Is Proterro the only company making sucrose photosynthetically from cyanobacteria?
Proterro's website states Proterro is the only company that makes sucrose instead of extracting it, lowering the cost of sugar for the economical and scalable production of biofuels and biobased chemicals.
Biofuels Digest wrote on 18 Dec. 2012: Where other companies are growing sugars via crop improvements, or extracting them, Proterro is making them via synthetic biology. Specifically, they have engineered a cyanobacteria to make sucrose (via a series of steps you can read all about here), ultimately from CO2, water, sunlight and nutrients – and secrete it into a sugarwater stream that can be purified into affordable sucrose.
However, Joule's US patent no. 8,227,237, issued July 24, 2012, months before the Biofuels Digest piece, has a first claim:
A method for the biogenic synthesis of sucrose for biofuel production, comprising:
culturing an engineered photosynthetic microbe in a culture medium, wherein said photosynthetic microbe comprises a glucose facilitated diffusion transporter, wherein said glucose facilitated diffusion transporter has an amino acid sequence identical to the sequence encoded by nucleotides 2680-4105 of SEQ ID NO: 11, and wherein one or more glycogen synthesis genes in said photosynthetic microbe are attenuated or absent by mutation; and exposing said engineered photosynthetic microbe to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said engineered photosynthetic microbe into sucrose, wherein the amount of sucrose exported by said microbe is at least two-fold greater than the amount of said sucrose exported by an otherwise identical photosynthetic microbe, cultured under identical conditions, but lacking said recombinant glucose facilitated diffusion transporter and lacking said mutation in said one or more glycogen synthesis genes which attenuates or renders absent the expression of said one or more glycogen synthesis genes.
Within the specification of US '237:
Example 57
Engineered Microorganisms Producing Glucose
pJB336 was constructed in the following manner. A synthetic DNA kan cassette (DNA2.0) was subcloned via flanking Pad and AscI restriction sites into vector pJB303. This kan cassette comprises a promoter, active in both E. coli and JCC1, driving expression of gene aph that confers resistance to kanamycin in both organisms. pJB303 contains a synthetic DNA region (DNA2.0) comprising an upstream homology region (UHR) and a downstream homology region (DHR) flanking a multiple cloning region that includes Pad and AscI sites. The UHR corresponds to coordinates 1615342 to 1615841, the DHR to coordinates 1617346 to 1617845, of the JCC1 genome (Genbank Accession NC.sub.--010475), respectively. The UHR and DHR mediate homologous recombinational integration of heterologous DNA flanked by these regions into the JCC1 chromosome. In the case of SfiI-linearized pJB336, recombination occurs in such a way that the JCC1 glgA1 gene encoding glycogen synthase 1 (SYNPCC7002_A1532; Genbank Accession YP.sub.--001734779) is deleted, and replaced by a kan cassette. The SfiI-flanked DNA sequence contained within pJB336 is shown as SEQ ID NO: 8. (...)
To fully segregate recombinants, a small population (10-20) of kanamycin-resistant colonies from this initial plate was streaked onto an A+50 .mu.g ml.sup.-1 kanamycin plate, and grown as above; a small population (10-20) of kanamycin-resistant colonies from this second plate was then streaked onto an A+75 .mu.g mY.sup.1 kanamycin plate, and grown as above. Genomic DNA was prepared from a single candidate JCC475 colony from this third plate and checked for complete segregation by PCR. For pJB336 and all other constructs, this involved checking for the presence of (i) the upstream recombinant junction, (ii) the downstream recombinant junction, and (iii) the heterologous gene(s), and the absence of the deleted wild-type gene (glgA1 or glgA2). Transformation of pJB342, pJB345, and pJB348 into JCC475 was carried out as described above except that kanamycin was included in all plates to maintain selection for the glgA1::kan disruption in JCC475, and spectinomycin was used as the selective antibiotic--first at 25 .mu.ml.sup.-1, then at 50 .mu.ml.sup.-1, and finally at 75 .mu.ml.sup.-1. (...)
Although GC-MS analysis indicated only basal levels of glucose were present in the growth medium of JCC547, it was apparent that there were several other ion chromatogram peaks only present, or present with larger areas, in this strain and not in JCC342c, JCC543, or JCC545. One of these peaks was positively identified as sucrose based on authentic standard analysis, as shown in Table 19 and FIG. 17. Based on the concentration of sucrose used in the authentic standard analysis, and assuming that the TIC peak area observed scales linearly with this known sucrose concentration, it was estimated that the JCC547 culture medium contained approximately 600 mg liter.sup.-1 sucrose, approximately 100 times that seen in JCC342c, JCC543, or JCC545. No maltose was observed in any of the four strains' culture media.
TABLE-US-00019 TABLE 19 Sucrose produced in the culture media of JCC342c, JCC547, JCC543, and JCC545, as determined by extracted ion chromatogram peak areas for the m/z 361 diagnostic disaccharide ion seen by GC-MS analysis Extracted ion chromatogram (EIC) peak area Strain Genotype for sucrose (m/z = 361 ion) JCC342c .DELTA.glgA1::kan .DELTA.glgA2::spec 63632 JCC547 .DELTA.glgA1::kan .DELTA.glgA2::lacI-P.sub.trc- 8892575 yihX-glf-spec JCC543 .DELTA.glgA1::kan .DELTA.glgA2::lacI-P.sub.trc- 50666 yihX-GLUT1-spec JCC545 .DELTA.glgA1::kan .DELTA.glgA2::lacI-P.sub.trc- 53940 yihX-GLUT1-spec
The P.sub.trc-yihX-glf cassette in JCC547 therefore resulted in significantly higher sucrose production than observed in an isogenic control strain. A possible reason for this is that the Glf transporter is able to mediate the export of sucrose that is naturally synthesized, and otherwise maintained, within the cell. There are no reports of Glf being able to mediate transport of disaccharides such as sucrose. Glf has been reported as being able to mediate transport of glucose and, to a much lesser degree, fructose. However, the notion of Glf-mediated disaccharide export was supported by the GC-MS analysis of the culture medium of JCC547. As mentioned above, GC-MS indicated several ion chromatogram peaks that were only present, or present with larger areas, in this strain and not in JCC342c, JCC543, or JCC545. Consistent with these peaks representing disaccharides, many of these peaks were characterized by a dominant m/z 361 ion, which is diagnostic of TMS-derivatized disaccharides [Molnar-Perl et al., Chem. Mater. Sci., 45:321-327 (1997)], as shown in Table 20. Because none of the authentic disaccharide standards that were used eluted at the times indicated in the table above, none of these corresponding peaks could be identified with certainty. However, given the presence of the m/z 361 ion in all, it is highly likely that these peaks represent disaccharide or disaccharide-like molecules, most likely synthesized naturally within the cell.
The issued Joule patent derived from application 13/174,580, file June 30, 2011. The cross-reference section of the issued patent notes:
This application is a continuation application of U.S. application Ser. No. 12/867,738, filed Aug. 13, 2010, now U.S. Pat. No. 7,981,647, issued Jul. 19, 2011, which is a national phase entry of PCT/US2009/035937, filed on Mar. 3, 2009, which claims the benefit of U.S. Provisional Application No. 61/033,411 filed Mar. 3, 2008; U.S. Provisional Application No. 61/033,402, filed Mar. 3, 2008; U.S. Provisional Application No. 61/044,419 filed Apr. 11, 2008; U.S. Provisional Application No. 61/056,999 filed May 29, 2008; U.S. Provisional Application No. 61/058,182 filed Jun. 2, 2008; U.S. Provisional Application No. 61/077,698 filed Jul. 2, 2008; U.S. Provisional Application No. 61/079,692 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,699 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,665 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,656 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,688 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,687 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,676 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,673 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,667 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,707 filed Jul. 10, 2008; U.S. Provisional Application No. 61/086,283 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,288 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,291 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,296 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,300 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,407 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,410 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,412 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,414 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,417 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,418 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,285 filed Aug. 5, 2008; U.S. Provisional Application No. 61/100,656 filed Sep. 26, 2008; U.S. Provisional Application No. 61/100,660 filed Sep. 26, 2008; U.S. Provisional Application No. 61/100,663 filed Sep. 26, 2008; U.S. Provisional Application No. 61/100,665, filed Sep. 26, 2008, U.S. Provisional Application No. 61/100,667 filed Sep. 26, 2008; U.S. Provisional Application No. 61/106,543 filed Oct. 17, 2008; and U.S. Provisional Application No. 61/121,532 filed Dec. 10, 2008, all of which are herein incorporated by reference in their entirety and for all purposes.
Biofuels Digest wrote on 18 Dec. 2012: Where other companies are growing sugars via crop improvements, or extracting them, Proterro is making them via synthetic biology. Specifically, they have engineered a cyanobacteria to make sucrose (via a series of steps you can read all about here), ultimately from CO2, water, sunlight and nutrients – and secrete it into a sugarwater stream that can be purified into affordable sucrose.
However, Joule's US patent no. 8,227,237, issued July 24, 2012, months before the Biofuels Digest piece, has a first claim:
A method for the biogenic synthesis of sucrose for biofuel production, comprising:
culturing an engineered photosynthetic microbe in a culture medium, wherein said photosynthetic microbe comprises a glucose facilitated diffusion transporter, wherein said glucose facilitated diffusion transporter has an amino acid sequence identical to the sequence encoded by nucleotides 2680-4105 of SEQ ID NO: 11, and wherein one or more glycogen synthesis genes in said photosynthetic microbe are attenuated or absent by mutation; and exposing said engineered photosynthetic microbe to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said engineered photosynthetic microbe into sucrose, wherein the amount of sucrose exported by said microbe is at least two-fold greater than the amount of said sucrose exported by an otherwise identical photosynthetic microbe, cultured under identical conditions, but lacking said recombinant glucose facilitated diffusion transporter and lacking said mutation in said one or more glycogen synthesis genes which attenuates or renders absent the expression of said one or more glycogen synthesis genes.
Within the specification of US '237:
Example 57
Engineered Microorganisms Producing Glucose
pJB336 was constructed in the following manner. A synthetic DNA kan cassette (DNA2.0) was subcloned via flanking Pad and AscI restriction sites into vector pJB303. This kan cassette comprises a promoter, active in both E. coli and JCC1, driving expression of gene aph that confers resistance to kanamycin in both organisms. pJB303 contains a synthetic DNA region (DNA2.0) comprising an upstream homology region (UHR) and a downstream homology region (DHR) flanking a multiple cloning region that includes Pad and AscI sites. The UHR corresponds to coordinates 1615342 to 1615841, the DHR to coordinates 1617346 to 1617845, of the JCC1 genome (Genbank Accession NC.sub.--010475), respectively. The UHR and DHR mediate homologous recombinational integration of heterologous DNA flanked by these regions into the JCC1 chromosome. In the case of SfiI-linearized pJB336, recombination occurs in such a way that the JCC1 glgA1 gene encoding glycogen synthase 1 (SYNPCC7002_A1532; Genbank Accession YP.sub.--001734779) is deleted, and replaced by a kan cassette. The SfiI-flanked DNA sequence contained within pJB336 is shown as SEQ ID NO: 8. (...)
To fully segregate recombinants, a small population (10-20) of kanamycin-resistant colonies from this initial plate was streaked onto an A+50 .mu.g ml.sup.-1 kanamycin plate, and grown as above; a small population (10-20) of kanamycin-resistant colonies from this second plate was then streaked onto an A+75 .mu.g mY.sup.1 kanamycin plate, and grown as above. Genomic DNA was prepared from a single candidate JCC475 colony from this third plate and checked for complete segregation by PCR. For pJB336 and all other constructs, this involved checking for the presence of (i) the upstream recombinant junction, (ii) the downstream recombinant junction, and (iii) the heterologous gene(s), and the absence of the deleted wild-type gene (glgA1 or glgA2). Transformation of pJB342, pJB345, and pJB348 into JCC475 was carried out as described above except that kanamycin was included in all plates to maintain selection for the glgA1::kan disruption in JCC475, and spectinomycin was used as the selective antibiotic--first at 25 .mu.ml.sup.-1, then at 50 .mu.ml.sup.-1, and finally at 75 .mu.ml.sup.-1. (...)
Although GC-MS analysis indicated only basal levels of glucose were present in the growth medium of JCC547, it was apparent that there were several other ion chromatogram peaks only present, or present with larger areas, in this strain and not in JCC342c, JCC543, or JCC545. One of these peaks was positively identified as sucrose based on authentic standard analysis, as shown in Table 19 and FIG. 17. Based on the concentration of sucrose used in the authentic standard analysis, and assuming that the TIC peak area observed scales linearly with this known sucrose concentration, it was estimated that the JCC547 culture medium contained approximately 600 mg liter.sup.-1 sucrose, approximately 100 times that seen in JCC342c, JCC543, or JCC545. No maltose was observed in any of the four strains' culture media.
TABLE-US-00019 TABLE 19 Sucrose produced in the culture media of JCC342c, JCC547, JCC543, and JCC545, as determined by extracted ion chromatogram peak areas for the m/z 361 diagnostic disaccharide ion seen by GC-MS analysis Extracted ion chromatogram (EIC) peak area Strain Genotype for sucrose (m/z = 361 ion) JCC342c .DELTA.glgA1::kan .DELTA.glgA2::spec 63632 JCC547 .DELTA.glgA1::kan .DELTA.glgA2::lacI-P.sub.trc- 8892575 yihX-glf-spec JCC543 .DELTA.glgA1::kan .DELTA.glgA2::lacI-P.sub.trc- 50666 yihX-GLUT1-spec JCC545 .DELTA.glgA1::kan .DELTA.glgA2::lacI-P.sub.trc- 53940 yihX-GLUT1-spec
The P.sub.trc-yihX-glf cassette in JCC547 therefore resulted in significantly higher sucrose production than observed in an isogenic control strain. A possible reason for this is that the Glf transporter is able to mediate the export of sucrose that is naturally synthesized, and otherwise maintained, within the cell. There are no reports of Glf being able to mediate transport of disaccharides such as sucrose. Glf has been reported as being able to mediate transport of glucose and, to a much lesser degree, fructose. However, the notion of Glf-mediated disaccharide export was supported by the GC-MS analysis of the culture medium of JCC547. As mentioned above, GC-MS indicated several ion chromatogram peaks that were only present, or present with larger areas, in this strain and not in JCC342c, JCC543, or JCC545. Consistent with these peaks representing disaccharides, many of these peaks were characterized by a dominant m/z 361 ion, which is diagnostic of TMS-derivatized disaccharides [Molnar-Perl et al., Chem. Mater. Sci., 45:321-327 (1997)], as shown in Table 20. Because none of the authentic disaccharide standards that were used eluted at the times indicated in the table above, none of these corresponding peaks could be identified with certainty. However, given the presence of the m/z 361 ion in all, it is highly likely that these peaks represent disaccharide or disaccharide-like molecules, most likely synthesized naturally within the cell.
The issued Joule patent derived from application 13/174,580, file June 30, 2011. The cross-reference section of the issued patent notes:
This application is a continuation application of U.S. application Ser. No. 12/867,738, filed Aug. 13, 2010, now U.S. Pat. No. 7,981,647, issued Jul. 19, 2011, which is a national phase entry of PCT/US2009/035937, filed on Mar. 3, 2009, which claims the benefit of U.S. Provisional Application No. 61/033,411 filed Mar. 3, 2008; U.S. Provisional Application No. 61/033,402, filed Mar. 3, 2008; U.S. Provisional Application No. 61/044,419 filed Apr. 11, 2008; U.S. Provisional Application No. 61/056,999 filed May 29, 2008; U.S. Provisional Application No. 61/058,182 filed Jun. 2, 2008; U.S. Provisional Application No. 61/077,698 filed Jul. 2, 2008; U.S. Provisional Application No. 61/079,692 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,699 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,665 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,656 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,688 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,687 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,676 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,673 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,667 filed Jul. 10, 2008; U.S. Provisional Application No. 61/079,707 filed Jul. 10, 2008; U.S. Provisional Application No. 61/086,283 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,288 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,291 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,296 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,300 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,407 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,410 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,412 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,414 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,417 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,418 filed Aug. 5, 2008; U.S. Provisional Application No. 61/086,285 filed Aug. 5, 2008; U.S. Provisional Application No. 61/100,656 filed Sep. 26, 2008; U.S. Provisional Application No. 61/100,660 filed Sep. 26, 2008; U.S. Provisional Application No. 61/100,663 filed Sep. 26, 2008; U.S. Provisional Application No. 61/100,665, filed Sep. 26, 2008, U.S. Provisional Application No. 61/100,667 filed Sep. 26, 2008; U.S. Provisional Application No. 61/106,543 filed Oct. 17, 2008; and U.S. Provisional Application No. 61/121,532 filed Dec. 10, 2008, all of which are herein incorporated by reference in their entirety and for all purposes.
posted by Lawrence B. Ebert at 5:01 PM
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