In vivo evolution of hydrogenases using a hydrogen-sensing system

A method for evolving a hydrogenase comprising:

• providing DNA comprising a HupSL promoter coupled with a reporter gene;
• transferring the DNA to a host microorganism lacking endogenous hydrogenase activity;
• transferring to the host microorganism one or more plasmids comprising a recombinant hydrogenase and associated assembly proteins;
• culturing the host microorganism under conditions permissive to production of H2 from the recombinant hydrogenase;
• measuring the level of the reporter gene product in vivo;
• comparing the level of production of H2 by the recombinant hydrogenase to the level of H2 produced by a wild-type hydrogenase cultured under the same conditions;
• and
• selecting a microorganism with increased H2 production over the H2 production by a microorganism having a wild-type hydrogenase;
• wherein production of H2 by the hydrogenase activates the hupSL promoter and the reporter gene product increases;
• and
• wherein the recombinant hydrogenase is evolved if the level of H2 produced is greater than that produced by the wild-type hydrogenase.

A method to evolve a hydrogenase comprising:

• (a) providing a library of recombinant hydrogenases;
• (b) transferring the recombinant hydrogenases to a pool of host microorganisms such that each host microorganism comprises one or more unique recombinant hydrogenases, each host microorganism further comprising hydrogenase support proteins, an H2 sensor, and a hupSL promoter coupled with a reporter gene;
• (c) culturing each of the host microorganisms under conditions permissive to production of H2 from the recombinant hydrogenases;
• (d) measuring the level of the reporter gene product in vivo produced by each microorganism;
• (e) comparing the level of production of H2 by each recombinant hydrogenase to the level of H2 produced by a wild-type hydrogenase cultured under the same conditions;
• and
• (f) selecting a microorganism with increased H2 production over the H2 production by a microorganism having a wild-type hydrogenase;
• (g) isolating the plasmid comprising the recombinant hydrogenase from the selected microorganism;
• (h) randomizing and/or shuffling the hydrogenase to generate a further library of recombinant hydrogenases;
• and
• (i) iterating as necessary steps (b) through (h) and stopping at step (g) when the recombinant hydrogenase fails to demonstrate further increased H2 production above the prior iteration;
• wherein production of H2 by the hydrogenase activates the hupSL promoter and the reporter gene product increases.

A method to identify an oxygen-resistant or a carbon dioxide-resistant hydrogenase, the method comprising:

• providing a foreign DNA comprising a hupSL promoter coupled with a reporter gene;
• transferring the foreign DNA to a host microorganism lacking endogenous hydrogenase activity and comprising a recombinant hydrogenase;
• culturing the host microorganism in the presence of oxygen or carbon monoxide levels inhibitory to H2 production by a wild-type hydrogenase;
• measuring the level of the reporter gene product in vivo;
• and
• comparing the level of production of H2 by the recombinant hydrogenase to the level of H2 produced by the wild-type hydrogenase cultured under the same conditions;
• wherein production of H2 by the hydrogenase activates the hupSL promoter and the reporter gene product increases;
• and
• wherein the recombinant hydrogenase is oxygen-resistant or carbon dioxide-resistant if the level of H2 produced is greater than that produced by the wild-type hydrogenase.

A method to evolve a H2-sensing system comprising:

• (a) providing a library of recombinant HupSL promoters and/or H2-sensing proteins;
• (b) transferring the recombinant HupSL promoters and/or H2-sensing proteins to a pool of host microorganisms such that each host microorganism comprises one or more unique recombinant H2-sensing systems, each host microorganism further comprising the lack of hydrogenases;
• (c) culturing each of the host microorganisms under conditions where H2 is not produced and H2 is not supplied;
• (d) measuring the level of the reporter gene product in vivo produced by each microorganism;
• (e) comparing the level of reporter gene product produced by each recombinant H2-sensing system;
• and,
• (f) selecting those microorganisms with the lowest reporter gene product;
• (g) re-culturing each of the selected host microorganisms under conditions where H2 is supplied exogenously;
• (h) re-measuring the level of the reporter gene product in vivo produced by each microorganism;
• (i) comparing the level of reporter gene product by each recombinant H2-sensing system;
• and,
• (j) selecting those microorganisms with the highest reporter gene product;
• (k) isolating the plasmid comprising the recombinant H2-sensing systems from the selected microorganisms;
• (l) randomizing and/or shuffling the recombinant H2-sensing systems to generate a further library of the recombinant H2-sensing systems;
• and
• (m) iterating as necessary steps (b) through (l) and stopping at step (k) when the recombinant H2-sensing systems fails to demonstrate further increased signal:noise ratio above the prior iteration.

A method for evolving a H2-sensing system comprising:

• providing DNA comprising a HupSL promoter coupled with a reporter gene;
• transferring the DNA to a host microorganism lacking endogenous hydrogenase activity;
• culturing the host microorganism under conditions where H2 is not produced and H2 is not added;
• measuring the level of the reporter gene product in vivo;
• selecting microorganisms with the lowest level of reporter gene product;
• re-culturing those selected microorganisms having the lowest basal level of reporter gene product in the presence of H2;
• selecting microorganisms with the highest level of reporter gene product;
• wherein the signal:noise ratio of the H2-sensing system is evolved if, in the absence of added H2, the level of H2 sensed is less than that sensed by the wild-type H2-sensing system, and/or, in the presence of added H2, the level of H2 sensed is greater than that sensed by the wild-type H2-sensing system.