Table 1.

Heterologous expression of hydrogenases in cyanobacteria and green algae.

Host	Implemented Strategy	Outcome and conclusion/Key findings	Reference
Cyanobacteria
Synechococcus elongatus PCC 7942	IPTG- inducible heterologous expression of [FeFe] hydrogenases (HydA) and psba1 controlled expression of [FeFe] maturation machinery, in vivo	2.8 μmol H2 · h−1· mg Chla−1, ferredoxin-hydrogenases compatibility is crucial for efficient coupling to native redox metabolism	Ducat et al. (2011)
Anabaena/Nostoc PCC 7120	Heterologous expression of [FeFe] hydrogenase operon from Shewanella oneidensis MR-1 controlled by heterocyst-specific promoter phetN, deletion of native NiFe hydrogenases, in situ	1 μmol H2 · h−1· mg Chla−1in in situ experiments (supplemented with dithionite and MV)	Gärtner et al. (2012)
Anabaena/Nostoc PCC 7120	Heterologous expression of [FeFe] hydrogenases (HydA) and maturation machinery in heterocyst controlled by late-phase promoter, cyanoglobin expression under control of patB promotor in vivo	400 μmol H2 · mg Chla−1 under argon atmosphere 13 μmol H2 · mg Chla−1 under aerobic conditions O2 level actively lowered by cyanoglobin GlbN in heterocyst, competition of nitrogenase and hydrogenase for reduced ferredoxin	Avilan et al. (2018)
Synechocstis PCC 6803	Expression of fusion protein PsaD-HoxYH (native NiFe hydrogenases and PSI subunit), deletion of hoxEFUYH and psaD	17-fold increase in H2 concentration (546 μM) compared to WT. Higher temporary H2 production rates than WT. H2-uptake circumvented by avoiding backwards electron transfer to PSI. Limited rate of H2 produced from oxygenic photosynthesis	Appel et al. (2020)
Green algae
Chlorella sp. DT	Heterologous overexpression of clostridial [FeFe] hydrogenase (HydA) under control of two different promoters in aerobic and sulfur-supplied conditions	Native maturation of heterologous [FeFe] hydrogenase, H2 production 10-fold higher than in WT strain, not proportional increase with protein expression level	Chien et al. (2012)
Chlamydomonas reinhardtii	Expression of a Fd-HydA1 fusion protein under control of the psaD promoter	Enhanced electron flow from PSI, leading to 4.5-fold higher hydrogen production than WT at low protein expression levels. Increased oxygen tolerance of the [FeFe] hydrogenase, probably due to Fd protection	Eilenberg et al. (2016)
Chlamydomonas reinhardtii	Screening for Chlamydomonas reinhardtii mutants overexpressing a Fd-HydA1 fusion integrated either in nuclear genome (psaD promoter) or chloroplasts chromosome (psaA promoter)	Sustained H2 production that correlates linearly with active enzyme abundance. Enzyme showed 4.5-fold increase in hydrogenase activity compared to WT, even at high expression levels. Fd fusion limited hydrogenase activation, where up to 85% of the overexpressed hydrogenase chimera remained in the apo-form	Weiner et al. (2018)
Chlamydomonas reinhardtii	Expression of two different Fd-HydA1 chimeric proteins harboring a mutant Fd (D19A, D58A) with limited affinity towards FNR under a sulfur-limitation induced promoter	Chimeric protein expressed at comparable levels of HydA1 in WT. The Fd-HydA1 harboring a 25 aa linker exhibited better performance. Average production of 111.7 ± 3.4 ml · H2 L−1, a 4.6-fold increase in H2 production compared to WT	Xiong et al. (2021)
Chlamydomonas reinhardtii	Design of a PSI-hydrogenase chimera by. Fusing the stromal PsaC subunit to either HydA1/HydA2 hydrogenases and in vivo expression in a ΔpsaC Chlamydomonas reinhardtii mutant strain	Fusion chimeras were successfully activated by HydEFG maturases (>90% active protein), additionally, up to 60% of O2-inactivated PSI-HydA chimeras could be reactivated after reducing O2 concentration. Enhanced H2 production was observed, mainly derived from indirect PETC regulation, lowered LET and O2 production Average production of 14.0 ± 1.7 μmol H2 · h−1· mg Chla−1 (for PSI-HydA2 chimera, expressed at 7 times lower levels than WT PSI, leading to reduced PSII activity and enabling sustaining H2 production due to limited O2 buildup). PSI-HydA1 chimera exhibited only 50% of PSI-HydA2 turnover rate. Its expression were 5 times higher than PSI-HydA2, restoring close-to-WT levels of PSII activity an increased O2 production, leading to a 10-fold overall H2 production compared to PSI-HydA2 chimera	Kanygin et al. (2020, 2022)