RECOMBINANT CELLS AND METHODS FOR BIOSYNTHESIS OF ENT-ATISERENOIC ACID
20170275659 · 2017-09-28
Inventors
Cpc classification
C12Y205/01039
CHEMISTRY; METALLURGY
C12P15/00
CHEMISTRY; METALLURGY
International classification
C12P15/00
CHEMISTRY; METALLURGY
Abstract
This disclosure describes recombinant cells and methods for microbial biosynthesis of ent-atiserenoic acid. Thus, in one aspect, this disclosure describes a recombinant cell genetically modified to exhibit increased biosynthesis of ent-atiserenoic acid compared to a comparable control cell. In some cases, the recombinant cell can include a host cell modified to include at least one heterologous polynucleotide encoding at least one enzyme in a biosynthetic pathway that produces ent-atiserenoic acid. In some cases, the recombinant cell can include a host cell and at least one heterologous enzyme in a biosynthetic pathway that produces ent-atiserenoic acid.
Claims
1. A recombinant cell genetically modified to exhibit increased biosynthesis of ent-atiserenoic acid compared to a comparable control cell.
2. A recombinant cell comprising: a host cell; and at least one heterologous polynucleotide encoding at least one enzyme in a biosynthetic pathway that produces ent-atiserenoic acid.
3. A recombinant cell comprising: a host cell; and at least one heterologous enzyme in a biosynthetic pathway that produces ent-atiserenoic acid.
4. The recombinant cell of claim 1 wherein the recombinant cell comprises a Streptomyces spp. host cell.
5. The recombinant cell of claim 1 wherein the comparable control cell comprises a wild-type cell.
6. A method of biosynthesizing ent-atiserenoic acid, the method comprising: culturing the recombinant cell of claim 1 under conditions effective for the recombinant cell to biosynthesize ent-atiserenoic acid.
7. The method of claim 6 further comprising isolating at least a portion of the ent-atiserenoic acid.
8. The method of claim 7 further comprising converting the ent-atiserenoic acid to serofendic acid.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0007]
[0008]
[0009]
[0010]
[0011]
[0012]
[0013]
[0014]
[0015]
[0016]
[0017]
[0018]
[0019]
[0020]
[0021]
[0022]
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0033] This disclosure describes recombinant cells and methods for microbial production of ent-atiserenoic acid (eAA), which is used as a source for serofendic acid (SA) synthesis. Serofendic acid is an endogenous factor found in fetal calf serum (FCS) that exhibits activity decreasing damage to neurons from radical insults. Thus, this disclosure also describes a method of biosynthesizing ent-atiserenoic acid. Generally, the method includes culturing a recombinant cell genetically modified to exhibit increased biosynthesis of ent-atiserenoic acid. In some cases, the method can further include converting the ent-atiserenoic acid to serofendic acid.
[0034] Serofendic acid exhibits activity at concentration as low as 1 nM that decreases cytotoxicity induced by reactive oxygen species in nerve cell culture, cardiomyocte culture, epithelial cell culture, and animal models. However, further assessment is limited by the limited availability of serofendic acid. Currently, serofendic acid can be obtained by extracting the compound from fetal calf serum or de novo chemical synthesis. It requires as much as 250 L of fetal calf serum to isolate 1.5 mg serofendic acid. Chemical synthesis of serofendic acid includes more than 15 steps with a low yields. Thus, neither method is sustainable for serofendic acid production.
[0035] This disclosure describes microbial fermentation to biosynthesize ent-atiserenoic acid using a genetically-modified microbe. In one exemplary embodiment, a recombinant Streptomyces spp. strain in engineered to include coding regions of nine enzymes required to biosynthesize ent-atiserenoic acid. The exemplary engineered strain produced 40 mg/L ent-atiserenoic acid. The ent-atiserenoic acid isolated from microbial fermentation can be used to synthesize serofendic acid. Additional embodiments also are described.
[0036] While described herein in the context of an exemplary embodiment in which the recombinant cell is, or is derived from, a Streptomyces spp., the recombinant cells and methods described herein can involve the use of other host cells that can be transformed with heterologous nucleic acids and express heterologous polypeptides encoded by the heterologous nucleic acids. As used herein “derived from” in connection with a microbe simply allows for the host cell to possess one or more genetic modifications before being modified to exhibit increased biosynthesis of ent-atiserenoic acid. Thus, the term “recombinant cell” encompasses a “host cell” that may contain nucleic acid material from more than one species before being modified to exhibit ent-atiserenoic acid. In various alternative exemplary embodiments, the host cell can be, or be derived from, Streptomyces coelicolor, Streptomyces sp. GS-93, or Streptomyces sp. 3211.
[0037] Also, while described herein in the context of an exemplary embodiment in which a host cell is genetically modified to include particular heterologous polynucleotides that encode particular heterologous enzymes involved in the biosynthesis of ent-atiserenoic acid, the recombinant cells and methods described herein can involve the use of other polynucleotides and/or other enzymes that perform similar or identical functions to those expressly exemplified. Alternative heterologous polynucleotides and alternative heterologous enzymes include those that, when transformed into a host cell, cause the host cell to exhibit increased biosynthesis of ent-atiserenoic acid.
[0038] Ent-atiserenoic acid (eAA) is a late stage intermediate in the synthesis of serofendic acid (SA). As shown in
[0039] Serofendic acid was originally isolated from the ether extract of fetal calf serum. While isolatable from fetal calf serum, serofendic acid is not detectable in adult bovine serum. The physiological concentration of serofendic acid is approximately in the tens of nanomolar level. The physiological role of serofendic acid is unclear.
[0040] Initial in vitro experiments showed serofendic acid protected cultured cortical neurons against acute glutamate neurotoxicity in a concentration-dependent and time-dependent manner at concentrations as low as 1 nM. The protective effect significantly increased when the cells were pretreated with serofendic acid for as short as 30 minutes prior to glutamate exposure.
[0041] Furthermore, serofendic acid decreased neurotoxic action of ionomycin, a calcium ionophore, and radical insults generated by NO and H.sub.2O.sub.2 donors. It was first speculated that the methylsulfoxide group of serofendic acid directly scavenges free radicals generated in neurotoxic events. However, it did not display direct radical scavenging activities against NO. In addition, serofendic acid does not directly inhibit glutamate receptors. Thus, serofendic acid may selectively attenuate toxicity without inhibiting the physiological roles of NO and glutamate receptors in signaling cascades.
[0042] Serofendic acid has also been tested in primary cultures of auditory neurons, astrocytes, cardiomyocytes as well as animal models of ischemic injuries. Serofendic acid is capable of protecting the cell types against oxidative stress-induced cell death. In vivo pharmacological studies indicated that serofendic acid afforded protection against cerebral ischemic-reperfusion injuries despite the low blood-brain barrier permeability (2.1 nmol/hour).
[0043] The activity of serofendic acid is still effective at the same level when RNA synthesis and de novo protein synthesis are inhibited. Without wishing to be bound by any particular theory or mechanism, these data suggest that serofendic acid's mode of action may be more likely to be mediated by protein interactions rather than transcriptional or translational regulations. Moreover, serofendic acid promotes neuron survival against neurotrophin deprivation, promotes stellation of astrocytes via cAMP-mediated and cGMP-mediated pathways, and inhibits TNF-α production in astrocytes. These observations revealed multifaceted effect of serofendic acid in promoting cell survival, but the underlying mechanisms were not entirely understood.
[0044] Current sources of serofendic acid are fetal calf serum and chemical synthesis. However, both sources are cost-prohibitive. 250 L of fetal calf serum are required to isolate 1.7 mg of serofendic acid. Moreover, harvesting fetal calf serum for biomedical research raises ethical questions. On the other hand, de novo chemical synthesis of serofendic acid requires more than steps with very low yield and thus requires much more initial precursors to generate sufficient amount of serofendic acid. Despite promising bioactivities of serofendic acid, the limited availability of serofendic acid has limited further therapeutic evaluation of serofendic acid.
[0045] This disclosure describes a microbial production source of ent-atiserenoic acid, a late intermediate that can be converted to serofendic acid and analogs. The microbial production of ent-atiserenoic acid allows for semi-synthesis of serofendic acid using ent-atiserenoic acid as a starting point for an abbreviated synthetic process compared to the current total synthesis process. Yield of serofendic acid is therefore increased because the microbial biosynthesis bypasses chemical synthesis steps that significantly decrease the final serofendic acid yield.
[0046]
[0047] In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0048] In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
[0049] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
[0050] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
EXAMPLES
Example 1
Construction of Engineered Pathway for Ent-Atiserenoic Acid Biosynthesis
[0051] The genomic DNA of Streptomyces NRRL S-1813 was extracted using standard extraction methods. A total of nine coding regions encoding enzymes in a synthetic pathway for ent-atiserenoic acid biosynthetic were cloned using PCR amplification. The coding sequences (CDSs) of nine genes were ptmM1, ptmM2, ptmM3, ptmT2, ptmT4, ptmO2, ptmO6, and ptmO9. The primer sequences (Table 1) contained AarI restriction recognition sites to make the AarI site flanking the CDSs. The internal restriction sites of AarI and SapI Type IIS restriction enzymes were removed during cloning. Each coding region was cloned into cloning vector pCDS using standard AarI reaction. The sequence of all DNA constructs were verified using Sanger sequencing.
TABLE-US-00001 TABLE 1 Primer sequences used in this study. SEQ ID Primer Sequence NO MSptmT1F ATTACACCTGCTTATA 1 ATGACGTCGGAACTGCCCGC MSptmT1rrs1R ATTACACCTGCTTAT 2 GTGCCGCGCAcGTGAACGCGGTGCG MSptmT1rrs1F ATTACACCTGCTTAT 3 GCACTGGAGATGGCGCTCGCCC MSptmT1R ATTACACCTGCTTATA 4 TCAGAGCGCGAGCAGCGGGATG MSptmO2F ATTACACCTGCTTATA 5 ATGACCACCCGCCCCACGGC MSptmO2R ATTACACCTGCTTATA 6 TCATGCTGGACACTCGGTCAC MSptmT2F ATTACACCTGCTTATA 7 ATGCTCGAAGTTCCCGCTCAG MSptmT2R ATTACACCTGCTTATA 8 TCAAGCACCTCCGGAAGCGGC MSptmO6F ATTACACCTGCTTATA 9 ATGTGGGAGCCGACGTCTGC MSptmO6R ATTACACCTGCTTATA 10 TCACGCGGCGGTGTTCTGCTG MSptmT4F ATTACACCTGCTTATA 11 ATGCACGCTGACACAGTCCAG MSptmT4rrs1R ATTACACCTGCTTAT 12 CGACGGGAATGgaCTTCTTCTTGTTGTG MSptmT4rrs1F ATTACACCTGCTTAT 13 GTCGTCGCCGCCCTCCACAGC MSptmT4R ATTACACCTGCTTATA 14 TCAGTGCTTCCTGAAGGCGAC MSptmM1F ATTACACCTGCTTATA 15 ATGACCGCCGCCATCGTGAG MSptmM1R ATTACACCTGCTTATA 16 TCAACAGCTGTCAACGGTCTTC MSptmM2F ATTACACCTGCTTATA 17 ATGCCGACGACACCGCTGCG MSptmM2rrs1R ATTACACCTGCTTAT 18 GTGCTCCGCGAAcAGCTCCGCCTC MSptmM2rrs1F ATTACACCTGCTTAT 19 GCACGACTTCCACGACTTCAAG MSptmM2R ATTACACCTGCTTATA 20 TCAGCCAGTCTCATCGAGGTC MSptmM3F ATTACACCTGCTTATA 21 ATGAGACTGGCTGACCTCAC MSptmM3rrs1R ATTACACCTGCTTAT 22 GTGGCCGGTGTCGAAcAGCAGGGTG MSptmM3rrs1F ATTACACCTGCTTAT 23 CCACCAGGCCTACGTCCACAAG MSptmM3R ATTACACCTGCTTATA 24 TCAGCGcGGGGCCGCTTGTG MSptmT2nestF GGCTCCCACACCAGCCCAGCCACCAGCCAG 25 MSptmT2nestR CATCGAGGGGCATGGGAAGGCCTTCTG 26 MSptmo9F ATTACACCTGCTTATA 27 ATGACGTACGTAATCGCGCAGC MSptmO9R ATTACACCTGCTTATA 28 TCATTCGCCCGCGGCTCCGGCC
CisREG and Monocistron Construction.
[0052] A library of CisREG cloning vectors that each contained a promoter, a ribosomal binding site, eGPF CDS, and a transcriptional terminator was assembled using BbsI type IIS assembly, PRC, and ligation. Then, the monocistrons were built using standard SapI Type IIS assembly reaction. Briefly, individual coding regions in the pCDS were mixed with equimolar of CisREG, with 10 U of T4 ligase and SapI restriction enzyme. The reaction was cycled between 37° C. and 16° C. for at least two hours, with last step of heat-inactivating the enzymes for 10 minutes. The CisTRON parts were transformed into E. coli DH5alpha and plated on an apramycin plate. The clones were verified using colony PCR with gene specific primers.
Partial Cluster Assembly.
[0053] Standard AarI type IIS assembly was used to construct a partial cluster from one, two, or three monocistronic parts. A total of three libraries of partial clusters (termed “M” clusters, “T” clusters, and “0” clusters) were assembled. The partial cluster constructs were transformed into E. coli DHSalpha and plated on a kanamycin plate. The clones were verified using colony PCR with gene specific primers
Full Cluster Assembly.
[0054] Full clusters were assembled using standard SapI type IIS assembly from libraries of three partial clusters. The full cluster constructs were transformed into E. coli DH5alpha and plated on apramycin plate. The clones were verified using colony PCR with gene specific primers
Heterologous Production in S. albus J1074 with Isolated Yields of 45 mg/L
Streptomyces Conjugation.
[0055] A total of eight full clusters were assembled and conjugated into Streptomyces using standard protocol. Briefly, individual full clusters were transformed into E. coli conjugation donor strain ET 12567 [pUCZS002]. Individual colonies were verified using colony PCR. Successful transformants were grown to confluence in 2 mL overnight cultures supplemented with apramycin. Frozen spore stock of S. albus J 1074 were quickly thawed, heat shocked at 50° C. for 10 minutes, supplemented with TSB medium with 10% sucrose, and incubated in 30° C. shaker for at least two hours. In the meantime, the E. coli donor strain containing the full cluster was washed with LB+20 mM magnesium chloride two times to remove antibiotics. After final centrifugation, the cell pellet was resuspended with 500 L of the Streptomyces germinated spore solution. The Streptomyces-E. coil solution was spotted on IWL-4 plates supplemented with 20 mM magnesium chloride, and dried completely over the surface. The plates were incubated overnight at 30° C. On the following day, the spot of Streptomyces-E. coli mixture was re-streaked on IWL-4 plates with 25 μg/mL nalidixic acid and 50 μg/mL apramycin plate to obtain single Streptomyces exconjugants. The plates were incubated for two to three days or until colonies appeared on the plate. The exconjugants were verified with colony PCR with gene-specific primers.
Small Scale Fermentation.
[0056] The exconjugants were grown in 3 mL seed culture in R2YE supplemented with apramycin at 30° C. shaker for 2-3 days. Once the culture reached confluence, 1:100 inoculation of each exconjugant was performed to start 3 mL seed cultures in ISM-3 medium supplemented with apramycin, incubating in 30° C. shaker for 2-3 days or until reached confluence. The ISM-3 seed cultures were used to inoculate 50 mL PCNM media (6 g/L yeast extract, 15 g/L malt extract, 6 g/L dextrose, 20 g/L MOPS sodium salt, 5 mL trace elements) with 1.5 g amberlite resin in 250 mL baffled flask. The fermentations were incubated in 30° C. shaker for 10 days.
Two-Liter Fermentation of Strain #4.
[0057] Single colonies of exconjugant containing #4 full cluster (strain 4) were grown in 3 mL seed culture in R2YE medium (Shepherd et al., 2010, Curr Protoc Microbiol. CHAPTER, Unit-10E.1. http://doi.org/10.1002/9780471729259.mc10e01s18) supplemented with apramycin at 30° C. shaker for 2-3 days. Once the culture reached confluence, 3 mL seed cultures in ISM-3 medium (15 g/L yeast extract, 10 g/L malt extract, 0.244 g/L MgSO.sub.4, 0.3 g/L FeCl.sub.3.6H.sub.2O, 20 g/L dextrose, pH 7.0) supplemented with apramycin were started by 1:100 inoculation from R2YE cultures and incubated in 30° C. shaker for 2-3 days or until reached confluence. The ISM-3 seed cultures were used to inoculate four 500 mL PCNM media with 15 g amberlite resin in two-liter baffled flasks. The fermentations were incubated in 30° C. shaker for 10 days.
Solid-Liquid Extraction.
[0058] The fermentations were poured into 50 mL conical tubes and centrifuged. The supernatants were saved in another 50 mL conical tube for later analysis. The resin and the cell pellets of the fermentation were washed with ddH.sub.2O three times. After the final centrifugation, the water was removed from the resin as much as possible, frozen at −80° C., and lyophilized overnight. Once the resin and the cell pellet were freeze-dried, they were extracted with 10 mL methanol for three times. The methanol extracts were dried down using vacuum distillation. The final samples were resuspended in 1 mL methanol to obtain concentrated extracts.
Purification of Ent-Atiserenoic Acid.
[0059] The concentrated extracts were subjected to column chromatography using silica gel as the solid phase, and a mixture of chloroform and methanol as the mobile phase. The samples were fractionated and each fraction was analyzed by TLC. The fractions containing compound with similar R.sub.f value to ent-atiserenoic acid were combined and dried down using vacuum distillation. The dried compounds were weighed and resuspended in 1-5 mL of methanol. The structure of eAA was further analyzed using NMR.
LC/MS analysis.
[0060] The injection samples were prepared by diluting each concentrated extracts 100-fold in methanol (10 μL concentrated extract in 990 μL methanol). The dilution samples were centrifuged in max speed, and the supernatants were transferred to LC/MS capped glass vials. The LC/MS buffer system used was 100% acetonitrile and 0.1% formic acid. The other buffer system was 100% acetonitrile and 10 mM ammonium formate (pH 8.3). The instrument used was plus single quadrupole Mass spectrometer. The total negative ion chromatogram was collected and analyzed in DIONEX Chromeleon Chromatography Data System (CDS) software, version 7.
Structure Determination of Ent-Atiserenoic Acid by NMR Spectroscopy
[0061] Purified material with a mass spectrum ([M-H]=301.2 amu) of a diterpene-acid was analyzed by 1D (.sup.1H and .sup.13C) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectroscopy to determine the chemical structure. The carbon spectrum showed chemical shifts for 20 carbon atoms, including three downfield shifts corresponding to a carboxylic acid (δC=184.91 ppm), and two olefinic carbons (δC=152.76 ppm and 104.58), the latter of which is shown to bear two protons by HSQC, and thus is an exocyclic methylene. In addition, the carbon spectrum and HSQC results show two methyls, nine methylenes, three methines, and three quaternary carbons. The proton spectrum showed two singlet methyl peaks, suggesting the methyl groups are connected to quaternary carbons, and these data together support that of an atiserene-based diterpene scaffold in which one of the methyl groups is oxidized to a carboxylic acid. This is confirmed by annotating COSY and HMBC correlations (
[0062] The three dimensional structure, coupled to the biosynthetic origin from a confirmed ent-atiserene synthase allow us to assign the absolute stereochemistry as ent-atiserenoic acid. The structures of shunt metabolites SH_B and SH_C were determined with similar 2 d correlations, and by comparison to reported chemical shifts reported in the REAXYS database.
Synthesis of Methylatiserenoate by Solvolysis
[0063] Heating ent-atiserenoic acid in methanol with 1% sulfuric acid afforded a new product, methylatiserenoate, with a longer retention time by HPLC.
Example 2
[0064] An ent-atiserenoic acid gene cluster, PB20 (genotype: scarA P[5] RBS[5] ptmM1 T1 scarB P[6] RBS[8] ptmM2 T[3] scarD P[12] RBS[10] ptmM3 T[2] scare P[9] RBS[7] dxr T4 scarF synP ispD T[5] scarA synP ispE T[6] scarB P[9] RBS[9] ispF T[7] scarD P[10] RBS[8]idi T[9] scarE; SEQ ID NO:30), was introduced into Streptomyces ceolicolor, Streptomyces sp. GS-93, or Streptomyces sp.3211 with standard intergenic conjugation protocol. The exconjugants were selected on apramycin supplemented IWL-4 solid media. A total of 10 colonies were inoculated in 2.4 mL R2YE and ISM3 media and fermented for four days in a 24-well plate. At the day of harvest, the entire 24-well plate was centrifuged in a tabletop centrifuge at top speed for 30 minutes. Ten-fold dilution of the extraction was prepared by adding 100 μL supernatant to 900 μL methanol. Individual extraction was analyzed by reverse phase LCMS (buffer system: 10 mM ammonium formate, pH 8.3; 100% acetonitrile). Ent-atiserenoic acid yield is shown in Table 2.
TABLE-US-00002 TABLE 2 Ent-atiserenoic acid yield using alternative Streptomyces spp. host cells ug/mL (10-fold Strain Medium AUC dilution) mg/L Streptomyces ceolicolor (1) ISM3 5062.2086 0.5620 5.6200 Streptomyces ceolicolor (2) ISM3 748.581 0.3352 3.3518 Streptomyces sp. GS-93 (3) ISM3 4197.0967 0.5165 5.1651 Streptomyces sp. GS-93 (4) ISM3 4379.9766 0.5261 5.2613 Streptomyces sp. GS-93 (5) ISM3 2128.1586 0.4077 4.0772 Streptomyces sp. GS-93 (6) ISM3 2915.8914 0.4491 4.4914 Streptomyces sp. 3211 (7) ISM3 13898.2826 1.0266 10.2662 Streptomyces sp. 3211 (8) ISM3 32368.5556 1.9978 19.9782 Streptomyces sp. 3211 (9) ISM3 27700.9468 1.7524 17.5238 Streptomyces sp. 3211 (10) ISM3 23643.9778 1.5391 15.3906 Streptomyces ceolicolor (1) R2YE 223.1946 0.3076 3.0756 Streptomyces ceolicolor (2) R2YE 610.3554 0.3279 3.2791 Streptomyces sp. GS-93 (3) R2YE 370.1540 0.3153 3.1528 Streptomyces sp. GS-93 (4) R2YE 668.9889 0.3310 3.3100 Streptomyces sp. GS-93 (5) R2YE 567.0887 0.3256 3.2564 Streptomyces sp. GS-93 (6) R2YE 991.7168 0.3480 3.4797 Streptomyces sp. 3211 (7) R2YE 5536.3695 0.5869 5.8693 Streptomyces sp. 3211 (8) R2YE 7982.4549 0.7156 7.1555 Streptomyces sp. 3211 (9) R2YE 7946.4374 0.7137 7.1366 Streptomyces sp. 3211 (10) R2YE 7645.9898 0.6979 6.9786
Example 3
[0065] An ent-atiserenoic acid gene cluster, PB La2 (genotype: P[11] RBS[10] ptmM1 T[1] P[4]RBS[6] ptmM2 T[3] P[4] RBS[6] ptmM3 T[2] P[12] RBS[9] dxr T[4] P[6] RBS[8] ispD T[5]P[5] RBS[5] ispE T[6] synP ispF T[7] synp idi T[9]; SEQ ID NO:31), was introduced into Streptomyces albus J1074 with standard intergenic conjugation protocol. A seed culture of the PB L2a-containing strain was grown in R2YE and then ISM3 for three days and used to inoculate 6×500 mL (total of 3 L) ISM3 cultures. Ent-atiserenoic acid production was assessed by collecting 5 mL of fermentation broth daily for seven days.
[0066] The cultures were separated into three groups and samples taken in duplicate: feed 0 (no feeding, take 5 mL sample daily); feed 1 (starting at 36 hours, supplement the culture with 5 g of glucose in the form of 10 mL glucose solution twice per day; take 5 mL sample daily); feed 2 (starting at 36 hours, supplement the culture with 10 g of glucose in the form of 20 mL glucose solution, 5 g yeast extract+malt extract, and 50 uL trace element per day; take 5 mL sample daily).
[0067] Each 5 mL sample was extracted by adding 5 mL methanol to 5 mL fermentation broth containing cell pellet and resin. The extraction mixture was incubated in a shaker for three hours. Subsequently, the extraction was diluted by 400-fold, then prepared as follows: the first dilution was prepared by adding 50 μL extraction to 950 μL methanol. The final dilution (400×) was made by taking 50 μL of first dilution to 950 μL methanol. The final dilution of the extract was analyzed by reverse phase LCMS (buffer system: 10 mM ammonium formate, pH 8.3; 100% acetonitrile.) The raw peak intensity of each sample was back calculated to concentration of ent-atiserenoic acid in the 400-fold diluted extraction by the standard curve (equation: [Peak intensity]=18793×[μg/mL ent-atiserenoic acid]). The original concentration of ent-atiserenoic acid production is calculated by diluted concentration×400. Results are shown in Table 3.
TABLE-US-00003 TABLE 3 Concentration of ent-atiserenoic acid by S. albus PB La2 Day Feed 0a Feed 0b Feed 1a Feed 1b Feed 2a Feed 2b 1 1.681477 11.70649 7.832704 0 4.831586 0 2 19.30506 94.14144 40.41931 32.26733 40.31288 16.55936 3 49.78449 59.55409 128.3669 41.46225 72.40994 15.47385 4 349.5983 199.7127 170.6806 147.7359 76.00702 34.43835 5 140.116 231.5969 133.9861 134.5395 126.4088 52.57277 6 925.3233 706.114 491.0977 524.2165 396.3816 406.5556 7 665.7372 385.4627 438.5463 503.8259 430.522 454.0414
Example 4
[0068] To identify enzymes that may be used in place of ent-atiserene synthase ptmM1 (accession number: ACO31274.1), the amino acid sequence of ent-atiserene synthase ptmM1 was obtained from Uniprot and subjected to BLAST analysis against the nr database (NCBI). The analysis identified the amino acid sequences identified below. A sequence alignment was performed by ClustalW multiple alignment application (1000 bootstraps) to calculate protein distance matrix. The phylogenetic analysis was performed using PHYLIP (University of Washington, Seattle, Wash.), and the phylogenetic tree was plotted in FigTree. A list of the sequences (accession number, protein name, species of origin) follows:
[0069] ACO3 1274.1 PtmT1 [Streptomyces platensis]
[0070] AHY18940.1 ent-atiserene synthase [Streptomyces platensis]
[0071] AHY18941.1 ent-atiserene synthase [Streptomyces platensis]
[0072] AHY18942.1 ent-atiserene synthase [Streptomyces platensis]
[0073] AHY18944.1 ent-atiserene synthase [Streptomyces platensis]
[0074] AHY18945.1 ent-atiserene synthase [Streptomyces platensis]
[0075] ADD83014.1 PtnT1 [Streptomyces platensis]
[0076] WP_030985278.1 hypothetical protein [Streptomyces sp. NRRL S-1813]
[0077] WP_030080417.1 hypothetical protein [Streptomyces decoyicus]
[0078] ACZ86631.1 hypothetical protein Sros_3708 [Streptosporangium roseum DSM 43021]
[0079] WP_005454079.1 4-hydroxybenzoate polyprenyltransferase [Saccharomonospora cyanea]
[0080] WP_005462072.1 4-hydroxybenzoate polyprenyltransferase [Saccharomonospora glauca]
[0081] WP_008028525.1 bacteriochlorophyll/chlorophyll synthetase [Rhodobacter sp. SW2]
[0082] WP_008280363.1 chlorophyll a synthase [Roseovarius sp. TM1035]
[0083] WP_009152840.1 4-hydroxybenzoate polyprenyltransferase-like prenyltransferase [Saccharomonospora marina]
[0084] WP_009455902.1 MULTISPECIES: homogentisate phytyltransferase [Fischerella]
[0085] WP_010997598.1 homogentisate phytyltransferase [Nostoc sp. PCC 7120]
[0086] WP_011908055.1 bacteriochlorophyll/chlorophyll a synthase [Rhodobacter sphaeroides]
[0087] WP_012616483.1 bacteriochlorophyll/chlorophyll synthetase [Chloroflexus aggregans]
[0088] WP_012796323.1 4-hydroxybenzoate polyprenyltransferase [Saccharomonospora viridis]
[0089] WP_016861644.1 homogentisate phytyltransferase [Fischerella muscicola]
[0090] WP_016874172.1 homogentisate phytyltransferase [Chlorogloeopsis fritschii]
[0091] WP_017310301.1 homogentisate phytyltransferase [Fischerella sp. PCC 9339]
[0092] WP_017319040.1 homogentisate phytyltransferase [Mastigocladopsis repens]
[0093] WP_017323227.1 homogentisate phytyltransferase [Cyanobacterium PCC 7702]
[0094] WP_017655775.1 homogentisate phytyltransferase [Fortiea contorta]
[0095] WP_017987983.1 hypothetical protein [Amycolatopsis methanolica]
[0096] WP_020420680.1 hypothetical protein [Amycolatopsis sp. ATCC 39116]
[0097] WP_022573875.1 chlorophyll synthase [Rhodobacteraceae bacterium HIMB 11]
[0098] ESW60080.1 bacteriochlorophyll/chlorophyll a synthase [Rhodobacter sp. CACIA14H1]
[0099] WP_024876751.1 4-hydroxybenzoate polyprenyltransferase [Saccharomonospora sp. CNQ490]
[0100] WP_026722928.1 homogentisate phytyltransferase [Fischerella sp. PCC 9431]
[0101] WP_027930452.1 hypothetical protein [Amycolatopsis thermoflava]
[0102] WP_029637774.1 homogentisate phytyltransferase [[Scytonema hofmanni] UTEX B 1581]
[0103] WP_030080417.1 hypothetical protein [Streptomyces decoyicus]
[0104] WP_030470347.1 hypothetical protein [Lechevalieria aerocolonigenes]
[0105] WP_031170430.1 hypothetical protein [Streptosporangium roseum]
[0106] KFL36038.1 hypothetical protein N788_05700 [Arenimonas donghaensis DSM 18148=HO3-R19]
[0107] WP_033358256.1 prenyltransferase [Dactylosporangium aurantiacum]
[0108] WP_034224802.1 4-hydroxybenzoate octaprenyltransferase [Arenimonas donghaensis]
[0109] WP_034260342.1 hypothetical protein [Actinospica robiniae]
[0110] WP_036015193.1 hypothetical protein [Lentzea albidocapillata]
[0111] WP_037261197.1 bacteriochlorophyll/chlorophyll a synthase [Roseivivax halodurans]
[0112] WP_037275502.1 prenyltransferase [Rhodococcus rhodnii]
[0113] WP_037308517.1 4-hydroxybenzoate polyprenyltransferase [Saccharomonospora viridis]
[0114] WP_007027364.1 4-hydroxybenzoate polyprenyltransferase-like prenyltransferase [Saccharomonospora paurometabolica]
[0115] WP_037571185.1 hypothetical protein [Streptacidiphilus oryzae]
[0116] WP_038007406.1 MULTISPECIES: bacteriochlorophyll/chlorophyll a synthase [Thalassobacter]
[0117] WP_039688771.1 bacteriochlorophyll/chlorophyll a synthase [Tateyamaria sp. ANG-S 1]
[0118] KIF37224.1 tocopherol phytyltransferase [Hassallia byssoidea VB512170]
[0119] WP_043143946.1 bacteriochlorophyll/chlorophyll a synthase [Mameliella alba]
[0120] WP_043655945.1 hypothetical protein [Streptosporangium roseum]
[0121] WP_043843101.1 hypothetical protein [Amycolatopsis taiwanensis]
[0122] WP_045313759.1 hypothetical protein [Lechevalieria aerocolonigenes]
[0123] WP_048599939.1 bacteriochlorophyll/chlorophyll a synthase [Nereida ignava]
[0124] WP_050530946.1 bacteriochlorophyll/chlorophyll a synthase [Aestuariivita atlantica]
[0125] WP_050930602.1 bacteriochlorophyll/chlorophyll a synthase [Aestuariivita boseongensis]
[0126] WP_053458409.1 homogentisate phytyltransferase [Hapalosiphon sp. MRB220]
[0127] WP_053738288.1 hypothetical protein [Nocardia sp. NRRL S-836]
[0128] KPP92134.1 chlorophyll synthase ChlG [Rhodobacteraceae bacterium HLUCCA08]
[0129] KPQ08812.1 chlorophyll synthase ChlG [Rhodobacteraceae bacterium HLUCCA09]
[0130] WP_057290696.1 4-hydroxybenzoate octaprenyltransferase [Noviherbaspirillum sp. Root 189]
[0131] WP_058123499.1 bacteriochlorophyll/chlorophyll a synthase [Thalassobacter stenotrophicus]
[0132] KU066555.1 cytochrome B562 [Alphaproteobacteria bacterium BRH_c36]
[0133] WP_062242826.1 homogentisate phytyltransferase [Fischerella sp. NIES-3754]
[0134] WP_062280982.1 MULTISPECIES: bacteriochlorophyll/chlorophyll a synthase [Rhizobium]
[0135] AOE07831.1 chlorophyll a synthase ChlG [uncultured bacterium]
[0136] WP_067765775.1 tocopherol phytyltransferase [Nostoc sp. NIES-3756]
[0137] WP_068353150.1 bacteriochlorophyll/chlorophyll a synthase [Rhodobacteraceae bacterium SB2]
[0138] WP_068361717.1 bacteriochlorophyll/chlorophyll a synthase [Jannaschia sp. EhC01]
[0139] WP_069210025.1 bacteriochlorophyll/chlorophyll a synthase [Ruegeria sp. PBVC088]
[0140] SDJ29593.1 chlorophyll synthase [Lutimaribacter saemankumensis]
[0141] SDC 18937.1 4-hydroxybenzoate polyprenyltransferase [Prauserella marina]
[0142] SDN46345.1 4-hydroxybenzoate polyprenyltransferase [Streptomyces guanduensis]
[0143] SDM79676.1 4-hydroxybenzoate polyprenyltransferase [Lentzea violacea]
[0144] SDX17745.1 4-hydroxybenzoate polyprenyltransferase [Amycolatopsis xylanica]
[0145] SES 11996.1 4-hydroxybenzoate polyprenyltransferase [Phycicoccus cremeus]
[0146] SER92449.1 4-hydroxybenzoate polyprenyltransferase [Lentzea flaviverrucosa]
[0147] SFJ85297.1 chlorophyll synthase [Jannaschia pohangensis]
[0148] SFA93839.1 4-hydroxybenzoate polyprenyltransferase [Amycolatopsis marina]
[0149] SFJ98404.1 4-hydroxybenzoate polyprenyltransferase [Amycolatopsis sacchari]
[0150] SFI62503.1 4-hydroxybenzoate polyprenyltransferase [Streptosporangium canum]
[0151] SFR25443.1 4-hydroxybenzoate polyprenyltransferase [Lentzea waywayandensis]
[0152] WP_071974050.1 bacteriochlorophyll/chlorophyll a synthase [Sulfitobacter sp. AMI-DI]
[0153] OJW42097.1 4-hydroxybenzoate polyprenyltransferase [Thiobacillus sp. 65-1059]
[0154] OJW84228.1 4-hydroxybenzoate polyprenyltransferase [Thiobacillus sp. 65-1402]
[0155] WP_072630499.1 bacteriochlorophyll/chlorophyll a synthase [Planktotalea frisia]
[0156] WP_072778091.1 bacteriochlorophyll/chlorophyll a synthase [Marivita hallyeonensis]
[0157] WP_072785392.1 4-hydroxybenzoate polyprenyltransferase [Duganella sacchari]
[0158] WP_052583874.1 4-hydroxybenzoate polyprenyltransferase, partial [Actinopolyspora iraqiensis]
[0159] WP_005443975.1 prenyltransferase UbiA [Saccharomonospora azurea]
[0160] EID53416.1 UbiA prenyltransferase family protein [Saccharomonospora xinjiangensis XJ-54]
[0161] EHK84863.1 4-hydroxybenzoate polyprenyltransferase-like prenyltransferase [Saccharomonospora azurea SZMC 14600]
[0162] EHK80706.1 4-hydroxybenzoate polyprenyltransferase-like prenyltransferase [Saccharomonospora azurea SZMC 14600]
[0163] EHY90523.1 4-hydroxybenzoate polyprenyltransferase-like prenyltransferase [Saccharomonospora azurea NA-128]
[0164] ABA23079.1 homogentisate phytyltransferase [Anabaena variabilis ATCC 29413]
[0165] WP_073555048.1 homogentisate phytyltransferase [Fischerella major]
[0166] WP_074201873.1 4-hydroxybenzoate polyprenyltransferase [Sulfurivirga caldicuralii]
[0167] WP_074621517.1 bacteriochlorophyll/chlorophyll a synthase [Mameliella alba]
[0168] WP_075787276.1 bacteriochlorophyll/chlorophyll a synthase [Rhodovulum sulfidophilum]
[0169] WP_076630627.1 bacteriochlorophyll/chlorophyll a synthase [Tateyamaria omphalii]
Example 5
Semi-Synthesis of Serofendic Acid
[0170] ##STR00001##
[0171] One molar equivalent of (PhSeO).sub.2O (1.19 mg/mg intermediate 1) was added to a 5 mM solution of intermediate 1 in benzene (0.66 mL/mg intermediate 1) and refluxed for four hours. Ethyl acetate was added prior to rotary evaporation, which was followed by flash chromatography using 10:1 hexane-ethyl acetate. Fractions containing the intermediate 2 were identified by TLC.
##STR00002##
[0172] One molar equivalent of NaSMe (15% in water, m/v; 1.48 μL/mg intermediate 2) was added to a 50 mM solution of intermediate 2 in THF (0.63 mL/mg intermediate 2) at room temperature. After 15 minutes, the reaction was diluted by a factor of five with NaCl brine. The product was extracted with ethyl acetate. The organic layer was washed with NaCl brine and dried with CaCl.sub.2. Solvent was removed by rotary evaporation, and the product was purified by flash chromatography using 10:1 hexane-ethyl acetate. Fractions containing the intermediates 3 were identified by TLC.
##STR00003##
[0173] A solution of intermediates 3 in THF was stirred at 0° C. Using a syringe, 370 μL of 1 M BH.sub.3 in THF was added over 10 minutes. After one hour, 1 mL methanol and 1 mL 10% HCl were sequentially added. The product was extracted with petroleum ether. The organic layer was dried with CaCl.sub.2, and solvent was removed by rotary evaporation. The product was purified by flash chromatography using 5:1 hexane-ethyl acetate. Fractions containing intermediates 4 were identified by TLC.
##STR00004##
[0174] A solution with 100 mM intermediate 4 and 130 mM Davis oxaziridine in chloroform was stirred at room temperature for one hour. One mL petroleum ether and 1 mL 10% HCl were sequentially added. The aqueous layer was removed and extracted with petroleum ether. The organic extract was combined with the organic layer from the reaction and dried with CaCl.sub.2, and solvent was removed by rotary evaporation. The product was purified by flash chromatography using a solvent gradient from ethyl acetate to 10:1 dichloromethane-methanol.
[0175] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
[0176] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[0177] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
[0178] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
TABLE-US-00004 Sequence Listing Free Text DNA sequence of Strain #3 (SEQ ID NO: 29) GCTCTTCggagCCGGGCGGCTTCCTCATGCTTGACTTGACTAGGATAAAGGGGCTACAAGGAAG AAGGAGGTTAACAaatgACCGCCGCCATCGTGAGTGTCGCCGCCCGCCCGCTGAGCACGGCGGT GCGCCACGGCGACGTGGTGCTCGCCGACCGGCTGCACCTGGCGTCGGGCGCCCGCGAGGGCCGG GTCCACCGGCTGCTCGCCGGGGCCCTCAGGGCACAGGGGCTGACGGTCCACACCGGCGGGGCCG CCGTCGCCGGCGCCGAGGCACCCCAGGGCGCCGACGCACCGCTGGTGGAGATCGCCGCGCCGGT CCGGCTCCCGGAGGCGGACGGTGACGCGCTGCTGTGCCTGGTGCGCGACGACACCGGATCGGAC GCCGTCGACCGCGCCGAGGAGGCCCTGGCCCGGGTCCTGGGCGACTGGGAAGCCGCGAAGGGGG ACCGAGCGGTGGCCCTGGCCGCCCCCCGGTCGTTCTGCGCCGGTGTGGACCGTGCCATCGAGAT CGTCGAACGCGCCCTCGACCAGTACGGCGCCCCCGTCTACGTACGCAAGCAGATCGTCCACAAC CGGCATGTCGTCGAGGACCTCGCCCGCCGCGGCGCGGTCTTCGTCGAGGAGCTTGACGAGGTGC CGGAGGGCGAGCTGGTCGTCTTCTCCGCCCACGGGGTCGCCCCGGCGGTACGTGACGCGGCGGG CGAGCGCGGTCTGCGAGTGATCGACGCGACCTGCCCGCTGGTCACCAAGGTCCATGCCGAGGCC AAGCGGTTCGCGGGCCGCGGCGACACCGTCGTCCTGATCGGGCACGCCGGTCACGAGGAGGTCG AGGGCACGCTGGGCGAGGCGCCCGACCGCACGGTGCTGGTGCAGAACGCGGCGGAGGCGGCGCG CCTGGAGGTCGAGGACCCCGAGGCGGTCTCCTTCCTCATGCAGACCACGCTGGCGATGTCCGAG GCCACCGAGGTGGCCCAGGCCCTGGCCGGCCGCTTCCCGTCGATCAAGGCACCGCAGTCCGAGG ACATCTGCTACGCCTCGACCAACCGGCAGCGCGCCGTCGAGGAGATCGCCGGGCGGGTCGATCT GCTCCTGGTGGTCGGATCGCCCAACTCCTCCAACTCCGTACGCCTGAAGGAACTCGCGGAGCGG ATGGGCACCCCCGCCCAGCTGGTGGACGACGCCTCGTACGTCGGTCTGGAGCAGCTGCACGGGG CCCGGCGGATCGGCCTGACGGCCGGCGCCTCGGCCCCGGACACCCTCGTCCAGGAAATCGTCGC CAATCTCCGTGCCCTCGGACCCGTCACGGTCACCGAGCACCAAGTCGCGACGGAGAACGTCACC TTCCAGCTGCCCAAAGAGCTGAGGAGCGCCCGGAAAGACCGCGCCCGCAAAGACTTGGAGAAGA CCGTTGACAGCTGTtgatCTCGGTACCAAACCAATTATTAAAGACGCTGAAAAGCGTCTTTTTT TGTTTCGGTCCtacttactCGCGTAGGCTAAAGATGACATCTTTAACATAGGATGTGCGCAAAA AAAAGGGAGAGGAGCGTATTTaatGCCGACGACACCGCTGCGTCGTCCCACCCGGCAGCTGATG CTCGGCGGCATGGGCGTCGGCAGCCGCCACCCCGTCTCGGTGCAGTCGATGACGACCACGGTGA CGGCCGATGCCCAGGCCACCCTTCAGCAGATAGCCGAACTCACCGCGGCGGGCTGCGACATCGT CCGGGTCGCCTGCCCCAGCCGGGACGACGCCGAAGCCCTCGCGGAGATCGCCCAGAAGTCGAAG ATCCCCGTCATCGCGGACATCCACTTCCAGCCGCGCTATGTCTTCGCCGCGATCGAGGCGGGCT GCGCCGGTGTCCGGGTCAACCCGGGCAACATCAAGGAATTCGACGACAAGATCAAGGAGATCGC GCAGGCCGCGAAGGAGGCCGGCACCCCGATCCGCATCGGGGTCAACGCGGGCTCGCTCGACCCG AGGATCCTGCGGAAGTTCGGCAAGGCGACCCCGGAGGCCCTGGCCGAATCGGCGCTGCGCGAGG CGGAGCTCTTCGCGGAGCACGACTTCCACGACTTCAAGATCTCGGTGAAGCACCACGACCCGAT GGTGATGATCCGGGCGTACGAGCTGCTGGCCGCGCAGTGCGACTACCCGCTCCACCTCGGCGTC ACCGAGGCGGGACCGGCCTTCCAGGGCACGGTCAAGTCCTCGGTGGCCTTCGGCGCGCTGCTGC GCCAGGGCATCGGCGACACCATCCGCGTCTCGCTGTCCGCACCGCCCGTGGAGGAGGTCAAGGT CGGCATCCAGATCCTCCAGTCGCTCGGCCTGCGCCCCCGGCGCCTGGAGATCGTCTCCTGCCCG TCCTGCGGCCGCGCCCAGGTGGACGTCTACAAGCTCGCCGAAGAGGTGAGCGCCGGGCTCGAAG GGCTTCCGGTGCCGCTGCGGGTCGCCGTCATGGGCTGTGTCGTCAACGGTCCCGGCGAGGCCCG CGAGGCCGACCTCGGTGTCGCCTCCGGCAACGGCAAGGGGCAGATCTTCGTCAAGGGCGAGGTC GTCAAGACCGTCCCCGAGTCGAAGATCGTCGAGACCCTCATCGAAGAGGCACTGCGCCTCGCGG ACGAGATGGGGGTGGACCTCGATGAGACTGGCTgatCTCGGTACCAAATTCCAGAAAAGAGACG CTGAAAAGCGTCTTTTTTCGTTTTGGTCCaggtTCAAATGGCTAGGATATAACCTACACGATTA GGATAGCCCCGCATCAATCAGAGAAGGAGGTACCAAaATGAGACTGGCTGACCTCACCGGTCCG GGCGATCTCGCCGCACTGACGGAGGATCAACTCCAGGCGCTGGCCGCGGACATCCGCTCCTTTC TCGTGGAGTCCGTTTCGAAGGTCGGCGGACACCTGGGCCCCAACCTCGGCGTCGTCGAGCTCAC CCTCGCCCTGCACCGGGTCTTCGAGTCCCCCAAGGACACCCTGCTCTTCGACACCGGCCACCAG GCCTACGTCCACAAGCTGCTCACCGGCCGGATGAAGGCCTTCTCGACGCTGCGCCAGGAAGGCG GGCTCTCCGGCTATCCCGACCGCAGCGAGTCCGAGCACGATGTCATCGAGAACTCCCATGCCTC CACGGCCCTTTCGTACGCAGACGGCATCGCCAAGGGCTTCGGCCTGGCCGGCGCCGCGCACCGC AGGGTGGTCGCCGTCGTCGGCGACGGCGCGCTGACCGGCGGGATGAGCTGGGAGGCGCTCAACA ACATCGGCGGCGCCCCGGACCGCCCGGTGATCATCGTCCTGAACGACAACGGCCGTTCCTACGC CCCCACCGCCGGCGCCCTCGCCACCCACCTCGGCGAGCTCCGGGCGGGCCGCGGCGGGGCCGGT CTCTTCGCGAACCTGGGCCTCGCCTACCTGGGCCCGGTCGACGGCCACGACCGCCCGGCACTCG AGCGCGCACTGCGCCGGGCGGCCGCACTCGACCGCCCCGTGGTGGTGCACTGTGTGACGCAGAA GGGCCACGGTTACGCACCGGCCGCCGAGGACCCCGACGACTGCTGGCACGCGGTGGGCACCTTC GACCCCGAGACCGGCGGCAAGTCCGCTTCCGGCGGCCGCTCCTGGACCGCGGTGTTCGGTGCGG AGATGACGGAACTCGGTGCGCAGCGGCCCGACGTGGTCGCCCTCACCGCCGCGATGCTCCAGCC CGTGGGCCTGGCCGACTTCGCCCGCCGCTTCCCCGACCGGGTCTTCGACGTCGGCATCGGCGAA CAGCATGCCGCCGTCTCCGCCGCCGGGCTGGCGCACACCGGGCTGCACCCCGTCGTGGCCGTCT ACTCCACCTTCCTCAACCGCGCCTTCGACCAGGTGCTGATGGATGTGGCGCTGCACCGGCAGCC GGTGACCTTCGTACTCGACCGGGCGGGTGTCACCGGCCCCGATGGCCCCAGCCATCACGGGATC TGGGACGCCTCCTGGCTGTCGCTGGTACCGGGGCTGCGCCTGGCGGTGCCGCGCGATGCCGAGG AACTCAGGACGCTGCTGCGGGAGGCGGTCGCCGTCACGGACGGGCCCACCGTCCTCCGCTTCCC GAAGGCACAGGCCGGCCCGGCCGTGCCGGCGCTCCGCCGGGAGGGGGGCATGGACGTGCTGCAC GAGGCGCCCGGCGCCCGGGTGCTGCTGGTCCCGACCGGTCCGCTCGCCGACCCGTGCCTTCAGG CCGCGGCCGCGCTGGACGCCCTGGGCATCCCGTCGACGGTGGTGGACCCGCGCTGGTCCGTCCC CGTACCGGAGGGGCTGCCGGAGCTGGCCGCACGGCACGAACTCGTGGTGACCGTCGAGGACAAC CTGAGCGACGGCGGGCTCGGCGCGCGCCTGCTGCGACAGCTGTCCGAGGCCGGCACGCCCACCC CCGTACGGACCGTCGGCCTGCCGACGGAGTTCCTTCCCCACGGCAGCAGGACGGCCCTCCTGCG CCGGCATGGGCTCACCGCCGACGGCCTGGTCGCACGGGTCGGCGGATGGCTGCCACAAGCGGCC CCCCGCTGACTCGGTACCAAACCAATTATTAAAGACGCTGAAAAGCGTCTTTTTTTGTTTCGGT CCgcttCAGGTCGGCTGGTTGGCTGACAAAGTGCGTAGGATGACTGTGCATCAATCAGAGAAGG AGGTACCAAATGACGTCGGAACTGCCCGCCGCACGCGGCGAAGGGCTGCGGAGCCTCGTCCGCA TCCACCGGCTGGAATACCCCTTTCCGGTCATCTATCTCTGCCATGTCCTGTGGGGCGCCTGCCT CGCGGCGACGGGTCCGGGCAGCCTGGCCGCCGCGCCCGTCCTGATCATGCTGTTCGCCAATATC GTCGCGATCATCTCCCAGAACCCGCTCAACGCCGGTCTGGACATCCGGGCGGACACCCACACCA GCGGCAAGGAGAGCATCGCCCGCGCCACCCAGCACCTCAGCGTCCGCACCGCGTTCACCTGCGC GGCACTGGAGATGGCGCTCGCCCTCGGGCTGTCCGTCTGGGTCGCGCTCTGGCTCGGCCGGCCG CTCGTCGCGGTGGGGGTGGCGCTGTCGATCGTGCTCCACCTCGCCTACAACCTGGAGCCGGTCC GGCTGAAGCGGCGCGGCTACGCCAACCCCGCCTACTTCGGGGCGACCTTCGCCTTCCTGCCGTC GCTGTCGACGTATGCGGCGGTGCGGGCGGACGTACCGCTCAGCGCGTGGCTGTTCCTCACCGGG CTCGGCATCCTGCTGTTCGGCCGCTCCCTGTGGTGGTGCATCCCGGACCTGATCGGTGACGCCA AGGCCGGGGACCGTACGCCCGCCGTACAGCACGGCCCGCGCCATGCGCTGGTGGTGGCGTGCCT GTGGACCGCGCTCGGGCTGCTGTTCATCGGCGCCGGGCTGTGGCCGTACGGCGTCCTCTGGGCG CTGCTCGGCATCCTGGCGAGCGCCGCCTTCCTCGTGGACAAGATCAAGCTGCTGCGGCACATCT CGCGGGAGAACCTCCCGCACGAGTCCACGATGCGCAAGCACAGCCTCTCGCTGGCGATGGGCGG CGACCTCCTGCTCTGCGCCATCCCGCTGCTCGCGCTCTGAtCAGATAAAAAAAATCCTTAGCTT TCGCTAAGGATGATTTCTcgctACCCGTGGCTACTCAGCGAATAACTCTTGTAGGATACATAAA AAAAAAAGGGAGAGGAGCGTATTTaATGCTCGAAGTTCCCGCTCAGCCCACGCCCGCCCCCCGC GAGGCCGAGGCGGCCGCGCTGCTCGCGGCGACCGTCCACGACCCCTGGGGCCTGGTCGCTCCGT CGGTGTACGACACCGCCCGGCTGGTCTCCCTCGCCCCGTGGCTCGACGGCCACCGGGAGCGTCT CGGCTATCTGGTCGAGGAGCAGAACCAGGACGGAAGCTGGGGCGCACCCGACGGGTACGGCCTG GTACCCACGCTCAGTGCGGTGGAGGCGCTGCTGACCGAACTCGCCCGGCCGGAATCCGGCGCGC CGCACCCGCCCCACGACGACCTCGCCGCGGCCTGCGCCGGCGGTCTGGGCGCCCTCCAGGACGG TCTGCTCGCCGGTCCGGTGCCCGACACCATCGGCGTCGAGTTCGTCGCGCCGTCCCTGCTCGCG GACATCAACACCCGGCTGGCCGCGCTGACCGAGCAGGCACCCGGCAAGCTCGGGGCATGGTCCG GCACCACCCTGACGTCACCGGCGCCCGACCTGGACGGTGCGCTGCTGGCCGGCGTCCGGGAGAT GACCGAGCAGGCGCCGCTGCCGGAGAAGCTGTGGCACACACTGGAGGCCATCACCCGCGACGGC ACCCGCGGTGCCCGGCCGCACGAGGGCGCACCGCCGCACAACGGCTCGGTCGGCTGCTCCCCCG CCGCCACCGCCGCCTGGCTGGGCGCCTCGCCCGATCCGGCCGCGCCGGGCGTCGCCTATCTCCG TGACGTCCAGGCGCGGTTCGGCGGGCCGGTGCCCTCGATCACCCCGATCGTCTACTTCGAGCAG GCGTGGGTCCTCAACTCGCTGGCCGCCTCCGGCCTGCGCTACGAGGCCCCGGCCGCGCTCCTCG ACAGCCTCGAAGCGGGTCTCACGGACGAGGGCACAGCCGCCGCCCCCGGTCTGCCGAGCGACTC CGACGACACCGCCGCCGTCCTCTTCGCCCTGGCGCAGCACGGCAGGACGCACCGCCCCGACAGC CTGATGCACTTCCGCCGGGACGGCTACTTCTCCTGCTTCGGCGTCGAGCGCACCCCCTCCACCA GCACCAACGCACACATCCTGGAGGCCCTCGGCCATCACGTCACGGTGCGCCCCGACGACGCGGG ACGCTATGGCGCGGAGATCCGGATGATCAGCGACTGGCTGCTGGACAACCAGCTGCCCGACGGC AGCTGGATGGACAAGTGGCACGCCTCGCCGTACTACGCCACGGCCTGCTGTGCGCTGGCGCTCG CCGAGTTCGGCGGCCCGTCCGCACGGGCCGCGGTCGGCCGGGCCGCCGCGTGGGCACTGGCGAC CCAGCGCGCCGACGGCTCCTGGGGACGCTGGCAGGGCACCACGGAGGAGACCGCGTACATGGTG CAGCTCCTGATGCGTACCCGTACCCCCGGGAGCCCGGGGACCGTCGCCCGGTCGGCGGCCCGCG GCTGCGACGCGCTGCTGGCCCACGACGACCCGGCCTCCTACCCCGGGCTCTGGCACGACAAGGA CATCTACGCGCCGGTGACCGTCATCCGGGCGGCGCGGCTCGCGGCACTGGCGCTCGGCGGCGCC GAGTCCGCCGCTTCCGGAGGTGCTTGAtTCCGGCAATTAAAAAAGCGGCTAACCACGCCGCTTT TTTTACGTCTGCAggagTAGCAGGGCTCCAAAACTAACGCCTGATGTAGGATCAGATGGCTACA AGGAAGAAGGAGGTTAACAaTGCACGCTGACACAGTCCAGCCCCTCGAGAGCAGTGTCGACCTG GCCCACCGCAACGCCTCGCGGGCCGCCGGCCTCGTCACGCCCGCCCTGCGGGCCACCGTCGACA CCTTCGACAACCGCATCCGCCCCATCGTCGCCTACCACTTCGGCTGGATGGACACCAGCGGACG CCCCACGGCGAACAGCGGCGGCAAGATGATCCGGGCGGCACTGACCATCCTTGCCGCCGAGGCC TGCGGCGGCGACGCCCAGCAGGCCGTACCCGGCGCCGCCGCCGTCGAACTGGTCCACAACTTCT CGCTGTTGCACGACGACGTCATGGACCGCGATCTGGAGCGGCGCGGCCGGCCCACGGTATGGAG CAAGTTCGGCACCCCCGCGGCGATCCTGGCGGGCGACATCCTGCTGGCGCGCGCCTGCGGCATG TTCGACGAGGCCTCCGGCCACCAGGGCTGGGCGACCAAGGCCCTGATCGACGCGATCGCCGAGC TGGCCGCGGGCCAGATGGCCGACCTCGCGCTGGAGCGCCGCGCCACGGTGACCCTGGAAGAGGC CCTCACCGTCTCCGAGCAGAAGACCGCGGCGCTGCTGCGCTGCGCCTGCACGCTGGGCGCGGGA CTCGTCGGCGCACCCGACGGGACCAGCCGCCGCTTCGGCGCCTTCGGTATGCACCTGGGCATGG CGTTCCAGCTGGTCGACGACGTACTCGGCATATGGGGCGACCCGGCCGTCACCGGCAAGCCGGT CCGCTCCGACCTGCACAACAAGAAGAAGAGCATTCCCGTCGTCGCCGCCCTCCACAGCGGCCGG CCCGGCTCCGCGGAACTGGCCGCGCTCTACGCGGACACCGACCCCATGACGGAGGACGGCGCCC GGCGCGCCGCCGAGCTGGTCGAGCTGGCCGGCGGCCGCGCCTGGACCGAGAGCGAGATCGAGCG GCACCGCGGCCTCGCCGTGGCGCAGCTCGACGCCCTCGGGCTGACGGAGGCGCAGCGGGCACCC CTGCTCGCCCTCGCCGACTACGTCGCCTTCAGGAAGCACTGAtGTCAGTTTCACCTGTTTTACG TAAAAACCCGCTTCGGCGGGTTTTTACTTTTGGtactCGCGTAGGCTAAAGATGACATCTTTAA CATAGGATGTGCGCAAAAAAAAGGGAGAGGAGCGTATTTaATGACCACCCGCCCCACGGCCGCC TCGCGCGGCAACAAGTACCGCTTCGCCTTCCGCACCCTCTCCTTCCTGGACGCCCACAAGAACG ACCGCTCCGGTGTGGCCGAGCTCAGCGGGCCGCCCGGCCGGGCGCTCCTGGTGTGGAAGCCGGA GATCATCAGCCAGGTCTTCCGCGGCGACCGGAACATGACGCTGGAGGGCTCCGACACGCTCGGC CCGCTGGTCGGCGACACCTCGCTGCTGTTCGCCAACGGGCCGCGGCACGCCGCGTACCGCCAGG TGATCGGCCCGCGGCTGCGCGGCCGTCCGCTGCGCGGCTACGAGGAGCTGATCGCCGAGGCCAC CCGGGCGGCGATCGACGAACTGCGGCCGGGCACCGACTTCCAGGTGCCCGACTGGACCCGCAGG CTCACCCTTCAGATCGTCAGCCAGATCATCCTCGGCCCGGTGGACCACGGTCTGCTGCACCGCT TCACCTCCTGGATCGAGGGGGTGCTGGGCTCGCGCGGCCGCACCCTCGCCTACCGCTATCTGCG GCTGCCGCACGCCCTGCCGTCTCCCTGGCGCACCTTTGTGCGGCAGCGCGAGAGCCTGGACAAG GAGCTGCTGTGCCCGGTGAGCGGCAAGAGCGCCGGCGGCGGTGCGGGTTCCGGTGCGCCGGAGC CGTCGCCGGCCACGCTCGCCGAGGTGCTGCGCAGCGGTGAGGAGCCGCTCGGCCCGCTGGGCGA CGGCGAACTGCGTGACCAGATCGTCTCGTTGCTGTTCGCGGGCCACGAGACGACCGCCTCGGCG ATCTCCTGGGCGCTGTTCTGGCTCGCGGAGCACGACGAGGTGCGCCGCGACATCATCGACGAGC TGAAGGCCACCTCCTCCAGCGGTGCGGCGGCGGAGGACGTACCGCTGCTGGACGCGGCCTGCCG GGAGGTGCTGCGGATCTCGCCGCCCGCCGTGGTGGCCGGCAACCGTGTGCTCAACGAGGGCCAG GAGATCGACGGGGTACCGCACGACGCGGGCACCCGGCTGACGCCGTGCATCTACCTGGCGCACC AGCAGCCGGATCTCTACCCGCAGCCGGAGCGCTTCGACCCGCACCGCTTCCTGGGCAAGCGCAA GTCCGCCCAGGAGTACCTGCCGTTCGGCGGCGGCACCCGCCGCTGTCTCGGTGCCGACCTCGCG ATGCTGGAGATGCGGATGGTGGTGGCGGCGGTGCTGCGCCGGCGAGAACTGAAGTGCGTCAATC CGGAGACCGGGGTACCGCAGCTGCGCGGTCCGGCGATGGGTCCGAGCGAAGACCTGAGAATGAC GGTGACCGAGTGTCCAGCATGAtCTCGGTACCAAATTCCAGAAAAGAGACGCTGAAAAGCGTCT TTTTTCGTTTTGGTCCaagtTCAAATGGCTAGGATATAACCTACACGATTAGGATAGCCCCGCA TCAATCAGAGAAGGAGGTACCAAaaTGTGGGAGCCGACGTCTGCGGGGTGGGGTGCGCGGGTCG AGGGGTGGGATCTGGGGGCGCCGTTGGGTGCGGAGTGTGCGGCGGCGTTGGTGGAGTTGTTCCG TGCGCGGCATCTGCTGGTGTTTTCGGGTCAGGGTTTCTCGTTGGAGGAGCAGATCCGGTTCATG GGGCATTTGGGTCCGGTGCTGCATGAGGAGGGTTCGGGGATCGGGTTTGTTTCGAATGTGAAGG AGGGGGCTGCTCTGGGGGTGAGTGAGCTGTCTTTCCATTCGGATACGGGGCATTGTGCGGTGCC GTTGGAGGCGGTGTCGCTTTTTGCTGAGGATGTTGAGGGGTGTGTGACGTCGACGCGGTTTGCG AATGTGGCGGCGGCGTATGGGCGTTTGCCGGCGGGTTTGCGGTCGCGGGTTGCGGGACTGGTGT GTGAGAACGCGATGCCGGTGTCGTTGGACGGCCGGAATGTGGGGTTGTCGGTGGCGGAGGGGAT GCCGCGGGCGGAGCATCCGGTGGTGTGGCGTCATCCGGTGTCGGGGGAGCCGGGGCTGATGGTG AATGCGAATCAGACGACGCGGATCGTGGGCCTTGAGGGTGAAGAGAGCCGGGGGTTGCTGGAGG AGTTGTTCTCGGTGATGTATGCCGAGGATGCGGTGTATGAGCATTCCTGGCAGCAGGGGGATGT GGTGATCTGGCACAACCTGGCGGTTCAGCATGCGCGGGGTGGCCTGGAAGGTAATGGCCGGCGG ACTCTGCGCCGGGTGGCGTTGGGTGAGAAGGGGTTTTGGGAGCAGTGCCCGACCCTGCGTTACG CCGATTTCAAAAACCAGCAGAACACCGCCGCGTgatAAAAAAAAAAAACACCCTAACGGGTGTT TTTTTTTTTTTGGTCTCCCgcttCCCGTCGGCTGGAAACACTTGATCCAACTTAGGATACTTCC TAATTCATTTTACTACAATTACGCGTTTACGTCAGaATGACGTACGTAATCGCGCAGCCCTGTG TCGACCTCAAGGACAAGGCGTGCATCGAGGAGTGCCCGGTCGACTGCATCTACGAGGGCAAGCG GTCCTTGTACATCCACCCGGACGAATGCGTCGACTGCGGTGCCTGTGAGCCGGTCTGCCCGGTC GAGGCCATCTTCTACGAGGACGACACACCGGAGGAGTGGAAGGACTACTACAAGGCGAATGTGG AGTTCTTCGACGAGCTCGGCTCGCCGGGCGGCGCCGCCCGGCTCGGCCTGATCGACCGCGACCA CCCCGTCGTCGCGGCGCAGCCGGTCCGTACGGCCGGAGCCGCGGGCGAAtgatCTCGGTACCAA AAAAAAAAAAAAAGACGCTGAAAAGCGTCTTTTTTTTTTTTGGTCCcgct