Synthetic rewiring of Chlamydomonas reinhardtii to improve biological H2 production

Venkanna D. Synthetic rewiring of Chlamydomonas reinhardtii to improve biological H2 production. Bielefeld: Universität Bielefeld; 2018.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. 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. 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. 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

Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in <i>Chlamydomonas reinhardtii</i>

Venkanna D, Südfeld C, Baier T, et al. Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in &lt;i&gt;Chlamydomonas reinhardtii&lt;/i&gt;. Frontiers in Plant Science. 2017;8: 1347.The protein superfamily of short-chain dehydrogenases/reductases (SDR), including members of the atypical type (aSDR), covers a huge range of catalyzed reactions and in vivo substrates. This superfamily also comprises isoflavone reductase-like (IRL) proteins, which are aSDRs highly homologous to isoflavone reductases from leguminous plants. The molecular function of IRLs in non-leguminous plants and green microalgae has not been identified as yet, but several lines of evidence point at their implication in reactive oxygen species homeostasis. The Chlamydomonas reinhardtii IRL protein IFR1 was identified in a previous study, analyzing the transcriptomic changes occurring during the acclimation to sulfur deprivation and anaerobiosis, a condition that triggers photobiological hydrogen production in this microalgae. Accumulation of the cytosolic IFR1 protein is induced by sulfur limitation as well as by the exposure of C. reinhardtii cells to reactive electrophile species (RES) such as reactive carbonyls. The latter has not been described for IRL proteins before. Over-accumulation of IFR1 in the singlet oxygen response 1 (sor1) mutant together with the presence of an electrophile response element, known to be required for SOR1-dependent gene activation as a response to RES, in the promoter of IFR1, indicate that IFR1 expression is controlled by the SOR1-dependent pathway. An implication of IFR1 into RES homeostasis, is further implied by a knock-down of IFR1, which results in a diminished tolerance toward RES. Intriguingly, IFR1 knock-down has a positive effect on photosystem II (PSII) stability under sulfur-deprived conditions used to trigger photobiological hydrogen production, by reducing PSII-dependent oxygen evolution, in C. reinhardtii. Reduced PSII photoinhibition in IFR1 knock-down strains prolongs the hydrogen production phase resulting in an almost doubled final hydrogen yield compared to the parental strain. Finally, IFR1 knock-down could be successfully used to further increase hydrogen yields of the high hydrogen-producing mutant stm6, demonstrating that IFR1 is a promising target for genetic engineering approaches aiming at an increased hydrogen production capacity of C. reinhardtii cells

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

BACKGROUND: High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. RESULTS: To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. CONCLUSIONS: The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation

An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

Abstract Background High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. Results To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. Conclusions The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation.</p

Neuartige Siliziumgele zur Einschlussimmobilisierung von Mikroalgen

Homburg SV, Venkanna D, Doebbe A, Kruse O, Patel A. Neuartige Siliziumgele zur Einschlussimmobilisierung von Mikroalgen. In: Special Issue: ProcessNet‐Jahrestagung und 31. DECHEMA‐Jahrestagung der Biotechnologen 2014. Chemie Ingenieur Technik. Vol 86. Wiley; 2014: 1536-1536

Entrapment and growth of <i>Chlamydomonas reinhardtii</i> in biocompatible silica hydrogels

Homburg SV, Venkanna D, Kraushaar K, Kruse O, Kroke E, Patel A. Entrapment and growth of &lt;i&gt;Chlamydomonas reinhardtii&lt;/i&gt; in biocompatible silica hydrogels. Colloids and Surfaces B: Biointerfaces. 2018;173:233-241.In this work, we aimed at improved viability and growth of the microalga *Chlamydomonas reinhardtii* in transparent silica hydrogels based on low-ethanol, low-sodium and low-propylamine synthesis. Investigation into replacement of conventional base KOH by buffers dipotassium phosphate and tris(hydroxymethyl)aminomethane along with increased precursor concentrations yielded an aqueous synthesis route which provided a gelation within 10 min, absorptions below 0.1 and elastic moduli of 0.04-4.23 kPa. The abrasion resistance enhanced by 41 % compared to calcium alginate hydrogels and increased to 70-85 % residual material on addition of chitosan. Entrapment of microalgae in low-sodium and low-propylamine silica hydrogels maintained the PSII quantum yield above 0.3 and growth rates of 0.23 ± 0.01 d-1, similarly to cells entrapped in calcium alginate. These promising results pave the way for the entrapment of sensitive, photosynthetically active and growing cells for in robust biotechnological applications

Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii

The protein superfamily of short-chain dehydrogenases/reductases (SDR), including members of the atypical type (aSDR), covers a huge range of catalyzed reactions and in vivo substrates. This superfamily also comprises isoflavone reductase-like (IRL) proteins, which are aSDRs highly homologous to isoflavone reductases from leguminous plants. The molecular function of IRLs in non-leguminous plants and green microalgae has not been identified as yet, but several lines of evidence point at their implication in reactive oxygen species homeostasis. The Chlamydomonas reinhardtii IRL protein IFR1 was identified in a previous study, analyzing the transcriptomic changes occurring during the acclimation to sulfur deprivation and anaerobiosis, a condition that triggers photobiological hydrogen production in this microalgae. Accumulation of the cytosolic IFR1 protein is induced by sulfur limitation as well as by the exposure of C. reinhardtii cells to reactive electrophile species (RES) such as reactive carbonyls. The latter has not been described for IRL proteins before. Over-accumulation of IFR1 in the singlet oxygen response 1 (sor1) mutant together with the presence of an electrophile response element, known to be required for SOR1-dependent gene activation as a response to RES, in the promoter of IFR1, indicate that IFR1 expression is controlled by the SOR1-dependent pathway. An implication of IFR1 into RES homeostasis, is further implied by a knock-down of IFR1, which results in a diminished tolerance toward RES. Intriguingly, IFR1 knock-down has a positive effect on photosystem II (PSII) stability under sulfur-deprived conditions used to trigger photobiological hydrogen production, by reducing PSII-dependent oxygen evolution, in C. reinhardtii. Reduced PSII photoinhibition in IFR1 knock-down strains prolongs the hydrogen production phase resulting in an almost doubled final hydrogen yield compared to the parental strain. Finally, IFR1 knock-down could be successfully used to further increase hydrogen yields of the high hydrogen-producing mutant stm6, demonstrating that IFR1 is a promising target for genetic engineering approaches aiming at an increased hydrogen production capacity of C. reinhardtii cells