The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms. The Sc2.0 project involved synthesis and debugging of 16 chromosomes, and a tRNA neochromosome. Here the authors descript the SynXVI project, accompanied by an analysis of how similar projects could operate with hindsight and newly available technologies, and lessons learned from Sc2.0.

Genome sequencing and assembly of the photosynthetic picoeukaryotic Picochlorum sp. SENEW3 revealed a compact genome with a reduced gene set, few repetitive sequences, and an organized Rabl-like chromatin structure. Hi-C chromosome conformation capture revealed evidence of possible chromosomal translocations, as well as putative centromere locations. Maintenance of a relatively few selenoproteins, as compared to similarly sized marine picoprasinophytes Mamiellales, and broad halotolerance compared to others in Trebouxiophyceae, suggests evolutionary adaptation to variable salinity environments. Such adaptation may have driven size and genome minimization and have been enabled by the retention of a high number of membrane transporters. Identification of required pathway genes for both CAM and C4 photosynthetic carbon fixation, known to exist in the marine mamiellale pico-prasinophytes and seaweed Ulva, but few other chlorophyte species, further highlights the unique adaptations of this robust alga. This high-quality assembly provides a significant advance in the resources available for genomic investigations of this and other photosynthetic picoeukaryotes.

With the global increase of infections caused by antibiotic-resistant bacterial strains, there is an urgent need for new methods of tackling the issue. Genomic analysis of bacterial strains can help to understand their virulence and antibiotic resistance profile. Bioinformatic skills are in great demand across the biological sciences. We designed a workshop that allows university students to learn the process of genome assembly using command-line tools within a virtual machine on a Linux operating system. We use Illumina and Nanopore short and long-read raw sequences to reveal the advantages and disadvantages of short, long, and hybrid assembly methods. The workshop teaches how to assess read and assembly quality, perform genome annotation, and analyze pathogenicity, antibiotic and phage resistance. The workshop is intended for a five-week teaching period and is concluded by a student poster presentation assessment.

Chlamydomonas utilizes the cyclic tetrapyrroles chlorophyll, heme, siroheme, vitamin B12, and linear tetrapyrrole (bilin) metabolites as cofactors for photosynthetic light harvesting, electron transport, redox reactions, mitigating oxidative stress, and signaling. With the exception of vitamin B12, Chlamydomonas can synthesize and degrade all of these tetrapyrroles. In this chapter, we review what is currently known about how Chlamydomonas biosynthesizes and metabolizes tetrapyrroles, how these processes are regulated, and the various functions of tetrapyrroles in Chlamydomonas.

Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H-2 is almost exclusively produced from coal and natural gas. As such, methods to produce H-2 that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H-2 may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 +/- 12,000 H-2 s(-1) for CaHydA from Clostridium acetobutyliticum). Yet to date, limitations in C. reinhardtii H-2 production pathways have hampered commercial scale implementation, in part due to O-2 sensitivity of hydrogenases and competing metabolic pathways, resulting in low H-2 production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H-2 production pathways in C. reinhardtii. We also summarize recent advancements of algal H-2 production using synthetic biology and describe valuable tools such as high-throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H-2 production.

Summary Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H 2 is almost exclusively produced from coal and natural gas. As such, methods to produce H 2 that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H 2 may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 ± 12,000 H 2 s −1 for Ca HydA from Clostridium acetobutyliticum ). Yet to date, limitations in C. reinhardtii H 2 production pathways have hampered commercial scale implementation, in part due to O 2 sensitivity of hydrogenases and competing metabolic pathways, resulting in low H 2 production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H 2 production pathways in C. reinhardtii . We also summarize recent advancements of algal H 2 production using synthetic biology and describe valuable tools such as high‐throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H 2 production.

Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4–10 cells/mL. Blinded analysis of each patient urine sample, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3–20 PCa cells per mL by TGiA, 4–13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine samples of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment.

Diverse microbial communities living in subsurface coal seams are responsible for important geochemical processes including the movement of carbon between the geosphere, biosphere and atmosphere. Microbial conversion of the organic matter in coal to methane involves a complex assemblage of bacteria and archaea working in syntrophic relationships. Despite the importance and value of this microbial process, very few of the microbial taxa have defined metabolic or ecological roles in these environments. Additionally, the genomic features mediating life in this chemically reduced, energy poor, deep subsurface environment are not well characterised. Here we describe the isolation and genomic and catabolic characterisation of three alphaproteobacterialStappia indicaspecies from three coal basins across Australia.S. indicagenomes from coal seams were compared with those from closely relatedS. indicaisolated from diverse surface waters, revealing a coal seam-specific suite of genes associated with life in the subsurface. These genes are linked to processes including viral defence, secondary metabolite production, polyamine metabolism, polypeptide uptake membrane transporters and putative energy neutral pressure-dependent CO(2)fixation. This indicates that subsurfaceStappiahave diverse metabolisms for biomass recycling and pressure-dependent CO(2)fixation and require a suite of defensive and competitive strategies relative to their surface-dwelling relatives.

Background Halomicronema hongdechloris was the first cyanobacterium to be identified that produces chlorophyll (Chl) f . It contains Chl a and uses phycobiliproteins as its major light-harvesting components under white light conditions. However, under far-red light conditions H. hongdechloris produces Chl f and red-shifted phycobiliprotein complexes to absorb and use far-red light. In this study, we report the genomic sequence of H. hongdechloris and use quantitative proteomic approaches to confirm the deduced metabolic pathways as well as metabolic and photosynthetic changes in response to different photo-autotrophic conditions. Results The whole genome of H. hongdechloris was sequenced using three different technologies and assembled into a single circular scaffold with a genome size of 5,577,845 bp. The assembled genome has 54.6% GC content and encodes 5273 proteins covering 83.5% of the DNA sequence. Using Tandem Mass Tag labelling, the total proteome of H. hongdechloris grown under different light conditions was analyzed. A total of 1816 proteins were identified, with photosynthetic proteins accounting for 24% of the total mass spectral readings, of which 35% are phycobiliproteins. The proteomic data showed that essential cellular metabolic reactions remain unchanged under shifted light conditions. The largest differences in protein content between white and far-red light conditions reflect the changes to photosynthetic complexes, shifting from a standard phycobilisome and Chl a- based light harvesting system under white light, to modified, red-shifted phycobilisomes and Chl f- containing photosystems under far-red light conditions. Conclusion We demonstrate that essential cellular metabolic reactions under different light conditions remain constant, including most of the enzymes in chlorophyll biosynthesis and photosynthetic carbon fixation. The changed light conditions cause significant changes in the make-up of photosynthetic protein complexes to improve photosynthetic light capture and reaction efficiencies. The integration of the global proteome with the genome sequence highlights that cyanobacterial adaptation strategies are focused on optimizing light capture and utilization, with minimal changes in other metabolic pathways. Our quantitative proteomic approach has enabled a deeper understanding of both the stability and the flexibility of cellular metabolic networks of H. hongdechloris in response to changes in its environment.

Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance maxima in the blue (Soret) and red (Qy) regions with Chl b, Chl d, and Chl f each possessing a single formyl group at a unique position. Relative to Chl a the Qy absorbance maximum of Chl b is blue-shifted while Chl d and Chl f are red-shifted with the shifts attributable to the relative positions of the formyl substitutions. Reduction of a formyl group of Chl b to form 7-hydroxymethyl Chl a, or oxidation of the vinyl group of Chl a into a formyl group to form Chl d was achieved using sodium borohydride (NaBH4) or β-mercaptoethanol (BME/O2), respectively. During the consecutive reactions of Chl b and Chl f using a three-step procedure (1. NaBH4, 2. BME/O2, and 3. NaBH4) two new 7-hydroxymethyl Chl a species were prepared possessing the 3-formyl or 3-hydroxymethyl groups and three new 2-hydroxymethyl Chl a species possessing the 3-vinyl, 3-formyl, or 3-hydroxymethyl groups, respectively. Identification of the spectral properties of 2-hydroxymethyl Chl a may be biologically significant for deducing the latter stages of Chl f biosynthesis if the mechanism parallels Chl b biosynthesis. The spectral features and chromatographic properties of these modified Chls are important for identifying potential intermediates in the biosynthesis of Chls such as Chl f and Chl d and for identification of any new Chls in nature.

Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species.

Chlorophyll biosynthesis and specific enzymes with the chlorophyll biosynthetic pathway have been reviewed extensively over the last 20 years. This chapter is intended to both summarize and update on previous reviews but it will primarily concentrate on the reaction mechanisms, enzymology and structure function relationships of the enzymes involved in synthesizing chlorophyll a from protoporphyrin IX.

Pulses are traditionally processed prior to consumption, providing opportunities for modifying nutritional composition, dependant on the type of pulse and method used. In this study, we investigated the effect of whole seed, dehulling (dahl), germination and roasting on changes in mungbean flour nutritional properties, protein composition and relative protein abundance. Processed flours were analysed and compared for protein content, moisture, fat, ash, dietary fibre, total starch and amylose. Significant differences were imparted on dietary fibre content, with roasting and germination increasing the ratio of insoluble/soluble fibre as well as resistant starch. Comparative proteomic analysis resulted in a combined total of 539 protein identifications, searching against the Mungbean reference genome (NCBI Vigna radiata Annotation Release 100). Normalised spectral abundance factors were used as a measure of relative abundance and statistical analysis was applied (Students’ T-Test), where proteins with a p-value of < 0.05 considered significantly different. Processing imparted considerable changes to nutritional composition and should be further exploited for food applications. The comparative proteomic analyses carried out in this study proved useful for investigating the effect of processing on subsequent changes in protein composition and relative abundance.

This study aimed to establish a mathematical modeling to evaluate the inhibitory effect of phenolic derivatives on acetone-butanol-ethanol (ABE) fermentation by Clostridium saccharoperbutylacetonicum N1-4. Vanillin, 4-hydroxybenzoic acid, and syringaldehyde were selected to represent guaiacyl, hydroxyphenyl, and syringyl phenols, respectively, to be examined in a series of fed-batch experiments. Results show the presence of phenolic derivatives blocked the pathway of the assimilation of organic acids and reduced cell growth and glucose utilization. The inhibition model projected that the levels of 0.13, 0.14, and 0.04 g L−1 for vanillin, 4-hydroxybenzoic acid, and syringaldehyde, respectively, resulted in 25% inhibition of butanol production, whereas 100% inhibition was predicted at the levels of 4.94, 4.37, and 4.20 g L−1 for vanillin, 4-hydroxybenzoic acid, and syringaldehyde, respectively. Syringaldehyde was more toxic than the other two compounds. The established model described that the phenolic compounds derived from different phenyl propane monomers of lignin severely obstructed biobutanol production.

Barley grain has been used as a source of food and malt production to make fermented beverages like beer and whisky for thousands of years. In this review we will focus on the proteome of barley, malt, and beer and how the detailed knowledge of the complexity of the barley proteome has advanced over the last 20–30 years.

Pulses are normally processed prior to consumption, are high in protein, providing opportunities for improving nutritional qualities of food. Processing methods can modify nutritional properties, affecting protein content, composition and functionality. In this study, we investigated the effect of processing methods on protein concentration and yield from protein isolates prepared by alkaline solubilisation and isoelectric precipitation, from whole seed flour, raw dahl flour, roasted dahl flour and germinated flour of mungbean. Protein isolates contained protein contents ranging from 87.3 to 90.4 g 100 g-1. The effect of processing methods on protein solubility characteristics was evident, as protein yields ranged from 75.7 (whole seed flour), 80.8 (raw dahl flour), 27.1 (roasted dahl flour) and 65.9 g 100 g-1 (germinated seed flour). Essential amino acid content was higher in protein isolates prepared from germinated flour, at 30.03 g 100 g-1 (39.1% of amino acids), compared to raw dahl flour, at 27.08 g 100 g-1 (38.3% of amino acids). A comparative proteomic analysis of protein isolates, prepared from raw dahl and germinated flour, resulted in the inferred identification of 214 proteins from protein sequence databases, in which, 72 proteins were classified as being common, 42 proteins specific to raw dahl flour and 28 proteins specific to germinated flour. Processing methods such as roasting and germination can significantly alter flour protein solubility, consequently, impacting on overall efficiency of protein extraction. The comparative proteomic analysis used in this study proved to be useful for investigating changes in protein composition and relative abundance, highlighting the potential in applying this technology for further characterisation of modified protein fractions for food applications.

Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme 'motor' complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.

Chlorophylls (Chls) are the most important cofactors for capturing solar energy to drive photosynthetic reactions. Five spectral types of Chls have been identified to date, with Chl f having the most red-shifted absorption maximum because of a C21-formyl group substitution of Chl f. However, the biochemical provenance of this formyl group is unknown. Here, we used a stable isotope labeling technique (18O and 2H) to determine the origin of the C21-formyl group of Chl f and to verify whether Chl f is synthesized from Chl a in the cyanobacterial species Halomicronema hongdechloris. In the presence of either H218O or 18O2, the origin of oxygen atoms in the newly synthesized chlorophylls was investigated. The pigments were isolated with HPLC, followed by MS analysis. We found that the oxygen atom of the C21-formyl group originates from molecular oxygen and not from H2O. Moreover, we examined the kinetics of the labeling of Chl a and Chl f from H. hongdechloris grown in 50% D2O-seawater medium under different light conditions. When cells were shifted from white light D2O-seawater medium to far-red light H2O-seawater medium, the observed deuteration in Chl f indicated that Chl(ide) a is the precursor of Chl f. Taken together, our results advance our understanding of the biosynthesis pathway of the chlorophylls and the formation of the formyl group in Chl f.