Recombinantly engineered diazotrophs for whole cell hydrocarbon production and methods for making and using them

Patent metadata

Field	Value
Publication number	US-2017306291-A1
Application number	US-201515536653-A
Country	US
Kind code	A1
Filing date	Dec 17, 2015
Priority date	Dec 18, 2014
Publication date	Oct 26, 2017
Grant date	—

Abstract

In alternative embodiments, provided are genetically or recombinantly engineered nitrogen-fixing, nitrogenase expressing bacteria capable of enzymatically synthesizing hydrocarbons, and methods for making and using them. In alternative embodiments, provided are genetically or recombinantly engineered nitrogen-fixing, nitrogenase expressing bacteria including nitrogen-fixing diazotrophs such as nitrogen-fixing bacteria of the family Pseudomonadaceae, or the genus Azotobacter, for the whole cell synthesis of hydrocarbons and carbon-carbon bonds. In alternative embodiments, nitrogen-fixing, nitrogenase-expressing bacteria used to practice the invention are genetically or recombinantly engineered to express an exogenous nitrogenase express more endogenous nitrogenase or have increased nitrogenase, activity. In alternative embodiments, nitrogen-fixing, nitrogenase-expressing bacteria used to practice the invention are genetically or recombinantly engineered to lack or have decreased molybdenum transporter activity. In alternative embodiments, provided are culture systems, fermenters and bioreactors using nitrogen-fixing, nitrogenase-expressing bacteria for enzymatically synthesizing hydrocarbons.

First claim

1 . A nitrogen-fixing, nitrogenase-expressing diazotroph bacteria capable of enzymatically synthesizing hydrocarbons genetically or recombinantly engineered:
to lack or have decreased molybdenum transporter activity.

2 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , wherein the bacteria are genetically or recombinantly engineered to express an exogenous nitrogenase, to express more endogenous nitrogenase, or to have increased nitrogenase activity, or any combination thereof.

3 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , further comprising deletion of or decreasing the activity of a gene or genes involved in the inhibition of nitrogenase by ammonia, wherein optionally the gene whose activity is deleted or decreased is Nitrogen fixation-L (NifL),
and optionally further comprising overexpressing nifA.

4 . A method for making a nitrogen-fixing, nitrogenase-expressing diazotroph bacteria capable of enzymatically synthesizing hydrocarbons, comprising genetically or recombinantly engineering the bacteria to lack or have decreased molybdenum transporter activity,
wherein optionally the lack or decreased molybdenum transporter activity is achieved by deletion of one or more, or all, molybdenum transporter genes in the bacteria, or inhibition of molybdenum transporter message (transcript) generation or expression, or inhibition of or decrease in molybdenum transporter protein expression, wherein optionally the molybdenum transporter gene comprises an Azotobacter vinelandii modA 1 , or a molybdenum transporter-periplasmic molybdate-binding protein encoding gene, or a molybdenum ATP-Binding Cassette (ABC) transporter encoding gene, and optionally the molybdenum transporter polypeptide comprises a molybdenum ATP-Binding Cassette (ABC) transporter protein, and optionally the nitrogen-fixing, nitrogenase-expressing diazotroph bacteria comprise bacteria of the family Pseudomonadaceae, or the genus Azotobacter, or Azotobacter vinelandii, and optionally the nitrogenase comprises a vanadium nitrogenase, or an Azotobacter vanadium nitrogenase or an Azotobacter vinelandii vanadium nitrogenase.

5 . The method of claim 4 , wherein the bacteria are genetically or recombinantly engineered to express an exogenous nitrogenase, to express more endogenous nitrogenase, or to have increased nitrogenase activity, or any combination thereof,
wherein optionally the exogenous or endogenous nitrogenase comprises a vanadium nitrogenase, or an Azotobacter vanadium nitrogenase or an Azotobacter vinelandii vanadium nitrogenase, and optionally the exogenous nitrogenase and/or endogenous nitrogenase is modified (e.g., by site-directed or oligonucleotide-directed mutagenesis (including cassette mutagenesis, PCR site-directed mutagenesis, Cre-Lox recombination and whole plasmid mutagenesis), site-specific recombination, or mutagenesis in vivo or in vitro): to have increased enzymatic activity or velocity (Vmax); to be resistant to inhibition by ammonia; to have an enlarged active site to allow the formation of larger hydrocarbons or to allow the formation of larger hydrocarbons at higher efficiency; or, any combination thereof, and optionally the exogenous nitrogenase comprises an affinity tag, wherein optionally the affinity tag comprises: a CREB binding protein (CBP); an avidin or a streptavidin, a biotin or a desthiobiotin, a maltose binding protein (MBP); a glutathione-S-transferase (GST); a poly(His) tag; polyanionic amino acids; a FLAG-tag; or, any combination thereof.

6 . The method of claim 4 , further comprising deletion of or decreasing the activity of a gene or genes involved in the inhibition of nitrogenase by ammonia, wherein optionally the gene whose activity is deleted or decreased is Nitrogen fixation-L (NifL),
and optionally further comprising overexpressing nifA.

7 . A whole cell method for enzymatically synthesizing hydrocarbons, comprising contacting a nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , with a carbon-containing compound under conditions suitable to enzymatically form a carbon-carbon bond-comprising product compound, or a plurality of carbon-carbon bond-comprising product compounds, in the bacteria.

8 . The whole cell method of claim 7 , wherein the carbon-containing compound comprises: a carbon monoxide (CO); a carbon dioxide (CO 2 ); or a C 2 , C 3 , C 4 , or C 5 comprising compound, and optionally the carbon-containing compound is input, or “fed” to the nitrogen-fixing, nitrogenase-expressing diazotroph bacteria as a gas.

9 . The whole cell method of claim 7 , wherein the carbon-carbon bond-comprising product or products comprises or is a C 2 , C 3 , C 4 , or C 5 product, or the carbon-carbon bond-comprising product comprises or contains at least one double bond, or the carbon-carbon bond-comprising product or products comprise or are methane, ethane, ethene, propane or butane.

10 . The whole cell method of claim 7 , wherein the contacting comprises conditions comprising high oxygen, low ammonia, or high oxygen and low ammonia.

11 . The whole cell method of claim 7 , wherein the nitrogen-fixing, nitrogenase-expressing diazotroph bacteria are cultured, or in a cultured media, or in a bioreactor, and optionally the carbon-containing compound is input or “fed” to the culture, culture media or bioreactor as a gas.

12 . The whole cell method of claim 7 , further comprising harvesting, purifying and/or isolating the carbon-carbon bond-comprising product compound.

13 . A bioreactor or fermenter comprising: a culture or a liquid system comprising a nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 .

14 . The bioreactor or fermenter of claim 13 , further comprising an inlet configured to provide a carbon-containing compound to the culture or liquid system in an amount effective to allow a nitrogenase in the nitrogen-fixing, nitrogenase-expressing diazotroph bacteria produce the carbon-carbon bond-comprising product compound, and optionally further comprising an outlet configured to remove the carbon-carbon bond-comprising product compound.

15 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , wherein the lack or decreased molybdenum transporter activity is achieved by deletion of one or more, or all, molybdenum transporter genes in the bacteria, or inhibition of molybdenum transporter message (transcript) generation or expression, or inhibition of or decrease in molybdenum transporter protein expression.

16 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , wherein the molybdenum transporter gene comprises an Azotobacter vinelandii modA 1 , or a molybdenum transporter-periplasmic molybdate-binding protein encoding gene, or a molybdenum ATP-Binding Cassette (ABC) transporter encoding gene.

17 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , wherein the molybdenum transporter polypeptide comprises a molybdenum ATP-Binding Cassette (ABC) transporter protein.

18 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , wherein the nitrogen-fixing, nitrogenase-expressing diazotroph bacteria comprise bacteria of the family Pseudomonadaceae, or the genus Azotobacter, or Azotobacter vinelandii.

19 . The nitrogen-fixing, nitrogenase-expressing diazotroph bacteria of claim 1 , wherein the nitrogenase comprises a vanadium nitrogenase, or an Azotobacter vanadium nitrogenase or an Azotobacter vinelan

Assignees

• Univ California

Inventors

• Ribbe Markus Walter
• Hu Yilin
• Rebelein Johannes

Classifications

• C12F3/04

  Recovery of volatile fermentation products from carbon dioxide · CPC title

• C12Q1/04

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• C12P3/00

  Preparation of elements or inorganic compounds except carbon dioxide {(recovery of carbon dioxides as by-products C12F3/02)} · CPC title

• C12P5/023

  Methane · CPC title

• C12P5/026

  Unsaturated compounds, i.e. alkenes, alkynes or allenes · CPC title