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Methods and Systems for Methylotrophic Production of Organic Compounds

Inactive Publication Date: 2015-11-05
GINKGO BIOWORKS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

The present invention provides systems and methods for efficiently producing carbon-based products, such as fuels and sugars, without using traditional methods that require petroleum and produce toxic by-products. The invention introduces carbon product biosynthetic pathways into organisms that can then efficiently produce these products. Additionally, the invention provides an engineered methylotrophic organism that has a reduced growth rate on electrolytically generated C1 compound relative to a non-evolved methylotrophic organism. This allows for more efficient production of carbon-based products. Overall, the invention promotes the use of renewable energy and the avoidance of harmful petroleum-derived by-products in the production of chemicals.

Problems solved by technology

However, the algae-based production of carbon-based products of interest relies on the relatively inefficient process of photosynthesis to supply the energy needed for production of organic compounds from carbon dioxide [Larkum, 2010].
Moreover, commercial production of carbon-based products of interest using photosynthetic organisms relies on reliable and consistent exposure to light to achieve the high productivities needed for economic feasibility; hence, photobioreactor design remains a significant technical challenge [Morweiser, 2010].
Most existing, naturally-occuring methylotrophs are poorly suited for industrial bio-processing and have therefore not demonstrated commercial viability for this purpose.
In addition, techniques for genetic manipulation (homologous recombination, transformation or transfection of nucleic acid molecules, and recombinant gene expression) are inefficient, time-consuming, laborious or non-existent.

Method used

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  • Methods and Systems for Methylotrophic Production of Organic Compounds
  • Methods and Systems for Methylotrophic Production of Organic Compounds
  • Methods and Systems for Methylotrophic Production of Organic Compounds

Examples

Experimental program
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Effect test

example 1

Optimization of Growth Medium for Paracoccus sp. when Using Formate as the C1 Compound

[0257]Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534 were obtained from the American Type Culture Collection (ATCC).

[0258]Strains were tested for the ability to grow aerobically on sodium formate as a sole source of carbon and / or energy using MOPS minimal medium (Teknova, Inc.) with sodium formate as a sole carbon source at 37C. Unlike previous media used to evaluate the formate-dependent growth of Paracoccus, this medium contains defined levels of trace elements molybdenum, boron, copper, zinc, manganese, and other trace metals.

[0259]Growth was conducted in various high-throughput machinery capable of monitoring growth by light scattering at 600 nm, including a Gemini SpectraNax plate reader (Molecular Devices, Inc.), a Tecan M3000 plate reader (Tecan, Inc.), and a BioLector device (m2p-labs, Inc.). For the BioLector, the CO2 gas c...

example 2

Automatable Protocol for Conjugative Transfer of Plasmids from E. coli Donors to Paracoccus sp

[0267]E. coli strain 517-1 was obtained from the Yale E. coli Genetic Stock Center. Paracoccus denarificans PD1222 was obtained from Stephen Spiro (University of Texas at Dallas). E. coli S17-1 strain is tra+, meaning it is able to mobilize for conjugative transfer those plasmids harboring a mob+genotype. Plasmid pDIY313K, obtained from Dariusz Bartosik (University of Warsaw, Poland) and described by his laboratory [J Microbiol Methods, 2011, 86(2):166-74, DOI: 10.1016 / j.mimet.2011.04.016], and introduced into E. coli 517-1 by standard methods.

[0268]E. coli S17-1 was grown on Luria broth with carbenicillin overnight. Paracoccus versutus ATCC 25364 was grown overnight on MOPS minimal medium (Teknova, Inc.) with 50 mM sucrose and 40 mM sodium nitrate.

[0269]The next day, E. coli S17-1 was subcultured in antibiotic-free Luria broth for >4 hr. Paracoccus strains were subcultured in identical MOP...

example 3

Genome Sequencing of Paracoccus Strains

[0272]Genomic DNA was isolated from Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534 using a Wizard Genomic DNA Isolation Kit (Promega, Inc.). The resulting DNA samples were fragmented and converted to paired-end libraries for whole-genome shotgun sequencing on a 454 pyrosequencing platform (Roche, Inc.).

[0273]For Paracoccus denitrificans, 37,585,886 paired reads, each 100 nt in length, were obtained. This represents approximately 3.7 gigabases of sequence data, or approximately 730-fold coverage of the 5.2 megabase genome of Paracoccus denitrificans PD1222 (Genbank accession numbers CP000489, CP000490, and CP000491 for chromosome 1, chromosome 2, and a 653,815 bp megaplasmid, respectively). Reads were assembled first by de-novo assembly and second by mapping the de-novo contigs to the published PD1222 genome.

[0274]The resulting reads could be assembled into a crude whole-genome a...

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Abstract

The present disclosure identifies pathways, mechanisms, systems and methods to confer production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in engineered and / or evolved methylotrophs such that these organisms efficiently convert C1 compounds, such as formate, formic acid, formaldehyde or methanol, to organic carbon-based products of interest, and in particular the use of organisms for the commercial production of various carbon-based products of interest.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to and the benefit of U.S. Provisional Application No. 61 / 734,472 filed Dec. 7, 2012, the disclosure of which is incorporated herein by its entirety.STATEMENT REGARDING GOVERNMENT LICENSE RIGHTS[0002]This invention was made with government support under contract number DE-AR0000091 awarded by U.S. Department of Energy, Office of ARPA-E. The government has certain rights in the invention.TECHNICAL FIELD[0003]The invention relates to systems, mechanisms and methods to confer production of carbon-based products to a methylotroph or a methylotrophic organism to efficiently convert C1 compounds into various carbon-based products, and in particular the use of such organism for the commercial production of various carbon-based products of interest. The invention also relates to systems, mechanisms and methods to confer additional and / or alternative pathways for energy conversion, methylotrophy and / or carbon fixat...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12P7/40C12P7/16C12N15/74C12N1/20
CPCC12N15/52C12N15/74C12P7/40C12P7/16C12N1/20C12N1/36Y02E50/10
Inventor SHETTY, RESHMA P.FISCHER, CURT P.
Owner GINKGO BIOWORKS INC
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