Monday, December 3, 2018

BacterioFiles 364 - Polyproteins Promote Producing Pabulum

Nitrogenase enzyme
By Jjsjjsjjs - Own work,
CC BY-SA 3.0
This episode: Engineering other organisms to fix nitrogen by combining the required enzyme components into giant proteins that then get cut into the regular-sized subunit components!

Download Episode (10.5 MB, 11.5 minutes)

Show notes:
Microbe of the episode: Blastochloris sulfoviridis

Journal commentary (paywall)

Journal Papers:
Yang J, Xie X, Xiang N, Tian Z-X, Dixon R, Wang Y-P. 2018. Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology. Proc Natl Acad Sci 115:E8509–E8517.

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Episode outline:
  • Background: Nitrogen fixation amazing process
    • So far unique to microbes, and human ingenuity
    • Human process – Haber Bosch – uses a lot more energy
    • But microbe process also pretty energetic and tricky
  • Haber Bosch greatly expanded agricultural capacities
    • Made today's population possible
    • But uses a lot of energy, too much consumption of resources
  • Ideal would be creating cereal crops that can fix own nitrogen
    • Legumes interface with microbes that do it, but most other crops don't
    • Engineering interface is pretty tricky; reworking plant immune system and such
  • Engineering plants to produce nitrogenase enzymes possibly easier
    • But complicated enzyme system, multiple different subunits together, in specific proportions
    • And sensitive to oxygen
  • What’s new: Now, scientists publishing in the Proceedings of the National Academy of Sciences have developed a system of producing these enzymes in plants, with giant proteins that get cut into smaller pieces to function!
  • Strategy can help make sure that components will be made at similar levels
  • Methods: Using nif system from Klebsiella oxytoca
    • 1st studied relative levels of production of different proteins
    • Derived 4 clumps: NifHDKJ (main structural and accessory), NifENBY, USV, and FM
      • Help with folding, cofactor synthesis, etc
    • Left out some genes (TXWZQ) that don't seem essential, based on E. coli studies
  • Put together these clumps into giant genes
    • Individual proteins separated by processing site for virus enzyme
    • Tobacco Etch Virus protease, cuts peptides at particular sequence
      • Virus uses to produce its proteins efficiently
  • Tested first in E. coli; 1st try didn't work too well, not much activity
    • Found including J with structural proteins HDK inhibited activity
    • Changed some other genes around too
    • Went from 5% activity to 89%
  • Then saw if clumps worked together well
    • Not much reduction from 1st three, but USV messed it up, at least in some way
    • Considered putting WZ back in for more activity; W helped but not Z
  • Proportions of proteins seemed pretty good, esp multi-subunit ones
  • Finally, tested function of system overall in E. coli
    • Allowed bacteria to grow using only nitrogen gas as nitrogen source
  • Summary: Putting nitrogen-fixing system in organism that didn't have it before works more simply when combining small single genes into large polygenes that make large peptides that get chopped into individual components after translation
  • Applications and implications: Step toward nitrogen fixation more broadly, less fertilizer
  • Also good demonstration of system in general, though not first
    • Seems like each needs specific troubleshooting and finetuning
    • Might even need more once in plants or in each plant
  • Clarifications if necessary: Not in plants, but step toward
    • Gotta see if system would work in eukaryotes, then other challenges (oxygen)
  • What do I think: Advanced genetic engineering made easier by discoveries in nature
    • Virus that makes polyproteins
  • Individual genes allows more specific regulation, but sometimes grouping is more useful
  • In biology, there's a way for almost anything

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