Wim Vermaas

Biological hydrogen production: prospects and challenges

Hydrogen gas provides exceptional value as an energy carrier and industrial feedstock, but currently is produced entirely by reforming fossil fuels. Biological hydrogen production (BioH(2)), which offers the possibility of being renewable and carbon neutral, can be achieved by photosynthesis, fermentation, and microbial electrolysis cells. This review introduces the principles, advantages and challenges of each approach to BioH(2). Photosynthetic BioH(2) is the ultimate renewable source, since it directly uses inexhaustible resources: sunlight energy and electrons from H(2)O. However, it presents major technical challenges, particularly due to oxygen sensitivity. Fermentative BioH(2) offers a high production rate, but poor conversion efficiency from the organic substrate to H(2). The microbial electrolysis cell can achieve high conversion efficiency, but is an emerging technology.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand

The world’s crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques to Synechocystis sp. Strain PCC 6803

ABSTRACT The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis inSynechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and theSynechocystis β-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP andcrtB, respectively) were introduced intoSynechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, andcrtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtOgene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.

Succinate dehydrogenase and other respiratory pathways in thylakoid membranes of Synechocystis sp. strain PCC 6803: Capacity comparisons and physiological function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.

A cyanobacterial gene family coding for single-helix proteins resembling part of the light-harvesting proteins from higher plants

In the cyanobacterium Synechocystis sp. PCC 6803 five genes were identified with significant sequence similarity to regions of members of the eukaryotic chlorophyll a/b binding gene family (Cab family) and to hliA, a gene coding for a small high-light-induced protein in Synechococcus sp. PCC 7942. Four of these five genes are 174-213 bp in length and code for small proteins predicted to have a single transmembrane helix. The fifth Cab-like gene in Synechocystis sp. PCC 6803 is much longer and codes for a protein of which the N-terminal 80% resemble ferrochelatase but the C-terminal domain has similarity to Cab regions. The small genes were expressed preferentially in the absence of photosystem I, but gene expression was not significantly enhanced at moderately high light intensity. Therefore they were not designated as hli (high-light-induced) as was done for the Synechococcus sp. PCC 7942 homolog. Instead, the genes have been named scp, as the corresponding polypeptides of Synechocystis sp. PCC 6803 are small Cab-like proteins (SCP). The scpA gene, which codes for ferrochelatase with a C-terminal Cab-like extension, was interrupted by the insertion of a kanamycin-resistance cassette between the ferrochelatase and Cab-like gene domains. In the PS I-less background, interruption of scpA was found to lead to increased tolerance to high light intensity and to the requirement of a slightly higher light intensity to drive photosystem II electron transfer, suggestive of decreased light-harvesting efficiency in the absence of the C-terminal extension of ScpA. Immunodetection of ScpC and ScpD indicated that either or both accumulated in PS I-less strains. These proteins were also detected in bands of more than 45 kDa on denaturing gels, raising the possibility that they may occur as stable oligomers. The SCPs represent a new group of cyanobacterial proteins that, in view of their primary structure and response to deletion of photosystem I, are likely to be involved in transient pigment binding.

In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (≈0.03-μm3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the short-wavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.

EPR measurements on the effects of bicarbonate and triazine resistance on the acceptor side of Photosystem II

CO2 depletion leads to an approximately 10‐fold increase in the light‐induced EPR signal at g = 1.82, attributed to the QA − · Fe2+ complex, in Photosystem II‐enriched thylakoid membrane fragments. Upon reconstitution with HCO3 −the signal decreases to the size in control samples. The split pheophytin− signal is broader in control or reconstituted than in CO2‐depleted samples. It is concluded that HCO2 − strongly influences the localization and conformation of the QA − · Fe+ complex. The QA − · Fe2+ and split pheophytirr− EPR signals from triazine‐resistant Brassica napus were virtually identical to those from triazine‐susceptible samples, indicating that the change in the 32‐kDa azidoatrazine‐binding protein does not lead to a confonnational change of the Qa − · Fe2+ complex.

Evolution of heliobacteria: Implications for photosynthetic reaction center complexes.

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein show rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to the core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein shows rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

Multiple deletions of small cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: COnsequences for pigment biosynthesis and accumulation

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway. Upon deletion of multiple SCPs, neither 77 K fluorescence emission properties of phycobilisomeless thylakoids from the PS I-less/PS II-less strain nor the energy trapping efficiency of PS II were affected, indicating that under steady-state conditions SCPs do not bind much chlorophyll and do not serve as PS II antenna. Under conditions where protochlorophyllide reduction and thus chlorophyll synthesis were inhibited, chlorophyll disappeared quickly in a mutant lacking all five SCPs. This implies a role of SCPs in stabilization of chlorophyll-binding proteins and/or in reuse of chlorophylls. Under these conditions of inhibited reduction of protochlorophyllide, the accumulation kinetics of this intermediate were greatly altered in the absence of the five SCPs. This indicates an alteration of tetrapyrrole biosynthesis kinetics by SCPs. Based on this and other evidence, we propose that SCPs bind carotenoids and transiently bind chlorophyll, aiding in the supply of chlorophyll to nascent or reassembling photosynthetic complexes, and regulate the tetrapyrrole biosynthesis pathway as a function of the demand for chlorophyll.