Transgenic microorganisms and synthesis of piperazic acid, piperazic acid containing products, and derivatives thereof
Inventors
Van Dyke-Blodgett, Joshua • Hu, Yifei
Assignees
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Publication Number
US-12577597-B2
Publication Date
2026-03-17
Expiration Date
Abstract
Among the various aspects of the present disclosure is the provision of a biological and biochemical production of piperazic acid derived from the newly discovered production pathway for L-piperazic acid. One aspect of the present disclosure includes a transgenic microorganism (e.g., bacteria) engineered to accumulate piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product. Another aspect of the present disclosure includes biochemical and biological methods for producing piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product. Another aspect of the present disclosure includes compositions and methods of using isotopically labeled piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.
Core Innovation
The invention provides a transgenic microorganism transformed with an artificial DNA construct that includes a promoter, a first polynucleotide and a second polynucleotide operably associated in a 5′ to 3′ direction of transcription, and a transcriptional termination sequence. The transgenic microorganism expresses the first and second polypeptides from the first and second polynucleotides, respectively, and overexpresses the first and second polypeptides compared to a microorganism not comprising the artificial DNA construct.
The first polynucleotide encodes a first polypeptide having L-Ornithine N5 hydroxylase activity. The second polynucleotide encodes a second polypeptide having at least one of L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity, and the microorganism accumulates increased levels of piperazic acid (Piz) produced from L-Ornithine compared to a microorganism not comprising the artificial DNA construct.
The described product scope extends to Piz-containing products, including sanglifehrin and related sanglifehrin A-D, piperazimycins, lydiamycins, dehydropiperazic acid, chloropiperazic acid, and hydroxypiperazic acid. The document further describes embodiments involving a compound of formula (I) with defined substituents R1–R5 and optional substitutions on the Piz-containing product scaffold, and host-related embodiments describe engineered microorganisms capable of accumulating L-Piz.
Claims Coverage
The claims coverage centers on one independent claim directed to a transgenic microorganism and an artificial DNA construct that overexpresses two specific enzyme activities to accumulate increased piperazic acid (Piz) from L-Ornithine. Dependent claims refine the microorganism type and constrain the output to particular Piz-containing products, including specific product types and a formula (I) substituent-defined compound class.
Transgenic microorganism with artificial DNA construct for enzyme overexpression
A transgenic microorganism transformed with an artificial DNA construct consisting of a promoter, a first polynucleotide encoding a first polypeptide with L-Ornithine N5 hydroxylase activity, a second polynucleotide encoding a second polypeptide with at least one of L-Ornithine N5 cyclase activity or L-Ornithine N5 dehydratase activity, and a transcriptional termination sequence, wherein the microorganism expresses the first and second polypeptides and overexpresses them compared to a microorganism not comprising the artificial DNA construct.
Increased accumulation of piperazic acid (Piz) from L-Ornithine
A transgenic microorganism that accumulates increased levels of piperazic acid (Piz) produced from L-Ornithine compared to a microorganism not comprising the artificial DNA construct.
Piz-containing product restricted to specific product types
A transgenic microorganism producing a Piz-containing product selected from dehydropiperazic acid, chloropiperazic acid, and hydroxypiperazic acid.
Piz-containing product comprising a compound of formula (I)
A transgenic microorganism producing a Piz-containing product comprising a compound of formula (I) with substituents R1–R5 defined by specified options and optional substitutions.
The claim coverage is directed to engineering a transgenic microorganism to express a first polypeptide with L-Ornithine N5 hydroxylase activity and a second polypeptide with at least one of L-Ornithine N5 cyclase or L-Ornithine N5 dehydratase activity, such that the microorganism accumulates increased Piz from L-Ornithine. Dependent refinements narrow the produced Piz-containing products to specific product types and to compounds defined by formula (I) substituent constraints.
Stated Advantages
Increases levels of piperazic acid (Piz) produced from L-Ornithine compared to a microorganism not comprising the artificial DNA construct.
Provides Piz-containing products that can serve as antibacterial, antiviral, immunomodulatory, and anticancer leads.
Provides Piz-containing products that are described as chronic Hepatitis B antivirals.
Documented Applications
Piz-containing products are described as antibacterial leads.
Piz-containing products are described as antiviral leads, including chronic Hepatitis B antivirals.
Piz-containing products are described as immunomodulatory leads.
Piz-containing products are described as anticancer leads.
Production of Piz in a heterologous transgenic microorganism, including co-expression of pzbA/pzbB in Streptomyces lividans.
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