Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase

Wells, M. J.; Mercer, James; Mott, Richard A; Pereira‐Medrano, Ana G.; Burja, Adam M.; Radianingtyas, Helia; Wright, Phillip C.

doi:10.1016/j.ymben.2011.01.004

Cited by 38 publications

(23 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After transforming the plasmid bearing the hydrogenase into Escherichia coli strain BL21ΔH 4 cells [8,32] and overnight colony outgrowth, individual colonies were picked and used to inoculate 1.7 mL of autoinduction media [33] in sterile 10 mL scintillation vials sealed with sterile natural rubber septa (Aldrich). Cultures were grown overnight (~24 hours) at 30°C, 200 rpm rotation.…”

Section: Methodsmentioning

confidence: 99%

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

et al. 2014

View full text Add to dashboard Cite

BackgroundIn order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys.ResultsAmong all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme.ConclusionsWe have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.

show abstract

Section: Methodsmentioning

confidence: 99%

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Expressing active A. macleodii hydrogenase in both E. coli and cyanobacteria requires the co-expression of at least nine additional maturation factors to properly assembly an active enzyme [17]. Due to the complex maturation requirements of [NiFe] hydrogenases, only a few other examples exist of heterologous expression in E. coli [18-20]. The dual expression system is ideal for rapidly generating variant hydrogenases using powerful E. coli genetic tools, quickly screening for desirable properties in E. coli , and confirming the persistence of the property in the final target cyanobacterial host.…”

Section: Introductionmentioning

confidence: 99%

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

et al. 2013

View full text Add to dashboard Cite

BackgroundPhotosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.ResultsWe adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.ConclusionsOur results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

show abstract

“…coli BL21 is a widely used expression system and has the ability to overexpress multi non-native genes, and has been metabolically engineered for the production of many substances like hydrogen and fatty acids (Akhtar and Jones, 2009;Lu et al, 2008;Wells et al, 2011). In this work, we constructed a recombinant E. coli BL21 as a safer host for heparosan production.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

Zhang

Liu

Teng

et al. 2012

Metabolic Engineering

View full text Add to dashboard Cite

Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase

Cited by 38 publications

References 51 publications

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

Contact Info

Product

Resources

About