RECOMBINANT HOST CELLS AND METHODS FOR THE PRODUCTION OF GLYCERIC ACID AND DOWNSTREAM PRODUCTS

Abstract

Methods and materials related to producing glyceric acid and downstream products are disclosed. Specifically, isolated nucleic acids. polypeptides, host cells, methods and materials for producing glycolic acid by direct fermentation from sugars are disclosed.

Claims

1. A recombinant host cell, comprising: a glyceric acid biosynthetic pathway, comprising heterologous nucleic acids encoding a 3-phosphoglycerate phosphatase and a 2-phosphoglycerate phosphatase; wherein the expression of the heterologous nucleic acids produces glyceric acid.

2. The recombinant host cell of claim 1, wherein the recombinant host cell is a yeast cell.

3. The recombinant host cell of claim 2, wherein the yeast cell is of the Issatchenkia orientalis/Pichia fermentans clade.

4. The recombinant host cell of claim 3, wherein the yeast cell belongs to the genus Pichia, Issatchenkia, or Candida.

5. The recombinant host cell of claim 4, wherein the yeast cell is Pichia kudriavzevii.

6. The recombinant host cell of claim 2, wherein the yeast cell of the Saccharomyces clade.

7. The recombinant host cell of claim 6, wherein the yeast cell is Saccharomyces cerevisiae.

8. The recombinant host cell of claim 1, wherein the recombinant host cell is a prokaryotic cell.

9. The recombinant host cell of claim 8, wherein the prokaryotic cell belongs to the genus Escherichia, Corynebacterium, Bacillus, or Lactococcus.

10. The recombinant host cell of claim 9, wherein the prokaryotic cell is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, or Lactococcus lactis.

11. The recombinant host cell of any one of claims 1-10, wherein the 3-phosphoglycerate phosphatase has an amino acid sequence selected from SEQ ID NO: 9 and an amino acid sequence having at least 90% identity with SEQ ID NO: 9.

12. The recombinant host cell of any one of claims 1-10, wherein the 3-phosphoglycerate phosphatase has an amino acid sequence selected from SEQ ID NO: 7 and an amino acid sequence having at least 90% amino acid identity with SEQ ID NO: 7.

13. The recombinant host cell of any one of claims 1-12, wherein the 2-phosphoglycerate phosphatase has an amino acid sequence selected from SEQ ID NO: 1 and an amino acid sequence having at least 90% amino acid identity with SEQ ID NO: 1.

14. The recombinant host cell of any one of claims 1-12, wherein the 2-phosphoglycerate phosphatase has an amino acid sequence selected from SEQ ID NO: 7 and an amino acid sequence having at least 90% amino acid identity with SEQ ID NO: 7.

15. The recombinant host cell of any one of claims 1-14, further comprising: a heterologous nucleic acid encoding a mitochondrial external NADH dehydrogenase; a nucleic acid encoding a water-forming NADH oxidase; a heterologous nucleic acid encoding a glyceric acid transporter; or combinations of the foregoing.

16. The recombinant host cell of claim 15, wherein the mitochondrial external NADH dehydrogenase has an amino acid sequence selected from SEQ ID NO: 20 and an amino acid sequence having at least 90% identity with SEQ ID NO: 20.

17. The recombinant host cell of claim 15 or claim 16, wherein the water-forming NADH dehydrogenase has an amino acid sequence selected from SEQ ID NO: 21 and an amino acid sequence having at least 90% identity with SEQ ID NO: 21.

18. The recombinant host cell of any one of claim 15-17, wherein the glyceric acid transporter has an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, a protein having 90% sequence identity to SEQ ID NO: 22, a protein having 90% sequence identity to SEQ ID NO: 23, a protein having 90% sequence identity to SEQ ID NO: 24, and a protein having 90% sequence identity to SEQ ID NO: 27.

19. The recombinant host cell of any one of claims 1-18, further comprising: a genetic disruption of an endogenous gene encoding a protein selected from phosphoglycerate mutase, phosphoglycerate dehydrogenase, enolase, glycerate 3-kinase, glycerate 2-kinase, glycerol-3-phosphate dehydrogenase, and combinations thereof.

20. The recombinant host cell of claim 19, wherein the phosphoglycerate mutase has an amino acid sequence selected from SEQ ID NO: 14 and an amino acid sequence having at least 90% identity with SEQ ID NO: 14.

21. The recombinant host cell of claim 19, wherein the phosphoglycerate mutase has an amino acid sequence selected from SEQ ID NO: 25 and an amino acid sequence having at least 90% identity with SEQ ID NO: 25.

22. The recombinant host cell of any one of claims 19-21, wherein the enolase has an amino acid sequence selected from SEQ ID NO: 26 and an amino acid sequence having at least 90% identity with SEQ ID NO: 26.

23. The recombinant host cell of any one of claims 19-21, wherein the enolase has an amino acid sequence selected from SEQ ID NO: 28 and an amino acid sequence having at least 90% identity with SEQ ID NO: 28.

24. The recombinant host cell of any one of claims 19-21, wherein the enolase has an amino acid sequence selected from SEQ ID NO: 29 and an amino acid sequence having at least 90% identity with SEQ ID NO: 29.

25. The recombinant host cell of any one of claims 19-24, wherein the glycerate 3-kinase has an amino acid sequence selected from SEQ ID NO: 16 and an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 16.

26. The recombinant host cell of any one of claims 19-25, wherein the glycerate 2-kinase has an amino acid sequence selected from SEQ ID NO: 18 and an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18.

27. The recombinant host cell of any one of claims 19-26, wherein the recombinant host cell produces less than 5 g/l of ethanol, acetate, pyruvate, or combinations thereof.

28. The recombinant host cell of any one of claims 19-27, wherein the phosphoglycerate dehydrogenase has an amino acid sequence selected from SEQ ID NO: 15 and an amino acid sequence having at least 90% identity with SEQ ID NO: 15.

29. The recombinant host cell of any one of claims 19-27, wherein the phosphoglycerate dehydrogenase has an amino acid sequence selected from SEQ ID NO: 17 and an amino acid sequence having at least 90% identity with SEQ ID NO: 17.

30. The recombinant host cell of claim 28 or claim 29, wherein the recombinant host cell produces less than 5 g/l of serine.

31. The recombinant host cell of any one of claims 19-30, wherein the glycerol-3-phosphate dehydrogenase has an amino acid sequence selected from SEQ ID NO: 19 and an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19.

32. The recombinant host cell of claim 28 or claim 29, wherein the recombinant host cell produces less than 5 g/l of glycerol.

33. A method of producing glyceric acid, comprising: culturing the recombinant host cell of claim 1 under conditions suitable to produce glyceric acid.

34. The method of claim 33, wherein the culturing is performed at an oxygen transfer rate greater than 10 mmol/l/hr.

35. The method of claim 33 or claim 34, wherein the culturing is performed at a temperature of about 25 C.-45 C.

36. The method of any one of claims 33-35, wherein the final pH of the fermentation broth is less than about pH 5.

37. The method of any one of claims 33-36, wherein the culturing produces at least 50 g/l glyceric acid.

38. The method of any one of claims 33-36, further comprising: providing at least 100 g/l glucose to the recombinant host cell; and producing at least 25% glyceric acid yield.

39. A method for producing glycerate esters, comprising: recovering glyceric acid and glycerate salts from a cell-containing fermentation broth; reacting the glyceric acid and glycerate salts with an alcohol in the presence of sulfuric acid to produce a glycerate ester; and isolating the glycerate ester by distillation.

40. A method for producing acrylate esters, comprising: reacting a glycerate ester with a reducing agent in the presence of a transition metal catalyst to produce an acrylate ester; and isolating the acrylate ester; wherein the transition metal catalyst is selected from vanadium compounds, molybdenum compounds, and rhenium compounds.

41. The method of claim 40, wherein the glycerate ester is selected from the group consisting of methyl glycerate, ethyl glycerate, butyl glycerate, and 2-ethylhexyl glycerate.

42. The method of claim 40 or claim 41, wherein the transition metal catalyst is methyltrioxorhenium or (NH.sub.4).sub.6Mo.sub.7O.sub.24.Math.4H.sub.2O.

43. The method of any one of claims 40-42, wherein the reducing agent is selected from H.sub.2, Na.sub.2SO.sub.3, benzene, toluene, 5-nonanol, 3-octanol, 2-octanol, 1-butanol, 3-pentanol, 2-methyl-1-butanol, isopropanol, and 2-ethylhexanol.

44. The method of any one of claims 40-43, wherein the acrylate ester is butyl acrylate or 2-ethylhexyl acrylate.

45. A poly(glycerate carbonate) compound comprising repeat units of formula (I): [OCH(C(O)OR.sup.1)CH.sub.2OC(O)] or a salt thereof; wherein each R.sup.1 is independently selected from hydrogen, optionally substituted C.sub.1-C.sub.5 alkyl, and optionally substituted aryl.

46. The compound of claim 45, which comprises about 25%. about 50%, about 75%, or about 90% non-petrochemical based components.

Description

EXAMPLES

Parent Strain Used in the Examples

[0311] The parent strain in Example 1 was a P. kudriavzevii strain auxotrophic for histidine and uracil due to genetic disruptions in URA2 and HIS3 (i.e., the strain cannot grow in media without histidine and uracil supplementation). Histidine auxotrophy in the parent strain enables selection of new, engineered strains that carry a HIS3 marker, enabling histidine prototrophy and indicating desired nucleic acid modification. Likewise, uracil auxotrophy in the parent strain enables selection of new, engineered strains that carry a URA2 marker, enabling uracil prototrophy and indicating desired nucleic acid modification. Thus, cells that were successfully modified with exogenous nucleic acids to comprise desired genetic modifications can grow in media without histidine and/or uracil supplementation, dependent on the selection marker included in the exogenous nucleic acid. Following confirmation of correct strain engineering, the selection marker(s) were removed by, for example, homologous recombination and marker loopout. Removing the marker enables subsequent rounds of strain engineering using the same selection markers.

Media Used in the Examples

[0312] Complete supplement mixture (CSM) medium. CSM medium comprised Adenine 10 mg/L; L-Arginine HCl 50 mg/L; L-Aspartic Acid 80 mg/L; L-Histidine HCl 20 mg/L; L-Isoleucine 50 mg/L; L-Leucine 100 mg/L; L-Lysine HCl 50 mg/L; L-Methionine 20 mg/L; L-Phenylalanine 50 mg/L; L-Threonine 100 mg/L; L-Tryptophan 50 mg/L; L-Tyrosine 50 mg/L; Uracil 20 mg/L; L-Valine 140 mg/L. The YNB used in the CSM comprised Ammonium sulfate 5.0 g/L, Biotin 2.0 g/L, Calcium pantothenate 400 g/L, Folic acid 2.0 g/L, Inositol 2.0 mg/L, Nicotinic acid 0-400 g/L, p-Aminobenzoic acid 200 g/L, Pyridoxine HC1 400 g/L, Riboflavin 200 g/L, Thiamine HCl 400 g/L, Boric acid 500 g/L, Copper sulfate 40 g/L, Potassium iodide 100 g/L, Ferric chloride 200 g/L, Manganese sulfate 400 g/L, Sodium molybdate 200 g/L, Zinc sulfate 400g/L, Potassium phosphate monobasic 1.0 g/L, Magnesium sulfate 0.5 g/L, Sodium chloride 0.1 g/L, and Calcium chloride 0.1 g/L.

[0313] Complete supplement mixture minus histidine (CSM-His) medium. CSM-His medium is identical to CSM medium with the exception that histidine was not included in the medium. Engineered strains auxotrophic for histidine are unable to grow on CSM-His medium while engineered strains containing exogenous nucleic acids comprising a histidine selectable marker (for example, HIS3) are capable of growth in CSM-His medium.

[0314] Complete supplement mixture minus uracil (CSM-Ura) medium. CSM-Ura medium is identical to CSM medium with the exception that uracil was not included in the medium. Engineered strains auxotrophic for uracil are unable to grow on CSM-Ura medium while engineered strains containing exogenous nucleic acids comprising a uracil selectable marker (for example, URA2) are capable of growth in CSM-Ura medium.

[0315] BM02 medium. BM02 medium is Glucose 125 g/l, K.sub.2SO.sub.4 0.816 g/l, Na.sub.2SO.sub.4 0.1236, MgSO.sub.4-7H.sub.2O 0.304 g/l, Urea 4.3 g/l, Myo-inositol 2 mg/l, Thiamin HC1 0.4 mg/l, Pyridoxal HCl 0.4 mg/l, Niacin 0.4 mg/l, Ca-Pantothenate 0.4 mg/l, Biotin g/1, Folic acid 2 g/1, PABA 200 g/l, Riboflavin 200 g/l, Boric acid 0.25 mg/l, Copper sulfate pentahydrate 393 g/1, Iron sulfate 11.0 mg/l, Manganese chloride 1.6 mg/l, Sodium molybdate 100 g/1, Zinc sulfate 4 mg/l, and EDTA 11 mg/l.

[0316] BM02-P medium. BM02-P medium is BM02 medium with 1 g/l potassium phosphate.

[0317] YPE medium. YPE medium is Bacto peptone 20 g/l, Yeast extract 10g/l, and Ethanol 2% (v/v).

Example 1

Construction of the Recombinant P. kudriavzevii Strain LPK151290 With Eliminated Expression of Phosphoglycerate Mutase

[0318] This example describes the construction of the P. kudriavzevii strain LPK151290 that comprises genetic disruption in both native copies of phosphoglycerate mutase, resulting in eliminated expression of phosphoglycerate mutase. The endogenous Gpm1 gene that encodes for phosphoglycerate mutase in P. kudriavzevii (PkGPM1; SEQ ID NO: 14) was disrupted to prevent the conversion of 3-PG to 2-PG in the glycolytic pathway. The deliberate, consequential buildup of 3-PG provided ample 3-PG substrate for the glyceric acid pathway, which was constructed as described in Example 3 (see below).

[0319] The parent P. kudriavzevii strain used in this example was LPK15775-it was diploid and had two copies of Gpm1, providing a GPM plus phenotype (i.e., native phenotype with respect to Gpm1 expression). The parent LPK15775 strain was also auxotrophic for uracil and histidine. Gpm1 in LPK15775 was disrupted by deletion of both gene copies to produce the GPM minus strain LPK151290.

[0320] Both copies of Gpm1 in LPK15775 were disrupted by insertion of a HIS3 selectable marker via homologous recombination. The HIS3 selectable marker, amplified by PCR, was provided to the parent LPK15775 strain to complement the histidine auxotrophic deficiency. The HIS3 selectable marker comprised unique upstream and downstream homologous regions for homologous recombination at the P. kudriavzevii Gpm1 locus, a transcriptional promoter, a HIS3 coding region, and a transcriptional terminator. The transcriptional promoter 5 of HIS3 was the P. kudriavzevii TEF1 promoter (pPKTEF1) and the transcriptional terminator 3 of HIS3 was the S. cerevisiae TDH3 terminator (tScTDH3).

[0321] The PCR product of the HIS3 selectable marker was purified and provided as exogenous nucleic acids to P. kudriavzevii. Transformation was carried out in a single step and gene deletion was achieved by homologous recombination. Transformants were selected on CSM-His medium and successful deletion of both copies of the gene encoding PkGPM1 was confirmed by genetic sequencing of this locus and the flanking regions.

[0322] This example produced the GPM minus, LPK151290 strain that cannot convert 3-PG to 2-PG via PkGPM1. Thus, all engineered P. kudriavzevii strains derived from this parental strain had the GPM minus phenotype, resulting in disrupted carbon flux through glycolysis downstream of 3-PG.

Example 2

Recombinant P. kudriavzevii Strain LPK151290 Produces Low Amounts of Glyceric Acid

[0323] This example describes the culturing and analysis of the GPM minus strain, LPK151290 (from Example 1), for basal level glyceric acid production before glyceric acid pathway strains were constructed (see Example 3 below). Recall that the parent strain of LPK151290 is LPK15775 (see Example 1). The parent LPK15775 strain expressed native levels of PkGPM1 (i.e., GPM plus) and was also cultured and analyzed for comparison.

[0324] In this example, LPK151290 (GPM minus) and LPK15775 (GPM plus) colonies were used to inoculate replicate tubes of 2 mL to 4 mL of YPE medium with 1% glycerol and incubated at 30 C. with shaking at 250 rpm for 20 hours. These replicate tubes of pre-cultures were each pelleted and resuspended in 0.5 mL of BM02-P media with 5% glucose, 1% ethanol and 50 g/mL uracil, and placed into 96-well plates. The 96-well plate cultures were then incubated at 30 C. with 80% humidity and shaking at 250 rpm. After 48 hours, 1 volume of 12 M HCl was added to 10 volume of culture for each culture sample. The HCl-culture sample mixtures were spin-filtered and frozen for storage. Samples were analyzed by HPLC within 48 hours of harvest.

[0325] For HPLC analysis, frozen samples were thawed analyzed by HPLC using a Bio-Rad Aminex 87H column (3007.8 mm) and a Bio-Rad Fermentation Monitoring column (#1250115; 1507.8 mm) installed in series, with an isocratic elution rate of 0.8 ml/min with water at pH 1.95 (with sulfuric acid) at 30 C. Refractive index and UV 210 nm measurements were acquired for 35 minutes.

[0326] While the LPK15775 GPM plus strain did not produce detectable amounts of glyceric acid, the LPK151290 GPM minus strain produced a yield of 6% (i.e., percentage of the amount of glyceric acid produced per amount of glucose consumed). This result demonstrated that disruption of GPM activity alone was sufficient to produce detectable, albeit low amounts of glyceric acid in a P. kudriavzevii strain background, and more generally in microbes. Thus, all engineered P. kudriavzevii strains without heterologous nucleic acids that encode the glyceric acid pathway of the present disclosure can produce appreciable amounts of glyceric acid. Incorporation of heterologous nucleic acids that encode the glyceric acid pathway should increase glyceric acid yields (Example 4).

Example 3

Analysis of Strains LPK15775 (GPM Plus) and LPK151290 (GPM Minus) for the Formation of Byproducts Pyruvate, Ethanol, and Acetate

[0327] Example 3 describes the culturing and analysis of LPK15775 (GPM plus; parent P. kudriavzevii strain) and LPK151290 (GPM minus; from Example 1) recombinant host cells for in vivo production of byproducts pyruvate, ethanol and acetate. Native P. kudriavzevii cells are capable of producing downstream metabolites pyruvate, ethanol and acetate. The presence of these metabolites indicates that 3-PG is siphoned from the glyceric acid pathway of the present disclosure, thereby causing decreased fermentation yield of glyceric acid and/or downstream product.

[0328] In this example, LPK15775 and LPK151290 are cultured and analyzed by HPLC as described above in Example 2. While the GPM plus LPK15775 produce low amounts of pyruvate, ethanol and acetate, the GPM minus LPK151290 produce relatively lower amounts of pyruvate, ethanol and acetate. This example demonstrates, in accordance with the present disclosure, the successful decrease of byproducts ethanol and acetate from fermentation by genetic disruption of PkGPM in recombinant P. kudriavzevii for glyceric acid and/or downstream product synthesis.

Example 4

Construction of Recombinant P. kudriavzevii Strains Expressing Glyceric Acid Pathway Enzymes With Decreased or Eliminated Expression of Phosphoglycerate Mutase

[0329] Example 4 describes the construction of recombinant host cells of the present disclosure that each comprise heterologous nucleic acids encoding a glyceric acid pathway of the present disclosure. The parent P. kudriavzevii strain LPK15775 used in this example was auxotrophic for uracil and histidine. Heterologous nucleic acids encoding 3-PG phosphatases of the glyceric acid pathway were integrated into the PkGPM1 locus. Thus, each strain described in this example not only comprised a glyceric acid pathway, but also comprised a GPM minus phenotype due to eliminated expression of phosphoglycerate mutase.

[0330] The heterologous nucleic acids used in this example encoded 3-PG phosphatases of the glyceric acid pathway that were derived from EcGPH; SEQ ID NO: 1, HsGPH; SEQ ID NO: 3, ScPHO13; SEQ ID NO: 9, PKPHO13; SEQ ID NO: 10, PKORF64; SEQ ID NO: 11, PKORF423; SEQ ID NO: 12, or ScYKR070W; SEQ ID NO: 13. Heterologous nucleic acids encoding HsGPH were codon-optimized for yeast and were synthesized and provided by Twist Bioscience. Heterologous nucleic acids encoding ScPHO13 and ScYKR070W were amplified form Saccharomyces cerevisiae genomic DNA. Heterologous nucleic acids encoding PKPHO13 and PKORF64, and PKORF423 were amplified from Pichia kudriavzevii genomic DNA. Heterologous nucleic acids encoding EcGPH was amplified form Escherichia coli genomic DNA. The transcriptional promoters cloned in front (5) of each gene were constitutive and derived from P. kudriavzevii. The promoters for EcGPH, HsGPH, ScPHO13, PKPHO13, PKORF64, PKORF423, and ScYKR070W were the P. kudriavzevii TDHI promoter (pPKTDH1). The transcriptional terminators cloned behind (3) of each gene were derived from S. cerevisiae. The terminators for EcGPH, HsGPH, ScPHO13, PKPHO13, PKORF64, PKORF423, and ScYKR070W were the Saccharomyces cerevisiae PYC2 terminator (tScPYC2). Additionally, a HIS3 marker was included in the heterologous expression cassette to complement the histidine auxotrophic deficiency in the background strain. This HIS3 marker comprised a transcriptional promoter, a HIS3 coding region, and a transcriptional terminator. The transcriptional promoter 5 of HIS3 was the P. kudriavzevii TEFI promoter (pPKTEF1) and the transcriptional terminator 3 of HIS3 was the S. cerevisiae TDH3 terminator (tScTDH3). All genetic elements were amplified from various templates with upstream and downstream homologous regions to neighboring genetic elements to drive correct assembly of the full-length pathway. The 5 and 3 ends of the expression cassette comprised regions homologous to the genomic sequences upstream and downstream of the P. kudriavzevii GPM1 locus, thereby facilitating integration of the heterologous nucleic acids encoding the glyceric acid pathway enzymes at the GPM1 locus in the P. kudriavzevii genome. Consequently, one or both copies of the PkGPM1 gene were deleted from the host genome; thus, genomic integration of the glyceric acid pathway simultaneously decreased or eliminated expression of PkGPM1.

[0331] All PCR products were purified and provided as exogenous nucleic acids to P. kudriavzevii. Transformation was carried out in a single step. Transformants were selected on CSM-His medium. Successful integration of all nucleic acids encoding glyceric acid pathway enzymes were confirmed by genetic sequencing of this locus and the flanking regions.

[0332] This example produced GPM minus strains which also comprised a glyceric acid pathway of the present disclosure with the following strain designations: LPK151910 (with EcGPH; SEQ ID NO: 1), LPK151909 (with HsGPH; SEQ ID NO: 3), LPK151954 (with ScPHO13; SEQ ID NO: 9), LPK152365 (with PkPHO13; SEQ ID NO: 10), LPK152367 (with PKORF64; SEQ ID NO: 11), LPK152366 (with PKORF423; SEQ ID NO:12), and LPK151912 (with ScYKR070W; SEQ ID NO: 13).

Example 5

Recombinant P. kudriavzevii Strains LPK151910, LPK151909, LPK151954, LPK152365, LPK152367, LPK152366, and LPK151912 Produced Increased Amounts of Glyceric Acid

[0333] Example 5 describes the culturing and analysis of recombinant host cells LPK151910, LPK151909, LPK151954, LPK152365, LPK152367, LPK152366, and LPK151912 from Example 4 (GPM minus, with genomic insertion of a glyceric acid pathway). All seven recombinant strains were cultured and analyzed by HPLC according to methods described above in Example 2.

[0334] In this example, all recombinant strains with a glyceric acid pathway produced a glyceric acid yield of 10-20% as compared to the lower, 6% product yield achieved when culturing the sister LPK151290 GPM minus strain that lacked a heterologous glyceric acid pathway. This example demonstrates, in accordance with the present disclosure, the expression of heterologous nucleic acids encoding a glyceric acid pathway in recombinant P. kudriavzevii increased glyceric acid yields as compared to a host cell lacking the heterologous glyceric acid pathway but otherwise genetically identical.

[0335] It should be noted that there are alternative ways of implementing the embodiments disclosed herein. Accordingly, the present embodiments are to be considered as illustrative and not restrictive; various modifications can be made without departing from the spirit of this disclosure. Furthermore, the claims are not to be limited to the details given herein, and are entitled their full scope and equivalents thereof.