehydrogenase activity is removed by homologous recombination
• the genetically engineered derivative of a moderately thermophilic Bacillus species that is facultative anaerobic and homolactic is a strain wherein the IdhL gene coding for S-lactate dehydrogenase activity is replaced with a gene coding for an NADH-dependent 2-hydroxyac ⁇ d dehydrogenase having R-lactate dehydrogenase activity by homologous recombination
• the construct that is used for homologous recombination contains a defective IdhL gene that does not encode a functional S-lactate dehydrogenase enzyme.
• a defective IdhL gene can be provided by a construct wherein part or all of the IdhL coding sequence is deleted.
• a defective IdhL gene may be created by replacing part or all of the IdhL gene with another gene, for instance a gene encoding a selection marker or a gene of interest.
• the IdhL gene coding for S-lactate dehydrogenase activity is replaced by a construct containing a gene coding for an NADH-dependent 2-hydroxyacid dehydrogenase having R-lactate dehydrogenase activity, including enzymes with EC number EC 1.1.1.28..
• Suitable genes coding for R-lactate dehydrogenase activity are those genes that are able to complement an E. coli IdhA mutant, such as E. coli FMJ 144 as described by Bernard et al. (See Bernard, N., T. Ferrain, D. Garmyn, P. Hols, and J. Delcour, 1991 : Cloning of the D- lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli. FEBS Lett. 290:61 -64).
• a suitable IdhA gene coding for R-lactate dehydrogenase activity for instance is the IdhA gene from Lactobacillus delbrueckii subsp. bulgaricus [see also Bernard et a!.) or the hdhD gene from the same species (Bernard, N., K. Johnsen, T. Ferain, D. Garmyn, P. Hols, J.J. Holbrook, and J. Delcour.
• Suitable genes coding for R-lactate dehydrogenase activity are those genes that encode an amino acid sequence of SEQ ID NO:2 or homologous genes that encode an amino acid sequence that displays a degree of identity of at least 30%, more preferably at least 40%, even more preferably at least 50%, 60%, 70%, 80%, 90%, to the amino acid sequence of
• homologous sequences may encompass polymorphisms that may exist in cells from different populations or within a population due to natural allelic or intra-strain variation.
• a homologue may further be derived from a Bacillus species other than the species where the specified DNA or amino acid sequence originates from, or may be artificially designed and synthesized.
• genetic engineering can be used to optimize the industrial fermentation, e.g. to enable the fermentation of cheap substrates like lignocellulose-derived sugars including xylose and arabinose and/or to remove undesired functionalities.
• the present invention provides a process for the production of a compound of interest comprising culturing the genetically engineered strain of the previous aspect under conditions conducive to production of said compound.
• Figure 1 shows restriction analyses of pNW33N isolated from three E. coli isolates that were transformed with plasmid material isolated from three transformed B. coagulans DSM 1 isolates. Lanes 1 , 4, and 7. pNW33N digested with EcoRI; 4217-bp fragment. Lanes 2, 5, and 8. pNW33N digested with EcoR ⁇ -Stu ⁇ ; 333-bp and 3884-bp fragments. Lanes 3 and 6.
• Kb DNA ladder 250 bp, 500 bp, 750 bp, 1.0 kb, 1.5 kb, 2.0 kb, 3.0 kb, 4.0 kb, 5.0 kb, 6.0 kb, 7.0 kb, 8.0 kb, 9.0 kb, 10.0 kb, and 12.0 kb.
• Figure 2 shows a plasmid map of pJS28.
• the replication gene (repB) and the chloramphenicol resistance gene (cat) are depicted by arrows.
• the IncP plasmid RK2 origin of transfer (oriT) and the E. coli origin of replication (ori) are depicted as boxed regions.
• Figure 3 shows the nucleotide sequence of synthetic B. coagulans ATCC 23498 amylase promoter region based on the sequence disclosed in US 5171673.
• BgIW AGATCT
• BamH ⁇ GGATCC
• CCATGG ⁇ /col
• the ⁇ /col site allows translational fusion to the amylase promoter.
• the ATG start codon of the amylase gene is depicted boldface.
• Figure 4 shows a plasmid map of pJS25. Replication genes (repA and repC), the chloramphenicol resistance gene without ⁇ /col site (cm * ) are indicated by arrows.
• the B. coagulans promoter region (P) is depicted as boxed region.
• Figure 5 shows a plasmid map of pJS26.
• Replication genes repA and repC
• the chloramphenicol resistance gene without ⁇ /col site cm *
• the LMG 6901 IdhA gene IdhA 6901
• the B. coagulans promoter region (P) is boxed.
• Figure 6 shows a plasmid map of pJS27.
• the replication gene (repB), the chloramphenicol resistance gene (cat), and the L. bulgaricus LMG 6901 IdhA gene (IdhA 6901 ) are depicted by arrows.
• the B. coagulans promoter region (P) and the E. coli origin of replication (ori) are depicted as boxed regions.
• Figure 7 shows a plasmid map of pRK1.
• the replication genes (repA and repC), the chloramphenicol resistance gene (cat), and the L. bulgaricus IdhA gene (IdhA) are depicted by arrows.
• the B. coagulans promoter region (Pamy ATTC) and B. coagulans IdhL upstream and downstream regions are depicted as boxed regions.
• Plasmid pNZ124 (Platteeuw, C 1 G. Simons, and W. M. de Vos. 1994. Use of the Escherichia coli ⁇ -qlucuronidase (QUSA) gene as a reporter gene for analyzing promoters in lactic acid bacteria. Appl. Environ. Microbiol. 60:587-593) was obtained from NIZO food research. It is based on the cloning vector pNZ12 disclosed in EP 0228726 B1.
• Plasmid pNW33N (Zeigler, D. R. 2001 : The genus Geobacillus; introduction and strain catalog. 7th ed., vol. 3. Bacillus Genetic Stock Center, www.bgsc.org) was obtained from the Bacillus Genetic Stock Center, Ohio State University, Columbus, Ohio, USA, and propagated in Escherichia coli DH5 ⁇ (Invitrogen Life Technologies). The nucleotide sequence of pNW33N is available under GenBank accession number AY237122. Plasmid pAT ⁇ S28 containing the IncP plasmid RK2 origin of transfer (oriT) (Namy, O., M. Mock, A.
• Lactococcus lactis subsp. cremoris MG1363 was described by Gasson (M. J. Gasson. 1983: Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154:1-9).
• Lactobacillus delbrueckii subsp. bulgaricus LMG 6901 was obtained from the BCCM/LMG bacteria collection, Gent, Belgium.
• E. coli HB101 harbouring pRK24 was described by Trieu-Cuot, et al. (Trieu-Cuot, P., C. Carlier, P. Martin, and P. Courvalin, 1987: Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria. FEMS Microbiol. Lett. 48:289-294).
• B. coagulans was routinely grown at 45°C under aerobic conditions in BC-broth (BC for S. coagulans) containing 10 g/L yeast extract, 2 g/L di-ammoniumphosphate, 3.5 g/L di- ammoniumsulphate, 10 g/L Bis-Tris buffer (bis[2-hydroxymethyl]iminotris[hydroxymethyl]- methane), 3 mg/L CaCI 2 , 5 mg/L MgCI 2 ; if appropriate the medium was supplemented with 50 g/L sucrose; pH was adjusted to 6.6 - 6.7 and medium was autoclaved (20 min 121 0 C) before use. For plates the medium was supplemented with 10 g/L Gelrite and 1 g/L MgCI 2 .
• Filter sterilised trace elements were added separately. Final concentrations were: 0.2 mg/L CoCI 2 • 6 H 2 O, 0.01 mg/L CuCI 2 • 2 H 2 O, 0.3 mg/L H 3 BO 3 , 0.03 mg/L Na 2 MoO 4 • 2 H 2 O, 0.02 mg/L NiSO 4 ⁇ 6 H 2 O, 0.03 mg/L MnCI2 • 4 H 2 O, 0.05 mg/L ZnCI 2 . If appropriate, the media were supplemented with filter sterilised chloramphenicol at 7 mg/L.
• Competence medium contained 0.05 g/L yeast extract, 2 g/L di-ammoniumphosphate, 3.5 g/L di- ammoniumsulphate, 10 g/L glucose, 10 mg/L CaCI 2 , 0.5 g/L KCI, 25 mg/L MgCI 2 ; pH was adjusted to 6.8 and medium was autoclaved (20 min 121°C) before use. Filter sterilised trace elements and vitamins were added separately.
• Transformation medium was C-broth with 0.025 g/L yeast extract instead of 0.05 g/L.
• E coli was routinely cultured in LB broth (Molecular Cloning, a laboratory manual. 3 rd edition. J. Sambrook and D. W. Russell. 2001. Cold Spring Harbor Laboratory Press, New York) at 37°C under aerobic conditions. When appropriate chloramphenicol and/or ampicillin were used at concentrations of 5 mg/L and 100 mg/L, respectively.
• L. bulgaricus was routinely cultured in MRS broth ® (BD Biosciences) at 37°C under anaerobic conditions.
• L. lactis was routinely cultured in M17 broth ® (BD Biosciences) supplemented with 0.5% glucose at 30 0 C under anaerobic conditions. When appropriate chloramphenicol was used at a concentration of 5 mg/L.
• E. coli plasmid DNA isolation from 100 mL culture was performed using the Jetstar 2.0 Plasmid Maxiprep Kit ® (Genomed) following the instructions of the manufacturer.
• Small-scale E. coli plasmid DNA isolation from 1 mL culture was performed using the Nudeospin Plasmid Quick Pure ® (Macherey-Nagel) kit following the instructions of the manufacturer.
• E. coli competent cells were prepared using calcium chloride and transformed by heat shock as described by Sambrook and Russell (Molecular Cloning, a laboratory manual. 3 rd edition. J. Sambrook and D. W. Russell. 2001. Cold Spring Harbor Laboratory Press, New York).
• L. lactis was transformed by electroporation as described by HoIo and Nes (High-freguency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. 1989. HoIo, H., and I. F. Nes. Appl. Environ. Microbiol. 55:3119-3123).
• PCR reactions for cloning purposes were performed with the high-fidelity Pwo polymerase (Roche) following the instructions of the manufacturer.
• Colony-PCR analysis was used to demonstrate the presence of pNW33N in the chloramphenicol resistant colonies.
• PCR primers were designed for identification of the pNW33N replication gene repB with the sequences ⁇ '-TCGCCTTCTTCTGTGTCATC-S' and ⁇ '-CTGGAGGAGAGCAATGAAAC-S'. Colonies were picked with a tooth pick and a little cell material was transferred to a 0.5 mL PCR reaction tube. The cells were disrupted by 1 min incubation at 1000 W in a microwave oven.
• PCR reaction mixtures of 50 ⁇ L with rTaq polymerase (Amersham Biosciences) and 0.5 ⁇ g of each primer were prepared as recommended by the manufacturer and added to the reaction tubes with the disrupted cells. PCR reactions were performed in a RoboCycler ® (Stratagene). 4 min of incubation at 94°C was followed by 25 cycles of 30 sec at 94°C for denaturation, 1 min at 58°C for primer annealing, and 1 min at 72°C for elongation. After the final cycle the reaction mixture was incubated at 72°C for another 5 min.
• Electroporation Preparation of electrocompetent cells required determination of the proper culture conditions, time of harvest, and composition of wash and electroporation buffers.
• B. coagulans DSM 1 is able to grow on LB broth, the developed BC broth increased electroporation efficiencies.
• Glycine was added to weaken the cell wall, and the concentrations were optimized. Glycine concentrations were between 0.5 and 2.0% and preferably between 1.0 and 1.5%. Cells were harvested at early to mid-log phase for optimal results.
• the electroporation buffer had a pH between 4.3 and 6.0 and preferably between 4.3 and 5.0. The optimal electroporation settings (voltage, resistance, capacitance) also needed to be determined.
• Voltage preferably was between 1.0 and 2.5 kV, and more preferably between 1.25 and 2.0 kV. Resistance was preferably between 100 and 800 ⁇ , and more preferably between 200 and 600 ⁇ . Recovery before plating on a selective medium is important and was at least 2 hours and preferably 3 hours. Electrocompetent cells of B. coagulans were prepared as follows. An overnight culture was used to inoculate (5% volume/volume) 50 ml_ of medium supplemented with 1 % glycine (resulting in a turbidity at 600nm of approximately 0.13-0.14). After 2.5 hrs of aerobic incubation at 45°C cells were harvested by centrifugation.
• Cell pellets were washed twice with 50 mL and 25 mL, respectively, of ice-cold electroporation buffer (5 mM KH 2 PO 4 , 0.4 M sorbitol, 10% glycerol, 4 mM MgCI 2 adjusted to pH 4.5), and resuspended in 1 mL of ice-cold electroporation buffer.
• ice-cold electroporation buffer 5 mM KH 2 PO 4 , 0.4 M sorbitol, 10% glycerol, 4 mM MgCI 2 adjusted to pH 4.5
• 100 ⁇ l_ of the cell suspension was mixed with 1 ⁇ g plasmid DNA and transferred to a 0.2 cm electroporation cuvet (Bio-Rad ®) that was precooled on ice.
• the sample was subjected to a 1.6 kV pulse at 200 ⁇ and 25 ⁇ F using a Gene Pulser and a Pulse Controller apparatus (Bio-Rad®). Immediately after electroporation 1 mL of medium was added and the cells were incubated for 3 hours at 45 0 C in a Thermomixer ® (Eppendorf) at 900 rpm, after which they were plated on piates supplemented with chloramphenicol. The plates were incubated at 45 0 C for 1 to 2 days under aerobic conditions.
• Filter matings were used for conjugal transfer of recombinant plasmids from E. coli to B. coagulans.
• Logarithmic growing cells of donor (2 mL) and recipient (2 mL) were pooled and using a syringe harvested on a 0.45 ⁇ m cellulose acetate filter (Schleiger & Schuell) sterilized in a plastic filter holder (Schleiger & Schuell). Cells were washed with 10 mL of BC-broth and the filters were dried by forcing air through the filter. Filters were placed on the surface of BC-plates without antibiotics and incubated overnight at 45°C.
• Enzyme overproduction in S coagulans using the B coagulans promoter was determined in exponentially growing cultures Cells were harvested by centrifugation Cell-free extracts were prepared using a FastPrep FP120 ® apparatus (Qbiogene) in two runs of 30 sec at speed 4 Cells were cooled on ice for 1 mm between the runs Protein content was determined by the method of Bradford (Bio-Rad) using Bovine Serum Albumine as a standard SDS-PAGE (12 5%) was performed as described by Sambrook and Russell (Molecular Cloning, a laboratory manual 3 rd edition J Sambrook and D W Russell 2001 Cold Spring Harbor Laboratory Press, New York) using the Protean Il electrophoresis system (Bio-Rad)
• R-lactate dehydrogenase-specific activities were determined spectrophotometrically at 340 nm and carried out at 50 0 C, using 1 ml of an assay mix containing 0 3 M glycylglycine buffer pH10, 0 25% (v/v) Triton X-100, 5 mM NAD, and 1 % R-lactate The reaction was started by the addition of 40 or 50 ⁇ l_ of cell-free extract Specific activities were expressed as ⁇ A mm 1 mg 1 where ⁇ A is the increase in absorbance at 340 nm for 1 cm path length and mg the amount of protein
• Organic acids (lactic acid, acetic acid, formic acid, succinic acid) were measured using a denvatisation and GLC R- and S-lactates were methylated to methyl-lactate and measured by headspace analysis on a chiral column.
• Bacillus coagulans DSM 1 was cultured overnight at 45°C in 5 ml BC-broth under aerobic conditions. This culture was used to inoculate 25 ml of prewarmed C-broth resulting in a turbidity at 600 nm of 0.15. The fresh culture was incubated aerobically at 45°C until a turbidity at 600 nm between 0.9 - 1.2 was reached (Table 1 ).
• coagulans DSM 1 was used as negative control and yielded no PCR products. Plasmid DNA of a single transformant was isolated from a cesium-chloride gradient. Plasmid DNA was digested with EcoRI and with EcoR ⁇ -Stu ⁇ to confirm the integrity of pNW33N. The restriction patterns were as expected (333-bp and 3884-bp fragments) demonstrating that plasmid pNW33N was transformed to B. coagulans DSM 1.
• the origin of transfer (or/7) of the IncP plasmid RK2 was cloned into pNW33N. This allows effective comobilization by any self-transferable IncP plasmid co-resident in E. coli donors (Plasmid transfer by conjugation from Escherichia coli Xo Gram-positive bacteria. Trieu-Cuot, P., Carlier, C, Martin, P., and Courvalin, P. 1987. FEMS Microbiol. Lett. 48:289-294).
• the RK2 oriT region was cloned as a blunted 0.5-kb Acc ⁇ -Ava ⁇ fragment derived from pAT ⁇ S28 in pNW33N digested with Sma ⁇ .
• pJS28 (Fig. 2)
• E. coli HB101 harbouring pRK24 This strain was used as donor in plate matings with B. coagulans DSM 1.
• a B coagulans ATCC 23498 nucleotide sequence fragment (disclosed in US 5171673) having promoter activity was produced as a synthetic DNA fragment (Fig 3) and cloned as Bg/ll-SamHI fragment into pMH3 digested with the same enzymes
• the resulting cloning vector, pJS25 (Fig 3), allows translational fusion to this promoter by the use of an ⁇ /col site overlapping the start codon
• first plasmid pMH3 was constructed from plasmid pNZ124 by removing the ⁇ /col site from the cat gene using a megaprimer Primers with the sequences ⁇ '-CTATTATTCCGTGGACTTC-S' and 5'-
• Plasmid pJS25 (Fig 4) can be used in various mesophilic Gram- positive organisms including Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, and Leuconostoc lactis, and the Gram-negative organism Escherichia coli (Use of the Escherichia coli ⁇ -qlucuronidase (QUSA) gene as a reporter gene for analyzing promoters in lactic acid bacteria.
• the PCR product was cloned as a blunt-Xibal fragment into pUC18 digested with Xba ⁇ -Sma ⁇ and its integrity was confirmed by nucleotide sequence analysis.
• the IdhA gene was cloned as Rca ⁇ -Xba ⁇ fragment into pJS25 digested with Nco ⁇ -Xba ⁇ .
• the resulting expression vector, pJS26 (Fig. 5), has the IdhA gene of L. bulga ⁇ cus translationally fused to the B. coagulans promoter. Subsequently, the complete fragment encompassing the B. coagulans promoter and L.
• delbrueckii IdhA gene was transferred as a Pst ⁇ -Bgl ⁇ fragment (by partial digestion with BgIW) to the thermophilic cloning vector pNW33N digested with Pst ⁇ -BamH ⁇ generating plasmid pJS27 (Fig. 6).
• Plasmid pJS27 was transformed to B. coagulans DSM 1 by electroporation. Specific R- lactate dehydrogenase enzyme activities were determined as decrease of absorbance at 340 nm per min per mg protein at 50°C. The specific activity of B. coagulans DSM 1 harbouring pNW33N was 0.45 ⁇ A min "1 mg protein "1 and that of B. coagulans DSM 1 harbouring pJS27 was 2.15 ⁇ A min "1 mg protein "1 demonstrating that the overproduction of R-lactate dehydrogenase in the modified strains containing the L. delbrueckii LMG 6901 IdhA gene resulted in a 4.8-times increased activity.
• B. coagulans DSM 1 harbouring pJS27 (EXAMPLE 4) was grown in a batch culture mimicking industrial conditions.
• B. coagulans DSM 1 harbouring pNW33N and B. coagulans DSM 1 without plasmids were used as a reference strains. After fermentation the concentrations of organic acids and the optical purity of the lactic acid were determined (Table 2). Lactate produced by B. coagulans DSM 1 and B. coagulans DSM 1 harbouring pNW33N was enantiopure in the S-form, while the lactate produced by B. coagulans DSM 1 harbouring pJS27 was for a significant part in the R-form. No differences in by-product formation were detected.
• a modified B. coagulans strain producing R-lactate is constructed by replacing the B. coagulans IdhL gene coding for the major S-lactate dehydrogenase activity by the L bulgaricus IdhA gene coding for R-lactate dehydrogenase. Replacement is achieved by homologous recombination in a two step process using a conditional cloning vector, e.g. a thermosensitive replicon which is functional at 45°C but not at 55°C.
• a conditional cloning vector e.g. a thermosensitive replicon which is functional at 45°C but not at 55°C.
• pSH71 replicon present in pNZ124 or pMH3, which was discovered to have such a thermosensitive nature in moderately thermophilic Bacillus species that are facultative anaerobic and homolactic.
• the integration vector pRK1 (Fig. 7) contains the pNZ124 replicon, two 1-kb regions flanking IdhL in the B. coagulans DSM 1 chromosome and now flanking the B. coagulans ATCC 23498 amy promoter fused to the L. bulgaricus IdhA gene and the cat gene encoding chloramphenicol resistance.
• the integration vector is constructed by ligation of the following cassettes: (i) a 1.8-kb Sal ⁇ -Xba ⁇ fragment, with the Sa/I site made blunt, containing the pNZ124 replicon; (ii) a 1.1-kb Xba ⁇ -BamH ⁇ fragment containing the upstream region of the IdhL gene cut from a PCR fragment generated using the primers 5'- GCGAGATCTAGAGGCCATCTGGGGGGCTTTCT-3' and 5'-
• Plasmid pRK1 is transformed to B. coagulans DSM 1 by electroporation. Transformants are obtained on BC plates supplemented with 7 mg/L chloramphenicol grown at 45°C. After confirmation of the transformation by plasmid isolation, a single colony is cultured in BC broth at 45°C to mid log-phase after which the temperature is shifted to 55°C and incubation is continued for 1 h. A dilution series is plated on BC plates and incubated overnight at 55°C. The colonies are pooled and plated on BC plates in a second dilution series. After overnight incubation at 55°C colonies are tested for integration by PCR analysis confirming a single crossover event.
• One colony is selected for continued cultivation by sequential transfer of 1/1000 dilutions (approximately 10 generations) in BC broth with antibiotics at 55°C. After approximately 100 generations a dilution series is plated on BC plates with antibiotics. After overnight incubation at 55°C colonies series are tested for the absence of the IdhL gene by colony PCR.
• a double crossover mutant can be screened for among the single crossover mutants of the first event by testing for the absence of the IdhL gene by colony PCR. Chromosomal DNA from a colony negative in these PCR reactions is isolated and further evaluated for the presence oUdhA and correct integration is confirmed by Southern blot analysis.
• a B. coagulans DSM 1 derivative was constructed with the IdhL gene replaced by a cassette containing the IdhA gene from an industrial L delbrueckii strain fused to the IdhL promoter from an industrial B. coagulans strain and the cat gene from pMH3.
• the resulting strain was designated RDSM 1.
• the RDSM 1 strain was grown in a batch culture mimicking industrial conditions. After fermentation the concentrations of organic acids and the optical purity of the lactic acid were determined (Table 3). Lactate produced by B. coagulans RDSM 1 was for 99.5% in the R- form. No differences in by-product formation were detected compared to B. coagulans DSM 1 (Example 5). Concentrations of 2-hydroxy butyric acid, acetic acid, butyric acid, formic acid, pyruvic acid, were below the detection limits ( ⁇ 0.02% for pyruvic acid; ⁇ 0.01% for others). Concentrations of succinic acid were below 0.1 % (v/v). These results demonstrate chromosomal deletion of native B. coagulans genes and chromosomal insertion and functional expression of (heterologous) genes is possible and can be applied for production of enantiopure R-lactate by ⁇ . coagulans.

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