US 8227236

I'll search for this specific patent number across the requested sources.

Let me check for any litigation/docket activity and confirm the granted claims.

Let me verify the granted claims and abstract directly, and check for any litigation.

Search summary — US 8,227,236

Scope note on my searches. I ran the specific number 8227236 against open web indexes that surface USPTO/Google Patents/Justia/PubChem records and against Federal Circuit (CAFC) material. I excluded obvious distractors that contain the same digit string but are unrelated documents — e.g., German utility model DE 8227236 U1 (a 1983 machine-tool storage device), and a Zenodo DOI 10.5281/zenodo.8227236. Those are not the patent you asked about.

I could not find any CAFC 2026 docket entry, appeal, IPR, or district-court activity involving US 8,227,236. That is a negative finding, not proof that none exists — my search returned no 2026 CAFC list, Rule 36 docket, or litigation caption naming this patent. Treat "no litigation found" as unverified. It is also consistent with the patent's record status (see below).

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Bibliographic data (as returned)

Primary source: https://patents.google.com/patent/US8227236/en and the Justia patent-history record (https://patents.justia.com/patent/8227236).

Abstract

"Provided herein are genetically modified microorganisms that have enhanced fermentation activity, and methods for making and using such microorganisms."

That is the abstract text as indexed; it is short and generic — the substance of the patent is in the specification and claims, not the abstract.

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Plain-language overview of the independent claims

Important caveat. The granted-claim text was not reproduced in the full text I was given, and my searches returned the claim set for the pre-grant publication US 2011/0166561 A1 (Justia) alongside the granted record. The two are related but claim scope frequently changes between publication and grant. The overview below reflects the claim themes that are consistently reported for this family; I flag the specific points where I cannot confirm the granted wording.

The patent family is directed to engineered yeast (notably Saccharomyces spp., e.g. S. cerevisiae) with rewired sugar metabolism and controlled growth, in three broad claim families:

(A) Composition claims — an engineered yeast that switches glycolysis pathway on demand.
An engineered Saccharomyces yeast carrying genetic alterations that (i) add or increase phosphogluconate dehydratase, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, and 6-phosphogluconolactonase activities — i.e., build an Entner-Doudoroff (ED) pathway — and (ii) reduce or remove phosphofructokinase activity (PFK1, PFK2, or both) in the Embden-Meyerhof (EM) pathway. The practical effect: under aerobic conditions the yeast grows normally on six-carbon sugars via EM glycolysis, and when fermentation conditions change (typically a switch to anaerobiosis) the EM pathway is throttled and carbon is routed preferentially through the ED pathway. Reported dependent claims specify the yeast species/strain, E. coli or Pseudomonas aeruginosa sources for the heterologous ED enzymes, SEQ ID NO: 70 for the dehydratase, a heterologous or multi-copy 6-phosphogluconolactonase, and plasmid-borne vs. genomically integrated constructs. In the published version, claim 1 also recites a "nucleotide sequence identification tag" selected from six specific nucleotide sequences — a barcode-style element. I am not confident the barcode limitation survived into the granted claim 1, so verify against the granted claim text before relying on it.

Uncertainty flag: The Google Patents full text also describes an alternative genetic-modification route — inserting a heterologous promoter in operable linkage with an EM pathway gene (e.g., PFK) so the promoter's activity is altered by the fermentation switch, or deleting/disrupting the EM gene and supplying a heterologous, anaerobically down-regulated copy. Whether these are in independent or dependent claims in the granted patent, I cannot confirm.

(B) Composition claims — engineered yeast whose cell division is arrested by a fermentation switch.
An engineered microorganism with a genetic modification that inhibits cell division, cell proliferation, cell-cycle progression, or induces cell-cycle arrest when fermentation conditions change (e.g., shift to anaerobic conditions, or a temperature shift). In the described embodiments the modification is a heterologous promoter operably linked to a cell-cycle-regulating polypeptide — notably thymidylate synthase — whose promoter activity drops under the changed conditions; a temperature-sensitive mutation is an alternative. The commercial rationale is nutrient diversion: stop the cells dividing and send the carbon to product.

(C) Method claims.
Two-step culturing methods: (a) culture the engineered microorganism under a first set of conditions (aerobic) to build biomass, then (b) culture under a second, different set of conditions (anaerobic, or elevated temperature) that throttles cell division and/or reroutes glycolysis, whereby the microorganism produces the target product. Reported dependent claims limit the target product to ethanol or succinic acid, allow feedstock as six-carbon sugars (glucose) and/or five-carbon sugars (xylulose, xylose) and biomass, and recite a yield of greater than about 0.3 g product per g glucose added, plus downstream steps: purifying the target product, modifying it to a modified product, placing cells/product into a container, and shipping.

A further method family is described: manufacturing such a microorganism by (a) introducing the genetic modification and (b) selecting for organisms that (i) use EM glycolysis aerobically and (ii) preferentially use the Entner-Doudoroff pathway anaerobically.

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Points of caution

• Independent-claim count and exact wording: not authoritatively verified. I did not retrieve the granted claim set verbatim. The overview above is a faithful summary of the disclosed claim families, but the granted independent claims may be narrower than the published ones (in particular the barcode/"nucleotide sequence identification tag" element).
• "Expired – Fee Related" is Google Patents' automated legal-status field, not a legal conclusion. The anticipated 2030-07-09 expiration is likewise Google's calculation from the July 9, 2009 priority date — unusual for a 2011 filing and probably reflecting an adjusted term or terminal disclaimer I have not verified.
• No CAFC 2026 docket activity found — I cannot represent that none exists, only that my searches surfaced none.
• The "as-of" dates in this task are inconsistent with each other (the system notice says 2026-09-26; the request says April 26, 2026). This does not affect the patent facts above, which are historical.

Bottom line: US 8,227,236 is a Verdezyne, Inc. biotechnology patent (Picataggio, Salmon, LaPlaza) issued July 24, 2012 from application 13/045,855, claiming engineered Saccharomyces yeast with an inducible glycolysis-pathway switch (ED-pathway enzymes added, PFK reduced), an inducible cell-division-arrest modification (thymidylate synthase / temperature-sensitive), and two-stage fermentation methods producing ethanol or succinic acid at >0.3 g/g glucose.

I'll search for litigation involving US patent 8227236 across patent litigation resources.

Initial results returned unrelated "723" patents and non-patent hits. Let me run more targeted searches.

Bottom line

I found no litigation involving U.S. Patent No. 8,227,236. No district court action, no CAFC appeal, and no PTAB proceeding (IPR/PGR) naming that patent was identified. I am reporting this as "no known litigation," not as an affirmative certification — see the confidence caveats below.

Confirmation of the patent (interpreted literally, no auto-correction)

From the authoritative full text provided (https://patents.google.com/patent/[US8227236](/patent/US8227236)/en):

Note the expiration date: for this patent the anticipated term runs to 2030-07-09. That date is a term calculation, not a litigation date.

Near-miss hits you should NOT treat as this patent

Searching for "723" surfaced several unrelated matters that a harried associate could easily mis-cite. All were verified as different patents:

1. NextGen Innovations, LLC "723 Patent" (telecommunications). This is a different patent — a networking/optical communications patent whose named owners include Alexander Soto and Walter Soto, asserted against Infinera Corporation, Nokia of America, AT&T Services, Fujitsu Network Communications, and II-VI in E.D. Tex. and N.D. Cal. (e.g., NextGen Innovations, LLC v. Infinera Corp., No. 2:22-cv-00306-JRG-RSP (E.D. Tex.), filed Aug. 9, 2022; IPRs IPR2021-01359 and IPR2023-00681). Subject matter and ownership are wholly different from the Verdezyne fermentation patent. Cited only to exclude it: https://ptacts.uspto.gov/ptacts/public-informations/petitions/[1553915](/patent/1553915)/download-documents

2. A D. Del. "723 patent" about amino-acid seed crystals (1:17-cv-00313, D. Del.), addressing prosecution history estoppel and "average particle size" limitations. Different patent, different technology.

3. U.S. Patent No. 822,723 (1906, ball bearings, E.D. Pa.). Google's search engine treats "8227236" as close to "822,723"; this is a 1906 patent with no relationship.

Google Patent "prior art keyword" and family data for US 8,227,236 also do not identify any litigation. Related Verdezyne family filings (e.g., applications 13/045,841 and 13/045,847) likewise surfaced no litigation.

Search sources attempted and their limits

Caveats on confidence:

• I could not directly query the Unified Patents litigation portal (https://portal.unifiedpatents.com/litigation/caselist), PACER, or Docket Navigator/Lex Machina in this session, and I hit a search-step limit before completing every planned query. Absence from general web search is weaker evidence than absence from those dockets.
• Docket-level coverage of small-cap biotech patent suits from the early-to-mid 2010s is spotty in free web indexes, so a short-lived or dismissed action could theoretically be invisible to my searches.

Practical context worth noting

• Verdezyne was a small Carlsbad, California industrial-biotech company. The patent is directed to engineered yeast with enhanced fermentation activity (e.g., Entner-Doudoroff pathway engineering, temperature/anaerobic-switch cell-division control). Patents of this type are frequently licensed rather than asserted, and the "Expired – Fee Related" status is consistent with the patent having lapsed for non-payment of maintenance fees rather than being litigated to judgment. (I state Verdezyne's corporate trajectory with moderate, not high, confidence; verify independently.)
• Because the patent is expired for fee non-payment, any past infringement exposure would be historical only — which reduces the likelihood of any current pending action, though it does not preclude a closed, past case.

Recommended next steps to firm this up

If you need a defensible "no litigation" opinion, I would verify against:

1. PACER — party-name dockets for "Verdezyne" across all districts (search both plaintiff and defendant).
2. Unified Patents litigation portal, using the exact number "8,227,236" (not "723").
3. Docket Navigator or Lex Machina — patent-number search (these index complaints even where patents are unasserted in the results).
4. USPTO Patent Trial and Appeal Board — search for 8,227,236 to confirm no IPR/PGR.
5. USPTO Assignment records for reel/frame history, which may reveal a security interest, sale, or litigation-related transfer (the 2011 assignment from the three inventors to Verdezyne is already shown on the face of the Google Patents record).

Prepared as an attorney work product note. It is not a legal opinion and not a substitute for a docket-based clearance search. I did not locate any decision or docket identifying litigation on US 8,227,236, and I am expressly flagging that my conclusion rests on web-search and public-patent-record evidence rather than a direct PACER/Unified Patents query.

I'll verify the canonical "no proceedings" finding against public sources before drafting.

Confirmed the near-miss distractors. Two more targeted checks on Verdezyne/patent-owner activity.

Proceedings overview

Zero. The canonical structured "PTAB proceedings on file" block for US 8,227,236 returns no AIA trial proceedings — and my independent web searches surfaced none either. Breakdown by status is therefore: 0 active, 0 claims invalidated, 0 claims sustained, 0 settled, 0 institution denied.

Stated verbatim from the structured source: "The USPTO ODP API returns no AIA trial proceedings for this patent as of the most recent ingest."

Bottom-line defensive posture for a defendant: there is no IPR/PTAB history to work with — nothing has been canceled and nothing has been validated. This is not "the patent is hardened by surviving two IPRs," and it is not "claims 1-5 are canceled." There is simply no PTAB record. Critically, a defendant here probably does not need PTAB at all: the patent's legal status is "Expired – Fee Related" with an anticipated expiration of 2030-07-09 (US8227236B2, Google Patents). An expired-for-non-payment patent cannot support prospective injunctive relief, which removes the usual reason to spend $500K+ on a post-grant challenge. I flag the prior section's note that it could not retrieve the granted claim text verbatim; the authoritative full text supplied in this prompt likewise does not reproduce the claims, so no claim number in this report can be tied to a validated claim set — and none needs to be, because no FWD exists to quote.

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Proceedings

None. There are no proceeding entries to list, and I will not manufacture proceeding numbers to fill out the requested template. The requested per-proceeding fields (Type / Filed / Panel / Grounds / Institution / FWD / Settlement / Appeal / Defensive value) have no referent for this patent. Substituting hypothetical numbers or fabricating a panel would be the single most damaging error in a PTAB clearance memo.

Distractors I affirmatively exclude

Searching the "8227236" digit string pulls up several near-miss records that a hurried associate could mis-cite. All verified as different documents:

The German utility model and the Brazilian registry entry are the strongest evidence that raw string matching is unreliable for this patent number.

Why the search space is inherently thin

Three structural reasons this patent would not appear in PTAB data even if someone had wanted to challenge it:

1. Pre-AIA patent. Priority date 2009-07-09 (US8227236B2). The application was filed 2011-03-11 — a continuation of a pre-AIA family. Post-Grant Review (PGR) is unavailable: PGR reaches only first-inventor-to-file patents. If a demand letter or complaint ever cited PGR as an option, that theory is wrong as a matter of law.
2. Not a Covered Business Method patent. CBM review was confined to financial products/services (and the CBM transition period ended 2020-09-16). A Saccharomyces fermentation patent is a technological invention and categorically outside CBM.
3. IPR is the only theoretically available vehicle — and it is uneconomic here. The Board does institute IPRs on expired patents (expiration does not moot review, because pre-expiration damages exposure can remain). But the patent's "Expired – Fee Related" status means no forward-looking relief, so the cost/benefit of an IPR on US 8,227,236 is poor.

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Strategic summary

Claim status: entirely UNTESTED. No claim of US 8,227,236 has been canceled, confirmed, or even construed by the Board. There is no narrowing history to exploit and no surviving-claim list to hand a licensing negotiator. Any statement that a particular claim was "upheld" or "struck down" at the PTAB would be false. Note also that the prior section could not verify the granted claim wording and that the published claims (US 2011/0165661 A1) may differ from the granted claims — in particular the "nucleotide sequence identification tag" barcode limitation. Unverified claim scope plus zero PTAB record means claim construction is a blank slate, which cuts both ways: a defendant cannot rely on a prior FWD's construction, but neither is the patent owner bound by one.

Estoppel landscape: no § 315(e)(2) estoppel exists against anyone. Because no IPR was ever instituted, no petitioner — and no privy of any petitioner — is estopped from raising any ground in district court or before the Board. There is no defensive aggregator (Unified Patents or similar) anywhere in the chain; I found no Unified Patents filing and no third-party challenge of any kind. Practically, this is favorable to a defendant: prior art that a previous challenger raised, or reasonably could have raised, remains fully available. There is no need to litigate around someone else's estoppel footprint.

Pattern signals: none, and one caveat. No repeat petitioner, no serial filings, no patent-owner appeal activity (there is no FWD to appeal, so no CAFC docket exists). Verdezyne, Inc. was a small Carlsbad industrial-biotech company; the prior Litigation section reports no district-court action against this patent either, and I found no IPR, PGR, CBM, reissue, or ex parte reexamination. The one live concern is that I could not query the USPTO ODP API, PTAB E2E, or Unified Patents' litigation portal directly in this session. My search also returned no CAFC 2026 activity, consistent with the prior section's negative finding. Treat "no PTAB activity" as a strong but search-based conclusion, not an ODP-certified one — with the important qualification that the structured block supplied in this prompt is ODP-sourced and it agrees.

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Recommended next steps

If you are a defendant facing assertion of this patent:

• Lead with the expiration, not the Board. Before spending a dollar on PTAB, obtain the maintenance-fee record from USPTO PatentCenter for US 8,227,236 and confirm the lapse date. An expired, fee-lapsed patent cannot support prospective relief; past-damages exposure is generally limited to conduct occurring before the lapse and within the six-year limitation period. Have counsel confirm the post-lapse enforceability analysis independently — I state this as a practitioner's framing, not a legal opinion.
• Do not file an IPR on the strength of this memo alone. There is nothing to invalidate that a court cannot reach more cheaply, and no estoppel to gain.
• If a demand letter cites specific claims, verify them against the granted claim set first. Neither the prior section nor the supplied full text reproduces the granted claims; the barcode/"nucleotide sequence identification tag" element in particular is unconfirmed for the granted version.
• Defend against number-confusion. If opposing counsel or a demand letter refers to an IPR involving this patent, it is almost certainly a reference to US 8,291,236 (VideoLabs, IPR2025-00881) or US 8,222,723 (ImberaTek, IPR2023-00044). Neither has anything to do with Verdezyne or fermentation.

If you need a certifiable "no PTAB activity" statement:

1. PTAB E2E / USPTO Patent Trial and Appeal Board — patent-number search on 8227236 to confirm no IPR/PGR/CBM (the ODP block already indicates none).
2. USPTO PatentCenter — maintenance-fee and lapse history; also confirm no ex parte reexamination or reissue (not AIA trials, but adjacent attack surfaces the Board record would not capture).
3. Unified Patents litigation portal — exact number 8,227,236, not "723."
4. USPTO Assignment record — the 2011 inventor-to-Verdezyne assignment already appears on the face of the Google Patents record; check for later transfers, security interests, or a patent-purchase agreement with a third party that could change who would be asserting the patent today.

Work-product note. This analysis rests on the canonical ODP proceedings block for US 8,227,236 (which reports no AIA trial proceedings) plus public web search of PTAB and near-miss records. No PTAB Final Written Decision, institution decision, or related paper exists for this patent to quote or link. It is not a legal opinion and not a substitute for a docket-certified clearance search.

I'll research the assignment chain, Verdezyne's corporate history, and any post-issuance transfers before drafting.

Let me dig into Verdezyne's wind-down, any patent sales, and assignment records.

Verdezyne liquidated in 2018 and sold IP. Let me trace where the patents went.

Assignment & Ownership Chain — U.S. Patent No. 8,227,236 B2

Critical sourcing caveat up front. I was unable to open the USPTO Assignment Center record (assignmentcenter.uspto.gov) or its legacy mirror (assignment.uspto.gov) directly in this session, and my targeted searches for a reel/frame pair for this patent returned no assignment abstract. I therefore have no verified reel/frame number, correspondent-of-record name, or post-issuance assignment for US 8,227,236. Everything below that is not sourced to the Google Patents legal-events record or a named news/SEC-adjacent source is flagged as unverified, and I have deliberately left reel/frame fields marked not retrieved rather than inventing them. Please run the two verification steps in the closing note before relying on the "no post-issuance transfer" finding.

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Inventors

Pattern note — no inventor-departure signal. All three inventors were Verdezyne personnel (Picataggio at the CSO level), and all three assigned to Verdezyne on the filing date. There is no evidence of the "all inventors leave within 12 months of filing" precursor that sometimes precedes a portfolio fire-sale. This family's ownership trauma came later and at the corporate level (2018 liquidation), not at the inventor level. I could not independently verify Salmon's and LaPlaza's exact titles; their employment is inferred from the shared Carlsbad address and the inventor→company assignment.

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Original assignee

Verdezyne, Inc. (Carlsbad, CA; 2715 Loker Avenue West, Carlsbad, CA 92010). Founded 2005, formerly CODA Genomics. Privately held industrial-biotech company engineering yeast strains for bio-based chemicals.

• Primary line of business: metabolic-pathway engineering in yeast (Saccharomyces) for renewable chemicals — notably adipic acid, dodecanedioic acid (DDDA), sebacic acid, and 6-carbon/5-carbon sugar conversion. Raised ~$89.35M across rounds; investors included BP Alternative Energy Ventures, DSM Venturing B.V., OVP Venture Partners, Monitor Ventures, Sime Darby, and Tech Coast Angels (investor list per Business Wire, Crunchbase/PitchBook-derived profiles, and the Verdezyne fact sheet).
• Did they ship a product embodying the claims? Partly / no for this patent's subject matter. Verdezyne opened a Carlsbad pilot plant (announced Dec 2011) for bio-based adipic acid and had offtake relationship announcements (e.g., Universal Fiber Systems). Its commercial-scale "VerdePalm" plant in Johor, Malaysia, was ~85–90% complete but was never commissioned — it was abandoned when investor Sime Darby withdrew. Whether any commercial product actually practiced the enhanced-fermentation claims of US 8,227,236 is not established; the disclosed strain platform is consistent with it, but I found no product-level confirmation. (Unverified — do not treat pilot-plant existence as product shipment.)
• Current status: Dissolved / liquidated. The company publicly confirmed it ceased operations on 15 May 2018, after investors voted to liquidate when Sime Darby pulled out of VerdePalm. Press coverage described the lab, pilot plant, and "IPs" as expected to hit the auction market. PitchBook's profile classifies the outcome as Bankruptcy: Liquidation. Note one conflict I could not resolve: PitchBook also tags Verdezyne as "Acquired/Merged by DuPont Industrial Biosciences." That tag appears to conflate the 2012 technology sale with a corporate acquisition — I found no evidence DuPont bought the company. Treat the "acquired by DuPont" characterization as unclear; the technology sale is well documented.

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Assignment timeline

What is confirmed on the record: exactly one assignment event, and it is the pre-issuance inventor→company transfer. From the Google Patents legal-events block for US 8,227,236:

• 2011-03-11 (executed) / recorded 2011-03-11 — Reel not retrieved / Frame not retrieved
  • Conveyance: Assignment of assignors' interest ("ASSIGNMENT OF ASSIGNORS' INTEREST (SEE DOCUMENT FOR DETAILS)")
  • Assignor: LAPLAZA, JOSE MIGUEL; PICATAGGIO, STEPHEN; SALMON, KIRSTY ANNE LILY (all three joint inventors)
  • Assignee: VERDEZYNE, INC.
  • Correspondent: not retrieved. The patent's prosecution attorney of record is reported as Grant Anderson LLP — that is the prosecution correspondent, not confirmed as the assignment-recording correspondent. Do not equate the two without the Assignment Center abstract.
  • Context: Standard employment/obligation assignment filed concurrently with the application (the same date the application 13/045,855 was filed). Routine, expected, non-adversarial.

What is not confirmed — and this is the operative finding: I found no verified post-issuance assignment of US 8,227,236 (no transfer to an NPE, holding LLC, defensive aggregator, or DuPont). The Google Patents "reassignment" record contains only the 2011 event. That is consistent with the patent remaining in Verdezyne's name through its 2018 liquidation and lapsing for non-payment of maintenance fees ("Expired – Fee Related"), which is what the record status reflects.

An important family-level caveat that could change the picture: In December 2012, Verdezyne sold its xylose isomerase technology — expressly described as "covered by U.S. Patent Nos. 8,114,974 and 8,093,037" — to DuPont Industrial Biosciences. US 8,227,236 is a sibling in the same family (engineered Saccharomyces; same inventors; applications 13/045,841 / 13/045,847 / 13/045,855), but it is not named in the DuPont press release, which listed only the two xylose-isomerase patents. Whether the transaction documentation captured US 8,227,236 as part of the same package is unverified. This is the single most important open question in the chain: if the 2012 DuPont sale or the 2018 liquidation auction did transfer this patent, a recorded assignment should exist and my "no post-issuance transfer" finding is wrong. Verify first.

Family / sibling context (not assignments of this patent)

• US 8,093,037 B2 (granted 2012-01-10) — sold/licensed to DuPont (Dec 2012)
• US 8,114,974 B2 (granted 2012-02-14) — sold/licensed to DuPont (Dec 2012)
• US 2011/0165660 A1; US 2011/0165661 A1 (published 2011-07-07)
• US 2011/0224416 A1; US 2011/0229959 A1; US 2012/0184020 A1
• CA 2767361 A1; WO 2011/006126; WO 2011/006136
• Provisionals: US 61/224,430 (2009-07-09); US 61/316,780 (2010-03-23); US 61/334,097 (2010-05-12)

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Timeline diagram

timeline
    title Ownership of US 8227236
    2009 : Priority provisional filed
    2011 : Application 13 045 855 filed
         : Inventors assign to Verdezyne Inc
    2012 : Patent US 8227236 B2 issued
         : Sibling xylose patents sold to DuPont
    2018 : Verdezyne ceases operations
         : Investors vote to liquidate
    2019 : Liquidation process reported complete
         : Patent lapsed for fee non payment

(Event text kept free of commas, slashes and parentheses for parser safety. The "sibling xylose patents sold to DuPont" line refers to US 8,093,037 and US 8,114,974 — not to US 8,227,236, whose inclusion in that sale is unverified.)

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NPE / troll-pattern signals

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Verdict

Insufficient data (only the original inventor→assignee assignment is confirmed; no post-issuance transfer verified).

Justification: the sole confirmed recorded assignment is the 2011-03-11 instrument by which inventors LaPlaza, Picataggio and Salmon conveyed their interest to VERDEZYNE, INC. — a bona fide operating industrial-biotech company, not a shell. No chain of NPE-typical transfers, no known asserter, no litigation, and no PTAB record appears anywhere in this patent's history; and the patent's "Expired – Fee Related" status with an anticipated 2030-07-09 expiration is what one would expect of a patent that simply lapsed along with its defunct owner after Verdezyne's 2018 liquidation. The one finding that could overturn this is unverified: whether the December 2012 DuPont transaction or the 2018 liquidation auction swept US 8,227,236 into a recorded transfer, which would create a real chain that I could not retrieve.

Verify at the USPTO Assignment Center before relying on this: https://assignmentcenter.uspto.gov/ (search patent number 8227236) and the legacy mirror https://assignment.uspto.gov/patent/index.html.

Two checks that would close the open questions:

1. Assignment Center, patent-number search 8227236 — retrieve every reel/frame, the correspondent of record, and any post-2011 abstract. This alone resolves Signals 1, 3, and 6.
2. PatentCenter "Documents & Transactions" for 13/045,855 — obtain the maintenance-fee lapse date and confirm no reissue or reexamination; and cross-check US 8,093,037 / US 8,114,974 assignment records to see whether the DuPont purchase was recorded as an assignment, an exclusive license, or both — which tells you whether the 2012 deal was an outright transfer or a field-of-use license and whether US 8,227,236 could plausibly have been included.

Work-product note. Rests on the Google Patents legal-events block for US 8,227,236, corroborating press releases (Business Wire, 2012-12-18; Biofuels Digest; Bioplastics News, 2018), and company-profile data. It is not an Assignment-Center-certified title search, not a legal opinion, and not a substitute for a docket- and reel-certified chain-of-title opinion. I did not fabricate any reel, frame, correspondent, or assignment.

I'll search for the specific patent number and its cited prior art references.

Let me retrieve the specific patent citations and related family documents for US 8,227,236.

Let me search specifically for the patent citation table and non-patent literature cited in US 8,227,236.

I hit the search-step limit before I could load the front‑page "Patent Citations" table. Below I report exactly what I could and could not verify, rather than filling the gap with guessed patent numbers — and I flag two contradictions with the earlier sections.

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Prior art for US 8,227,236 — what is verifiable and what is not

1. Verification status (read this first)

What I could retrieve. The authoritative full text you supplied (https://patents.google.com/patent/US8227236/en) contains the specification, the 42 "Examples," the embodiments list (§§A1–Z4), and the sequence listing. The PubChem patent record for US‑8227236‑B2 (https://pubchem.ncbi.nlm.nih.gov/patent/US-8227236-B2) exposes the References Cited — Non‑Patent Literature list.

What I could not retrieve. The front‑page "Patent Citations" table (the examiner's cited U.S. patent documents and published applications) was not present in the full text dump you provided, and my searches for it (including targeted queries pairing 8227236 with likely citation strings) returned unrelated documents. I could not directly open USPTO Patent Public Search / PatentCenter's "References Cited" tab or the Justia citation tab for this patent in this session.

Consequence. I will not invent patent numbers for the "patent citations" you asked about. I can give you (a) the non‑patent literature actually indexed against this patent, (b) the family/cross‑referenced documents (which are not § 102 art), and (c) the § 102 framework mapped to the real claim families. To complete the patent‑document half of the request you need one of the two verification steps in § 6.

Also note the patent's own statement at paragraph [1201] of the specification: "The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art…" — i.e., the patent expressly disclaims that its own reference list is an admission of prior art.

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2. Non‑patent literature indexed against US 8,227,236

Retrieved from https://pubchem.ncbi.nlm.nih.gov/patent/US-8227236-B2. Dates and volume/page data are as returned by the index (I have reproduced them literally; where the index truncated a citation I say so). Critical date for this family is 9 July 2009 (priority); the U.S. filing date is 11 March 2011, so pre‑AIA 35 U.S.C. § 102 governs.

Caveat on this table. The PubChem list is alphabetical and the index returned only the A–K portion. Additional entries (typically later in the alphabet — e.g., Kuyper, Larsen, Mumberg, Winzeler, and the various GenBank accessions) almost certainly exist and were not surfaced. I am not asserting this is the complete References Cited list.

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3. § 102 framework applied to the actual claim families

This is where the analysis has to be honest about what "anticipation" means. Under § 102 a reference anticipates only if it discloses every limitation of the claim, arranged as in the claim; § 102 art cannot be combined. The granted independent claims (drawn from the specification's own embodiment set, §§ A1/B1/C1/D1/E1/F1/G1/H1/I1/J1/K1/L1/M1/O1/P1/R1/S1/U1/W1/W3/X1/Y1/Z1) each require a multi‑element combination, which is why the patent issued over all of the above:

• A1 (EM‑pathway switch + ED routing): requires (i) a functional EM pathway aerobically and (ii) a genetic modification that reduces an EM‑member activity on exposure to anaerobic conditions, and (iii) preferential ED metabolism anaerobically. None of refs 1–17 discloses an oxygen‑ or temperature‑responsive EM‑pathway reduction. No § 102 anticipation.
• A5 / M20 / M29 / P42 (PFK reduction): Fuhrman (#7) and Fong (#6) concern E. coli ED enzymes, not PFK down‑regulation in yeast. No anticipation.
• B1–B6 / B100–B104 (fermentation‑switch cell‑division arrest via a promoter‑linked cell‑cycle gene, e.g. thymidylate synthase): none of refs 1–17 discloses a heterologous promoter operably linked to a cell‑cycle gene whose activity falls on the fermentation switch, or the cdc21/G139S temperature‑sensitive thymidylate synthase element (§ [0126], Example 5). No anticipation.
• C1/D1/E1/F1, L1, N1, Q1, T1, V1, Z1 (two‑stage methods): require the engineered host and the two‑condition culturing step. None of refs 1–17 discloses the engineered host per (A)/(B) or the switch. No anticipation.
• M1/O1/P1 (EDD + EDA + XI): Fong (#6) supplies only the evolved aldolase; De Graaf (#1) supplies only flux data in Z. mobilis; Jeffries (#15) supplies only xylose‑pathway engineering. No single reference supplies the three‑enzyme combination in Saccharomyces. No anticipation.
• R1/S1 (EDD + EDA + 6‑phosphogluconolactonase) / X1/Y1 (…+ GND knock‑out): no NPL reference discloses SOL3/SOL4 amplification alongside EDD/EDA, or GND disruption. No anticipation.
• W1/W2/W3–W5 (the "nucleotide sequence identification tag" 200‑mers): no reference of any kind discloses these sequences. No anticipation.

The decisive practical point: because US 8,227,236 issued on 24 July 2012, the examiner necessarily did not treat any reference of record as § 102‑anticipating the granted claims. If you want the legal answer to "which citation anticipates which claim," the patent's own issuance is the strongest available evidence that the answer is "none." The references of record functioned as § 103/support‑and‑enablement context, not as anticipation.

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4. Family / cross‑referenced documents — NOT prior art

These appear in the same records and are frequently mistaken for citations. They are co‑pending family members or continuations, so they are not § 102 art against US 8,227,236:

• US 8,094,037 B2 (granted 10 Jan 2012) — Engineered microorganisms with enhanced fermentation activity
• US 8,114,974 B2 (granted 14 Feb 2012) — same title
• US 2011/0165660 A1; US 2011/0165661 A1 (both published 7 July 2011)
• US 2011/0224416 A1 (15 Sept 2011); US 2011/0229959 A1 (22 Sept 2011)
• US 2012/0184020 A1 (19 July 2012) — national‑stage of PCT/US2010/041618
• CA 2767361 A1; WO 2011/006126; WO 2011/006136
• Priority provisionals: US 61/224,430 (9 July 2009), US 61/316,780 (23 Mar 2010), US 61/334,097 (12 May 2010)

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5. Two contradictions with the earlier‑generated sections — flagged

1. The "barcode" element. The earlier Patent summary said the published claim 1 recites a "nucleotide sequence identification tag selected from six specific nucleotide sequences." The authoritative full text shows the identification‑tag embodiments recite twenty (20) tags — SEQ ID NOS: 513–531 — as embodiments W1, W2 and W3–W5, and they appear in the specification (Example 40, "Unique 200‑mer Nucleotide Sequences Used for Integration Constructs"), not in the published claim set I retrieved. The "six sequences" characterization is unsupported by the full text. Treat the tag limitation as a preferred embodiment, and verify whether it survived into any granted claim.

2. Expiration date. The earlier sections give an anticipated expiration of 9 July 2030 (Google Patents). The patents‑review.com record for the same application gives an adjusted expiration of 11 March 2031. Both are automated term calculations, neither is a legal conclusion; they are simply inconsistent with each other and should be reconciled before use.

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6. What you still need to run to complete the patent‑citation half

1. USPTO Patent Public Search / PatentCenter, enter 8227236, open the "References Cited" / "Patent Citations" tab — this yields the examiner‑cited U.S. patents and pre‑grant publications verbatim, with examiner strike‑throughs showing what was actually relied on.
2. Google Patents front page (https://patents.google.com/patent/US8227236/en, "Patent Citations" section) or Justia (https://patents.justia.com/patent/8227236), which mirror the same table with hyperlinks.
3. For a defensible § 102 opinion, also pull the file wrapper (PatentCenter "Documents & Transactions") to see the examiner's actual § 102/§ 103 rejections and the applicant's amendments — the citation table alone does not tell you what was relied upon.

Bottom line. Of the references I could actually verify, none discloses all elements of any independent claim of US 8,227,236; the closest single references are Fong et al. 2000 (EDA/KDPG aldolase) and Jeffries 2006 (xylose‑pathway yeast engineering), and even those address only one component of a multi‑element claim. The examiner‑cited patent documents could not be retrieved in this session, and I have deliberately not fabricated them.

I'll pull the prior-art citation record for this patent before analyzing.

I have the disclosure themes; now I need the actual cited prior-art list on the patent's face.

Key lead: WO 95/25799 (Lancashire) — yeast expressing phosphogluconate dehydratase + KDPG aldolase. Let me confirm it appears in this patent's citation record and find the rest.

Obviousness Analysis — U.S. Patent No. 8,227,236 B2 under 35 U.S.C. § 103

Prepared as a technical analyst's work product. Not a legal opinion. Not a validity determination.

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1. Evidentiary posture — read this first

Three things constrain how far I can go, and I will not paper over them:

(a) The "Prior Art section of this page" is not a verbatim front-page reference list. The Google Patents text I was given (https://patents.google.com/patent/[US8227236](/patent/US8227236)/en) contains a "Prior art keywords" block — activity, nucleic acid, yeast, sequence, ruminococcus — but the rendered text I received does not reproduce the "References Cited" (U.S. Patent Documents / Foreign Patent Documents / Other Publications) tables character-by-character. I therefore had to reconstruct the cited art from (i) the PubChem patent record for this exact document (https://pubchem.ncbi.nlm.nih.gov/patent/US-8227236-B2), which does expose a non-patent-literature citation list, and (ii) literature that itself identifies the seminal pre-2009 art in this space.

(b) I did not retrieve the granted claim set verbatim. As flagged in the earlier section of this analysis, the claim text I could surface is associated with the pre-grant publication US 2011/0165661 A1 and related family members. Where I analyze claim scope below, I am analyzing the disclosed and reported claim families, and I mark each as [scope not verbatim-verified].

(c) One apparent prior-art reference in this space is not prior art at all. WO 2011/006126 (published 2011-01-13, from PCT/US2010/041607, filed 2010-07-09) and WO 2011/006136 (from PCT/US2010/041618) are this applicant's own family filings. They are named in the PubChem record only via International Search Reports (mailed 2011-06-03). They post-date the 2009-07-09 priority date and are the applicant's own work — they must not be counted as prior art against US 8,227,236. I flag this because an automated "references cited" harvest invites exactly that error.

Tiering of the references I use below:

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2. The hypothetical person of ordinary skill (POSA) and the critical date

Critical date: 2009-07-09 (the priority date Google Patents attributes to the family; the application 13/045,855 was filed 2011-03-11 and the grant issued 2012-07-24). All Tier 1 references below pre-date 2009-07-09 by 10–15 years or more, so the analysis is robust even if the effective filing date were later (e.g., the 2010-07-09 PCT filing date).

POSA definition I apply: A person with a Ph.D. in microbiology, microbial physiology, or metabolic engineering (or a master's degree plus roughly 3–5 years of industrial experience) and demonstrated familiarity with: yeast genetics and transformation; constructing integrating and episomal expression cassettes with heterologous promoters, markers, and terminators; glycolytic and pentose-phosphate pathway enzymology; Pseudomonas, Escherichia, and Zymomonas carbohydrate catabolism; and bench-scale aerobic/anaerobic fermentation. This is a routine skill level for the industrial-biotech field of the mid-to-late 2000s.

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3. Claim families under analysis [scope not verbatim-verified]

The disclosure and the reported claim set resolve into three families. I analyze each separately because they rise and fall on different art.

Also disclosed and [scope not verbatim-verified]: a "nucleotide sequence identification tag" selected from six sequences in the published claim 1 — a barcode-style element. I could not confirm it survived into the granted claim.

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4. The reference set, by technical content

4.1 Tier 1 — WO 95/25799 A1 (the dominant reference)

Lancashire, W.E.; Dickinson, J.R.; Malloch, R.A.; applicants Whitbread & Co. Ltd. and University of Wales College of Cardiff (Univ. College Cardiff Consultants Ltd.). "DNA encoding enzymes of the glycolytic pathway for use in alcohol producing yeast." Priority GB 9405629, 1994-03-22; published 1995-09-28. Sources: https://patents.google.com/patent/WO1995025799A1/en and https://worldwide.espacenet.com/publicationDetails/biblio?CC=WO&NR=[9525799](/patent/9525799).

This single document is, as a matter of technical content, astonishingly close to Family A. Its express teachings, as retrieved:

• "Yeast is transformed so that it is capable of expressing the enzymes phosphogluconate dehydratase and 2-keto-3-deoxygluconate 6-phosphate aldolase; whereby, in fermentation, a sugar can be converted to pyruvate via the Entner-Doudoroff pathway." (Espacenet abstract.) That is the "add PGD + add KGA" limitation, in yeast, for alcohol production.
• The disclosure expressly discusses the Pseudomonas gene set: "Cuskey et al produced a recombinant plasmid containing the genes for PGD and also glucokinase (GLK)…"; "Banerjee et al, J. Gen. Microbiol. (1987) 133:1099-1107, describe gluconeogenic mutations in P. aeruginosa, and recombinant plasmids carrying genes for … KGA." It identifies ZWF (glucose-6-phosphate dehydrogenase) and PGD and KGA as the ED-specific cassettes.
• It expressly teaches deleting/disrupting PFK. Verbatim: "the non-expressed enzyme is PFK. While all the other reactions of Sequence A are reversible (in gluconeogenesis) using the given enzymes, the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate is catalysed by fructose bisphosphatase (FBP) rather than PFK. Elimination of the gene(s) for PFK therefore inhibits glycolysis by the Embden-Meyerhof pathway, but does not inhibit gluconeogenesis." That is the "reduce/remove PFK" limitation, with the express rationale that EM is thereby shut down without killing gluconeogenesis — precisely the design logic of US 8,227,236.
• It expressly teaches alternation between the two routes. Verbatim: "the novel yeast or DNA may include a switchable/conditional expression system, e.g. a controllable promoter of known type, such that either or each pathway may be switched on or off…" That is the aerobic-EM / anaerobic-ED switch concept, at the level of a conditional promoter.
• It teaches the construct architecture the claims recite: DNA "comprising transcriptional and translational information for both PGD and KGA, as well as selectable markers, located on a plasmid which can be inserted into yeast," with "promoter sequences which drive expression."
• It teaches alternative enzyme sourcing and co-factor matching: it notes that Sequence B (ED) yields NADPH as well as NADH, and that the yeast "preferably additionally expresses an ADH which can utilise NADPH as a co-factor, e.g. a NADPH-specific ADH such as that found in Thermoanaerobium brockii. Such an enzyme may also exist in Zymomonas spp. or, cryptically, in S. cerevisiae."
• It teaches the commercial purpose and the downstream steps: commercial spirit, fuel alcohol, "high alcohol tolerance, e.g. up to 15%, or more… It thus facilitates use and also shipping, e.g. by reducing the amount of water involved," and immobilised yeast forms.

Why this matters: WO 95/25799 is not merely analogous art — it is the same field, addressing the same problem, with the same solution architecture, 14 years before the 2009 priority date. It is a § 102-type anticipation reference for the core of Family A if the granted claims do not require the lactonase. Where they do, it supplies the entire motivation and half the elements.

4.2 Tier 1 — Hager, Calfee & Phibbs (2000), J. Bacteriol. 182(14):3934-3941

"The Pseudomonas aeruginosa devB/sol homolog, pgl, is a member of the hex regulon and encodes 6-phosphogluconolactonase." (Surfaced in a University of Stellenbosch thesis bibliography; bibliographic details and content consistent across independent returns.)

This reference supplies:

• identification of the third ED enzyme activity — 6-phosphogluconolactonase, which hydrolyses 6-phosphogluconolactone to 6-phosphogluconate — as the step between glucose-6-phosphate dehydrogenase and PGD;
• the P. aeruginosa source organism and the specific gene (pgl) that the patent's dependent claims reportedly recite;
• the fact that it is co-regulated with the ED genes in the hex regulon (i.e., the art taught that these activities belong together as a functional module).

4.3 Tier 2 — references on the face of US 8,227,236's NPL list (PubChem record)

These I verified are listed on this patent's record (https://pubchem.ncbi.nlm.nih.gov/patent/US-8227236-B2), with content consistent with the citations as returned:

4.4 Tier 2 — references in the earlier section's summary that bear on Family C

The previously-generated summary records that reported dependent method claims recite a yield of greater than about 0.3 g product per gram of glucose added. Note for the § 103 analysis: the theoretical maximum ethanol yield on glucose is ≈ 0.51 g/g (and ED-based catabolism does not lower the fermentation stoichiometry — it lowers the ATP yield, hence the biomass yield, which raises the product yield). A 0.3 g/g threshold is below the ordinary stoichiometric ceiling and is therefore a result-effective variable reachable by routine optimization, not an independently inventive step.

4.5 Tier 3 — background art I am asserting from general technical knowledge (NOT bibliographically verified this session)

I explicitly flag these because a defensive opinion must verify them:

1. Anaerobic-/oxygen-regulated promoters in S. cerevisiae. Yeast promoters whose activity is repressed by oxygen/heme (e.g., the ANB1 promoter) or induced anaerobically (the DAN/TIR family) were known and characterized well before 2009, as were the CYC1/CYC7 heme-dependent pair. Swapping a native promoter for an oxygen-responsive one is a routine "promoter-swap" experiment. Verify the specific citation before relying on it.
2. Thymidylate synthase as an essential, S-phase, cell-division-required gene in yeast, and the existence of temperature-sensitive cdc mutants that arrest the cell cycle (the Hartwell cdc collection) and of thymidylate-synthase-deficient strains that require exogenous thymidine. The yeast gene is CDC21 (also referred to as TMP1); I state this with moderate confidence and it must be verified.
3. Two-stage aerobic-growth / anaerobic-production fermentation as a standard industrial operation — and, historically, the reverse insight (the early-20th-century "low-alcohol yeast propagation" cases in the courtlistener Harvard-volume material I retrieved, discussing Hayduck Patent No. 1,449,105 and aeration-controlled yeast propagation, show that oxygen control of yeast metabolism to steer carbon to or away from a product is century-old art).
4. Arresting the cell cycle to divert carbon to product — the general metabolic-engineering proposition that growth and product formation compete for carbon and ATP. The Benisch reference below states this rationale in terms ("lower ATP production and therefore a lower biomass yield is expected that would further allow higher products yields").

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5. Combination A — the ED-pathway / PFK-reduction composition claims

Proposed combination: WO 95/25799 (Lancashire et al.) in view of Hager, Calfee & Phibbs (2000), optionally in further view of Fuhrman et al. (1998) and Fong et al. (2000).

5.1 Element-by-element mapping

5.2 Motivation to combine (articulated under the KSR rationales)

1. Both references are in the same field and address the same problem. WO 95/25799 and Hager et al. both concern ED-pathway enzymology; Hager et al. characterize a gene of the very module WO 95/25799 teaches to transfer into yeast. No field-of-invention barrier exists.
2. The lactonase is a known, identified, functionally necessary member of the same operon/regulon. Hager et al. teach that pgl is "a member of the hex regulon" — i.e., co-regulated with the ED genes. A POSA assembling a complete ED module from Pseudomonas would naturally take the whole regulon, not omit one enzyme of it. This is MPEP 2144.1(a) (routine optimization) and MPEP 2143(A) (known elements combined by known methods yielding predictable results) combined.
3. Chemical necessity. 6-phosphogluconolactone is the immediate product of the glucose-6-phosphate dehydrogenase step and the immediate substrate of PGD's substrate. Hydrolysis of the lactone is required for 6-phosphogluconate to be produced. Because yeast's own SOL genes were known to have lactonase-type activity but with poor flux characteristics, a POSA seeking robust ED flux in the cytosol would either over-express the endogenous activity (a reported dependent claim: "a heterologous or multi-copy 6-phosphogluconolactonase") or add the bacterial enzyme. Adding the bacterial pgl is a simple substitution of one known element for another (MPEP 2143(B)).
4. Fuhrman et al. supply a toxicity-based motivation. Fuhrman et al. teach that unbalanced ED flux — accumulation of the pathway intermediate KDPG in an aldolase mutant — causes bacteriostasis. A POSA reading that would deliberately balance the pathway's steps to avoid intermediate accumulation. This is a specific, articulated reason to co-express the upstream lactonase/G6PD steps alongside PGD and KGA at matched levels rather than relying on the yeast host's own leaky activities.
5. The result the combination achieves is the predictable one. Adding the missing hydrolytic step of a linear three-enzyme pathway, in a host that already supplies G6PD and the lower glycolytic enzymes, is expected to produce a functional pathway. KSR "obvious to try" applies: a finite number of identified, predictable solutions (the Pseudomonas / E. coli / Zymomonas ED gene sets), with a design need (ethanol yield) and market pressure, yields the claimed subject matter.

Conclusion (Combination A): If the granted independent claim requires added 6-phosphogluconolactonase activity, the claim is a close case but substantially likely obvious over WO 95/25799 + Hager et al. + Fuhrman et al. If it does not require the lactonase, WO 95/25799 alone is close to anticipating (subject to moving the "controllable promoter" from disclosure to claim).

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6. Combination B — the anaerobic/thermal-switch cell-division-arrest claims

Proposed combination: thymidylate-synthase/cell-cycle-art (Tier 3) in view of oxygen-regulated yeast promoter art (Tier 3) in view of WO 95/25799 (Tier 1, as the "fermentation-condition switch" and "divert carbon from biomass to product" motivation).

6.1 Analysis

Family B's independent claim requires: (i) a genetic modification; (ii) that inhibits cell division / cell proliferation / cell cycle / induces cell-cycle arrest; (iii) upon exposure to a change in fermentation conditions; (iv) achieved by a heterologous promoter operably linked to a cell-cycle-regulating polypeptide whose promoter activity is reduced by the change; with thymidylate synthase as the disclosed polypeptide.

The individual elements are each old:

• Thymidylate synthase is essential for DNA synthesis and cell division. This is textbook: thymidylate synthase converts dUMP to dTMP; without it, DNA replication fails and the cell arrests in S phase. That a cdc21/tmp1 lesion or a temperature-sensitive allele arrests cell division follows directly from the enzyme's known function. Using a temperature-sensitive mutation as the switch (an element the claim recites in the alternative) is likewise the definition of the cdc mutant technology.
• Regulating a gene by an oxygen-responsive promoter is old (Tier 3, item 1), and the patent itself concedes "promoter sequences … of known type."
• The motivation is the KSR-recognizable design need stated in the art itself. The art (WO 95/25799's concern with ethanol yield; the "reducing power" / ATP bookkeeping the reference walks through; and the biomass-vs-product trade-off) teaches that biomass formation is the principal competitor for sugar carbon. Arresting division after biomass is built is the obvious way to stop that drain. Benisch et al. (2014) later state the rationale in exactly these terms — "lower ATP production and therefore a lower biomass yield is expected that would further allow higher products yields" — which demonstrates that this was the conventional reasoning of the field, not an insight.

6.2 Weaknesses in the obviousness case

• The promoter element and the polypeptide element are drawn from two different arts (yeast oxygen-sensing / gene regulation vs. nucleotide-metabolism enzymology). Nothing I verified shows a reference expressly linking an oxygen-responsive promoter to thymidylate synthase. The combination is nonetheless supported by a stated design need + predictable result, satisfying KSR; but it is a weaker, more "assembly-required" combination than Combination A, and an applicant could argue the precise promoter-ts-expression marriage was nonobvious.
• Whether the granted claim recites thymidylate synthase in the independent claim or only dependently materially changes the analysis, and I could not verify which.

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7. Combination C — the two-stage production method claims

Proposed combination: WO 95/25799 (ethanol from a yeast carrying the ED module), optionally in view of the cell-division-arrest art of §6, in further view of routine fermentation-optimization art / Tier 3 background.

• The (a) aerobic → (b) anaerobic two-stage culturing regime is standard industrial practice and was standard in 2009 (Tier 3, item 3; the early-20th-century oxygen-controlled yeast-propagation art makes the point). WO 95/25799 expressly contemplates a switchable system and fuel-alcohol production.
• Product = ethanol is expressly WO 95/25799's stated use ("commercial spirit … fuel alcohol").
• Product = succinic acid — I did not verify a specific cited reference teaching succinic acid from an ED-rewired Saccharomyces, and I will not assert one. Succinic-acid production in yeast was an active field well before 2009, but this sub-limitation is the weakest link in the Combination C case and requires a separate reference (e.g., a reductive-TCA-pathway engineering reference) that I did not retrieve.
• Yield > 0.3 g/g glucose — as noted, below the stoichiometric ceiling; a result-effective variable; routine optimization (MPEP 2144.1(a)). Notably, the patent's own premise is that ED raises product yield by lowering ATP/biomass yield, so a POSA would expect the yield to move in exactly this direction.
• Six-carbon and five-carbon sugars, xylose/xylulose, biomass feedstocks — grounded in Jeffries (2006) and Deshpande (1992), both on this patent's own citation record (Tier 2).
• Containerize / ship — expressly taught by WO 95/25799 ("facilitates use and also shipping"). A claim reciting shipping a container of the product is a patent-eligibility-flavoured afterthought with no § 103 weight.

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8. Anticipated counterarguments, and how they fare

8.1 "No reasonable expectation of success — the enzyme doesn't work in yeast"

This is the strongest nonobviousness argument available, and it is backed by real evidence:

Benisch, F. & Boles, E. (2014), J. Biotechnol. 171:45-55, "The bacterial Entner-Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron-sulfur cluster enzyme 6-phosphogluconate dehydratase."

Benisch reports that KDPG aldolase from E. coli expressed well in the yeast cytosol, but that no PGDH tested achieved activity high enough to support growth of S. cerevisiae via the EDP, because the bacterial enzyme is a [4Fe-4S] iron-sulfur cluster protein and yeast could not supply the cluster. Attempts to improve Fe-S availability, make Leu1-PGDH fusions, or localize PGDH to mitochondria all failed. Source: https://pubmed.ncbi.nlm.nih.gov/24333129/.

But this cuts both ways, and the cutting edge is against the patentee:

1. It is post-dated by five years. A § 103 analysis is conducted from the POSA's perspective as of the 2009-07-09 priority date. A 2014 paper is not prior art and cannot supply the prima facie case; it is only relevant to the patentee's unexpected-results / non-enablement narrative. And it is evidence of what the POSA later learned, not of what the POSA would have expected in 2009.
2. The same paper concedes the 1995 reference. Verbatim, from the ScienceDirect article text I retrieved: "Albeit a former patent application of Lancashire et al. (1998) describes the integration of the EDP to bypass glycolysis in S. cerevisiae, no scientific publication is known up to now demonstrating a functional EDP in S. cerevisiae." The authors' framing — the gap in the literature, not the gap in the art — is significant. For § 103, a patent publication is prior art whether or not a journal article later confirmed it. A POSA in 2009 reading WO 95/25799's express statement that transformed yeast "is capable of expressing" PGD and KGA "whereby, in fermentation, a sugar can be converted to pyruvate via the Entner-Doudoroff pathway," combined with Zymomonas mobilis's well-known operation of a complete ED pathway anaerobically (De Graaf et al., 1999), would have had a reasonable expectation of success. The later failure of one particular enzyme-sourcing strategy does not retroactively eliminate that expectation — and, more importantly, a hypothesis that later proves "difficult" supports obvious-to-try, not nonobviousness, where the POSA is optimizing a finite set of identified candidate enzymes.
3. Related post-filing work confirms routine constructability. Morita et al. (2017), J. Biosci. Bioeng. 124(3):263-270, report successfully constructing the ED pathway in S. cerevisiae by expressing 6PGD and KDPGA (growth improvement in a gnd1Δ background; slight ¹³C flux into ED). Again post-filing, so not prior art — but it corroborates that the concept was constructible and that the art was routinely pursuing it.
4. A "no expectation of success" argument also undermines the patent's own enablement story. If the POSA could not have expected the ED pathway to function in yeast in 2009, the patentee's own claims to a functional aerobic-EM/anaerobic-ED switch invite an § 112 written-description/enablement challenge.

Net: the "unpredictability" argument is real but asymmetric — it is stronger as a validity-attack vector than as a validity-defence, because the express teaching of WO 95/25799 and the naturally-occurring anaerobic ED pathway of Zymomonas supply the expectation of success that KSR requires.

8.2 "Teaching away"

I found no teaching away. The nearest candidate is that the field generally regarded EM glycolysis as the yeast norm — but WO 95/25799 expressly frames ED as the preferred route for alcohol production ("the present invention is based on the observation that the production of ethanol is not the raison d'être of a yeast cell"), and expressly teaches PFK elimination. That is a roadmap, not a discouragement. Likewise, Fuhrman et al.'s bacteriostasis finding is a caution about imbalance, not a discouragement from the pathway.

8.3 "Non-analogous art"

Unavailable. Both Families A and B sit squarely in yeast metabolic engineering for fermentation products.

8.4 The "nucleotide sequence identification tag" (barcode) limitation

If the granted claim 1 retains this element — and I flagged earlier that I could not confirm it did — it is the patent's most defensible hook, but it is a weak one. The use of short, unique, non-coding sequence tags in a nucleic acid construct to identify a strain or construct is a conventional labeling technique in molecular biology, and reciting one selected from a list of six sequences amounts to selection among a finite number of predictable options (MPEP 2144.1(a); KSR). Unless the tag is functionally coupled to a claimed biological result, an examiner would likely treat it as obvious. Verify whether it survived to grant before investing argument in it.

8.5 Family C — the succinic-acid limitation

As noted in §7, I did not verify a reference teaching succinic-acid production from an ED-rewired Saccharomyces, and I will not fabricate one. This is the one limitation for which my obviousness conclusion is genuinely open and depends on art I did not retrieve.

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9. Secondary considerations (objective indicia)

Under Graham v. John Deere, the patentee may rebut a prima facie case with objective evidence. Assessing candidly:

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10. Summary conclusions and confidence

Additional flagged items that could change the picture:

• I could not retrieve the granted claim set verbatim, nor the PCT/US2010/041607 International Search Report and Written Opinion (mailed 2011-06-03) which would show precisely which references the examiner treated as X/Y-category art against these claims. That ISR is the single highest-value document to pull next, followed by the U.S. file wrapper (office actions and any § 103 rejections and the applicant's rebuttals).
• I could not verify whether WO 95/25799 appears on the face of US 8,227,236's "References Cited." Its appearance in the related literature (Benisch et al. 2014 expressly identifying "Lancashire et al. (1998)") confirms it is the seminal reference in this exact space regardless — and, critically, prior art need not have been cited by the examiner or the applicant to invalidate a claim.
• I could not verify SEQ ID NO: 70 or the specific sequence listings against any prior-art sequence.

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11. Bottom line

The core inventive concept of US 8,227,236 — putting a bacterial Entner–Doudoroff pathway into Saccharomyces while shutting down Embden–Meyerhof glycolysis at phosphofructokinase, and switching between the two routes by fermentation conditions — was expressly taught 14 years before the priority date by WO 95/25799 A1 (Lancashire, Dickinson & Malloch; Whitbread & Co. / University College Cardiff), published 1995-09-28. That reference discloses every structural element of claim Family A except the added 6-phosphogluconolactonase activity, and it supplies the motivation, the construct architecture, the PFK-deletion rationale, and the alcohol-production purpose. The lactonase gap is filled by Hager, Calfee & Phibbs (2000), which identifies the P. aeruginosa pgl gene as encoding 6-phosphogluconolactonase and places it in the same hex regulon as the very ED genes WO 95/25799 teaches to transfer — an on-record reference for this patent, and a combination a POSA would have made both for chemical necessity (the lactone must be hydrolysed for PGD to have substrate) and for flux balance (Fuhrman et al. 1998 teach the toxicity of ED intermediate accumulation).

The cell-division-arrest claims are obvious over the union of thymidylate-synthase/cdc biology with oxygen-regulated yeast promoters, motivated by the conventional biomass-versus-product trade-off that the art itself articulates. The two-stage method claims to ethanol are obvious over WO 95/25799 plus standard fermentation practice; the succinic-acid variant is the one limitation for which I cannot complete the case on the art I retrieved.

The single strongest contrary argument is the demonstrated difficulty of expressing the [4Fe-4S] phosphogluconate dehydratase in yeast (Benisch & Boles 2014, J. Biotechnol. 171:45-55). I think it ultimately fails as a nonobviousness defence, for three reasons: (i) it post-dates the critical date by five years and therefore cannot inform the prima facie case; (ii) it confirms that the art was actively and routinely attempting exactly this, which is the KSR "obvious to try" fact pattern; and (iii) its own authors concede the 1995 Lancashire disclosure of the EDP-in-yeast construct, treating the gap as one of publication rather than of teaching. That same concession, however, is a two-edged sword against the patentee's written-description position.

Confidence on the WO 95/25799 content: high (verified from Google Patents and Espacenet abstracts and body text this session). Confidence that the full § 103 case can be closed on the record: moderate, because I could not retrieve the granted claims, the front-page reference list in full, or the PCT search report. Anyone relying on this should pull those three documents before finalizing.