The green algae, Chlamydomonas reinhardtii is capable of harvesting sunlight to synthesize energy needs and also evolve hydrogen under stress conditions. Photolysis of water giving rise to protons and electrons as substrates for hydrogen producing enzyme (hydrogenase) backed by cellular respiration ensures establishment of anaerobiosis, which is a pre-requisite for hydrogen production. Due to the properties of hydrogen, it has gained widespread attention as a clean fuel which has also set forth a development in the Chlamydomonas community. Photobiological hydrogen production from green algae is currently not economically viable due to low efficeincy of light to H2 conversion. It has been shown that using a systematic approach towards genetically engineering strains can improve hydrogen yields. The aim of the following work was to improve hydrogen production via strain egineering. A previous study of transcriptome and metabolome of hydrogen producing culture served as a basis for the following work.<br />

In the following study C. reinhardtii wild type CC124, mutant stm6 and stm6glc4 were used. CC124 is routinely used as a hydrogen producing wild type strain whereas stm6 is a high hydrogen producing mutant with a manipulated state transition. stm6glc4 is a derivative of stm6 which is capable of taking up glucose and synthesize more starch that can fuel indirect pathway of hydrogen production. Hydrogen production was induced in air tight cultures of Chlamydomonas via sulfur deprivation. Potential target genes such as isoflavone reductase like protein (IFR1) and sulfite reductase (SIR1) were identified to be upregulated during H2 production. A comparison between a high hydrogen producer (stm6glc4) and its parental (low hydrogen producing wild type, CC406) showed that the expression of IFR1 was higher in the wild type. The role of IFR1 has been associated with stress tolerance in maize, rice, etc. but its function in Chlamydomonas is still unknown. SIR1 helps in sulfur assimilation process but by doing so it poses a competition for hydrogenase under sulfur deprived anaerobic hydrogen production conditions. Hence, a reverse genetic approach was adapted to counter these potential target genes.<br />

Artificial microRNA (amiRNA) was used to create IFR1 and SIR1 knockdowns. The phenotype of the knockdowns was studied and their positive implication on H2 production was established. IFR1 knockdown was first created in CC124 wild type strain. Two knockdown mutants IFR1-1 and IFR1-6 with 35% and 5% of control level proteins were identified and confirmed by western blots. The phenotype of IFR1 knockdown mutants was analyzed by performing growth studies such as sulfur and nitrogen starvation, high light stress, ROS and RES stress. An electrophile response element was found in the promoter region of IFR1 which is believed to be under the control of singlet oxygen resistant (sor1) protein. IFR1::YFP fusion protein was done to confirm the cytosolic localization of IFR1. The knockdown mutants were found to be sensitive to RES due to a perturbed RES homeostasis but interestingly showed a prolonged PSII activity (Fv/Fm) under sulfur depletion. The sustained PSII activity resulted in a prolonged phase of hydrogen production (~2fold more hydrogen). The contribution of electrons (~80%) for a direct pathway of hydrogen production from a sustained PSII activity was confirmed by applying a PSII inhibitor (DCMU). Based on these findings, benefits of IFR1 knockdown was extended to the mutant strain stm6. This again resulted in a sustained PSII activity which translated to ~70% more hydrogen production.<br />

The competition for electrons between hydrogenase and SIR1 was overcome by applying amiRNAs in the mutant stm6glc4. The amiRNAs were fused to a luciferase reporter to influence the knockdown screening. Two knockdown mutants sgh2 and sgh3 with ~20-30% reduced levels of SIR1 transcript were identified via RTqPCR and later confirmed by westernblot. The growth phenotype of the mutants were analyzed under photoautotrophic and photomixotrophic growths. The knockdown mutants were found to be slightly retarded in growth as compared to parental strain due to perturbed sulfur assimilation. Analysis of the hydrogen production phase showed that the knockdown mutants attained anaerobiosis faster than the parental strain and also had an increased rate of H2 production (~17-35% higher rates compared to parental strain). The mutants retained the ability to take up glucose which contributed to an increase in hydrogen produced via indirect pathway. Though the mutants were more susceptible to sulfur starvation, the higher H2 production rates boosted the overall H2 productivity by ~35-55%. This study showed that molecular target such as IFR1 and SIR1 could be manipulated genetically to improve biohydrogen production.