Calvin-Benson-Bassham Research Articles

Identifying the major metabolic potentials of microbial-driven carbon, nitrogen and sulfur cycling on stone cultural heritage worldwide

Microbial activities and biochemical reactions are responsible for the biodeterioration of stone cultural heritage, but information on microbial metabolic potentials remains elusive. Here we profiled microbial community signatures and its functional traits on stone cultural heritage from different climate zones globally using sequencing datasets available publicly. Bacterial community on stone cultural heritage shows a significant separation between BSk (cold semi-arid climate) and Cfb (temperate oceanic climate) with Aw (tropical savanna climate) as a transition region. Importantly, the ubiquity of ammonia oxidizers and nitrite oxidizers on stone cultural heritage under different climates supports the active production and accumulation of nitrates while ammonia/ammonium can be supplied by dinitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA), together with the hydrolysis of urea, arginine, formamide and cyanate. Sulfate accumulation on stone cultural heritage is mainly resulted from the microbial-driven transformation of organosulfur and thiosulfate, with little dissimilatory reduction of sulfate. Pseudorhodoplanes was identified and reported in elemental sulfur turnover for the first time. Notably, carbon sequestration via the reductive tricarboxylic acid (rTCA) cycle and an incomplete 3-hydroxypropionate/4-hydroxybutynate (HP/HB) cycle other than the Calvin Benson-Bassham (CBB) cycle is also significant on stone cultural heritage under relatively humid climate. These results advance our understanding of microbial metabolic potentials and their genetical partitioning patterns on stone cultural heritage of different climate zones globally.

The energy metabolism of Cupriavidus necator in different trophic conditions.

The "knallgas" bacterium Cupriavidus necator is attracting interest due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO2 via the Calvin-Benson-Bassham (CBB) cycle, and grows on organic acids and sugars. Its tripartite genome is notable for its size and duplications of key genes (CBB cycle, hydrogenases, and nitrate reductases). Little is known about which of these isoenzymes and their cofactors are actually utilized for growth on different substrates. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on succinate, fructose, hydrogen (H2/CO2), and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that (i) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. (ii) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. (iii) For hydrogenases, both soluble and membrane-bound enzymes were utilized for lithoautotrophic growth. (iv) Of the six terminal respiratory complexes in C. necator H16, only some are utilized, and utilization depends on the energy source. (v) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necator's genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium and inspire future engineering strategies.IMPORTANCEThe soil bacterium Cupriavidus necator can grow on gas mixtures of CO2, H2, and O2. It also consumes formic acid as carbon and energy source and various other substrates. This metabolic flexibility comes at a price, for example, a comparatively large genome (6.6 Mb) and a significant background expression of lowly utilized genes. In this study, we mutated every non-essential gene in C. necator using barcoded transposons in order to determine their effect on fitness. We grew the mutant library in various trophic conditions including hydrogen and formate as the sole energy source. Fitness analysis revealed which of the various energy-generating iso-enzymes are actually utilized in which condition. For example, only a few of the six terminal respiratory complexes are used, and utilization depends on the substrate. We also show that the protein cost for the various lowly utilized enzymes represents a significant growth disadvantage in specific conditions, offering a route to rational engineering of the genome. All fitness data are available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/.

Reversed oxidative TCA (roTCA) for carbon fixation by an Acidimicrobiia strain from a saline lake.

Acidimicrobiia are widely distributed in nature and suggested to be autotrophic via the Calvin-Benson-Bassham (CBB) cycle. However, direct evidence of chemolithoautotrophy in Acidimicrobiia is lacking. Here, we report a chemolithoautotrophic enrichment from a saline lake, and the subsequent isolation and characterization of a chemolithoautotroph, Salinilacustristhrix flava EGI L10123T, which belongs to a new Acidimicrobiia family. Although strain EGI L10123T is autotrophic, neither its genome nor Acidimicrobiia metagenome-assembled genomes (MAGs) from the enrichment culture encode genes necessary for the CBB cycle. Instead, genomic, transcriptomic, enzymatic, and stable-isotope probing data hinted at the activity of the reversed oxidative TCA (roTCA) coupled with the oxidation of sulfide as the electron donor. Phylogenetic analysis and ancestral character reconstructions of Acidimicrobiia suggested that the essential CBB gene rbcL was acquired through multiple horizontal gene transfer events from diverse microbial taxa. In contrast, genes responsible for sulfide- or hydrogen-dependent roTCA carbon fixation were already present in the last common ancestor of extant Acidimicrobiia. These findings imply the possibility of roTCA carbon fixation in Acidimicrobiia and the ecological importance of Acidimicrobiia. Further research in the future is necessary to confirm whether these characteristics are truly widespread across the clade.

The primary carbon metabolism in cyanobacteria and its regulation.

Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis. Many cyanobacterial strains can live in different trophic modes, ranging from photoautotrophic and heterotrophic to mixotrophic growth. However, the regulatory mechanisms allowing a flexible switch between these lifestyles are poorly understood. As anabolic fixation of CO2 in the Calvin-Benson-Bassham (CBB) cycle and catabolic sugar-degradation pathways share intermediates and enzymatic capacity, a tight regulatory network is required to enable simultaneous opposed metabolic fluxes. The Entner-Doudoroff (ED) pathway was recently predicted as one glycolytic route, which cooperates with other pathways in glycogen breakdown. Despite low carbon flux through the ED pathway, metabolite analyses of mutants deficient in the ED pathway revealed a distinct phenotype pointing at a strong regulatory impact of this route. The small Cp12 protein downregulates the CBB cycle in darkness by inhibiting phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase. New results of metabolomic and redox level analyses on strains with Cp12 variants extend the known role of Cp12 regulation towards the acclimation to external glucose supply under diurnal conditions as well as to fluctuations in CO2 levels in the light. Moreover, carbon and nitrogen metabolism are closely linked to maintain an essential C/N homeostasis. The small protein PirC was shown to be an important regulator of phosphoglycerate mutase, which identified this enzyme as central branching point for carbon allocation from CBB cycle towards lower glycolysis. Altered metabolite levels in the mutant ΔpirC during nitrogen starvation experiments confirm this regulatory mechanism. The elucidation of novel mechanisms regulating carbon allocation at crucial metabolic branching points could identify ways for targeted redirection of carbon flow towards desired compounds, and thus help to further establish cyanobacteria as green cell factories for biotechnological applications with concurrent utilization of sunlight and CO2.

Co-expression analysis reveals distinct alliances around two carbon fixation pathways in hydrothermal vent symbionts

Most autotrophic organisms possess a single carbon fixation pathway. The chemoautotrophic symbionts of the hydrothermal vent tubeworm Riftia pachyptila, however, possess two functional pathways: the Calvin–Benson–Bassham (CBB) and the reductive tricarboxylic acid (rTCA) cycles. How these two pathways are coordinated is unknown. Here we measured net carbon fixation rates, transcriptional/metabolic responses and transcriptional co-expression patterns of Riftia pachyptila endosymbionts by incubating tubeworms collected from the East Pacific Rise at environmental pressures, temperature and geochemistry. Results showed that rTCA and CBB transcriptional patterns varied in response to different geochemical regimes and that each pathway is allied to specific metabolic processes; the rTCA is allied to hydrogenases and dissimilatory nitrate reduction, whereas the CBB is allied to sulfide oxidation and assimilatory nitrate reduction, suggesting distinctive yet complementary roles in metabolic function. Furthermore, our network analysis implicates the rTCA and a group 1e hydrogenase as key players in the physiological response to limitation of sulfide and oxygen. Net carbon fixation rates were also exemplary, and accordingly, we propose that co-activity of CBB and rTCA may be an adaptation for maintaining high carbon fixation rates, conferring a fitness advantage in dynamic vent environments.

Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris

Pichia pastoris, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the P. pastoris cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-gene biosynthetic pathways. To broaden its application in the field of metabolic engineering, this study identified and screened 15 novel integration sites in P. pastoris using CRISPR-Cpf1 genome editing technology, with EGFP serving the reporter protein. These integration sites have integration efficiencies of 10–100 % and varying expression strengths, which allow for selection based on the expression levels of genes as needed. Additionally, these integrated sites are applied in the heterologous biosynthesis of P. pastoris, such as the astaxanthin biosynthetic pathway and the carbon dioxide fixation pathway of the Calvin-Benson-Bassham (CBB) cycle. During the three-site integration process, the 8 genes of the CBB cycle were integrated into the genome of P. pastoris. This indicates the potential of these integration sites for integrating large fragments and suggests their successful application in metabolic engineering of P. pastoris. This may lead to improved efficiency of genetic engineering in P. pastoris.

Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase carbon dioxide (CO2) concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

Removal of phosphoglycolate in hyperthermophilic archaea

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Preserved particulate organic carbon is likely derived from the subsurface sulfidic photic zone of the Proterozoic Ocean: evidence from a modern, oxygen-deficient lake.

Biological processes in the Proterozoic Ocean are often inferred from modern oxygen-deficient environments (MODEs) or from stable isotopes in preserved sediment. To date, few MODE studies have simultaneously quantified carbon fixation genes and attendant stable isotopic signatures. Consequently, how carbon isotope patterns reflect these pathways has not been thoroughly vetted. Addressing this, we profiled planktonic productivity and quantified carbon fixation pathway genes and associated organic carbon isotope values (δ13 CPOC ) of size-fractionated (0.2-2.7 and >2.7 μm) particulate matter from meromictic Fayetteville Green Lake, NY, USA. The high-O2 Calvin-Benson-Bassham (CBB) gene (cbbL) was most abundant in the <2.7 μm size fraction in shallow oxic and deep hypoxic waters, corresponding with cyanobacterial and eukaryote algal populations. The low-O2 CBB gene (cbbM) was most abundant near the lower oxycline boundary in the larger size fraction, coincident with purple sulfur bacteria populations. The reverse citric acid cycle gene (aclB) was equally abundant in both size fractions in the deepest photic zone, coinciding with green sulfur bacteria populations. Methane coenzyme reductase A (mcrA), of anaerobic methane cyclers, was most abundant at the lower oxycline boundary in both size fractions, coinciding with Methanoregula populations. δ13 CPOC values overlapped with the high-O2 CBB fixation range except for two negative excursions near the lower oxycline boundary, likely reflecting assimilation of isotopically-depleted groundwater-derived carbon by autotrophs and sulfate-reducers. Throughout aphotic waters, δ13 CPOC values of the large size fraction became 13 C-enriched, likely reflecting abundant purple sulfur bacterial aggregates. Eukaryote algae- or cyanobacteria-like isotopic signatures corresponded with increases in cbbL, cbbM, and aclB, and enrichment of exopolymer-rich prokaryotic photoautotrophs aggregates. Results suggest that δ13 CPOC values of preserved sediments from areas of the Proterozoic Ocean with sulfidic photic zones may reflect a mixture of alternate carbon-fixing populations exported from the deep photic zone, challenging the paradigm that sedimentary stable carbon isotope values predominantly reflect oxygenic photosynthesis from surface waters.

Machine learning predicts system-wide metabolic flux control in cyanobacteria

Metabolic fluxes and their control mechanisms are fundamental in cellular metabolism, offering insights for the study of biological systems and biotechnological applications. However, quantitative and predictive understanding of controlling biochemical reactions in microbial cell factories, especially at the system level, is limited. In this work, we present ARCTICA, a computational framework that integrates constraint-based modelling with machine learning tools to address this challenge. Using the model cyanobacterium Synechocystis sp. PCC 6803 as chassis, we demonstrate that ARCTICA effectively simulates global-scale metabolic flux control. Key findings are that (i) the photosynthetic bioproduction is mainly governed by enzymes within the Calvin–Benson–Bassham (CBB) cycle, rather than by those involve in the biosynthesis of the end-product, (ii) the catalytic capacity of the CBB cycle limits the photosynthetic activity and downstream pathways and (iii) ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a major, but not the most, limiting step within the CBB cycle. Predicted metabolic reactions qualitatively align with prior experimental observations, validating our modelling approach. ARCTICA serves as a valuable pipeline for understanding cellular physiology and predicting rate-limiting steps in genome-scale metabolic networks, and thus provides guidance for bioengineering of cyanobacteria.

Metagenomic analysis revealed highly diverse carbon fixation microorganisms in a petroleum-hydrocarbon-contaminated aquifer

As one of the ultimate products of hydrocarbon biodegradation, inorganic carbon always be used to evaluate hydrocarbon biodegradation rates in petroleum-hydrocarbon-contaminated (PHC) aquifers. The evaluation method was challenged because of the existence of carbon fixation microorganisms, which may uptake inorganic carbons and consequently cause the biodegradation rates to be underestimated. We wonder if there are carbon fixation microorganisms in PHC aquifers. Although an extremely limited number of carbon fixation microorganisms in PHC sites have been studied in previous studies, the vast majority of microorganisms that participate in carbon fixation have not been systematically identified. To systematically reveal carbon fixation microorganisms and their survival environmental conditions, high-throughput metagenomic sequencing technologies, which are characterized by culture-independent, unbiased, and comprehensive methods for the detection and taxonomic characterization of microorganisms, were introduced to analyze the groundwater samples collected from a PHC aquifer. Results showed that 1041 genera were annotated as carbon fixation microorganisms, which accounted for 49% of the total number of genera in the PHC aquifer. Carbon fixation genes involved in Calvin-Benson-Bassham (CBB), 3-hydroxy propionate (3HP), reductive tricarboxylic acid (rTCA), and Wood-Ljungdahl (WL) cycles accounted for 2%, 41%, 34%, and 23% of the total carbon fixation genes, respectively, and 3HP, rTCA, and WL can be deemed as the dominant carbon fixation pathways. Most of the identified carbon fixation microorganisms are potential hydrocarbon biodegraders, and the most abundant carbon fixation microorganisms, such as Microbacterium, Novosphingobium, and Reyranella, were just the most abundant microorganisms in the aquifer system. It's deduced that most of the microorganisms in the aquifer were facultative autotrophic, and undertaking the dual responsibilities of degrading hydrocarbons to inorganic carbon and uptaking inorganic carbon to biomass.

Changes in soil microbial functions involved in the carbon cycle response to conventional and biodegradable microplastics

Biodegradable mulch film has become an encouraging alternative to conventional petroleum–based plastic, but it is likely to generate more microplastics (MPs) than conventional films with lower resistance and thus profoundly affects the soil environment. However, the effects of biodegradable (BMPs) and conventional polyethylene MPs (PE–MPs) on soil microbial carbon (C) cycle remain unclear. Here, metagenomic sequencing was used to investigate microbial functional and relative taxonomic traits in response to PE–MP and BMP addition (5 % w/w) via a soil microcosm experiment. The results showed that the presence of BMPs, rather than PE–MPs, significantly changed soil microbial C cycling patterns at the functional and taxonomic levels. In soils, BMP addition significantly increased the abundances of genes regulating aerobic respiration, C decomposition (intracellular), C fixation, fermentation and CO oxidation pathways compared to those in the control and PE–MP added soils. Specifically, BMPs promoted soil starch and PHA (liable C) degradation, acetate and ethanol fermentation, Calvin–Benson–Bassham (CBB) cycle and 3–hydroxypropionate/4–hydroxybutyrate (3HP/4HB) associated with C fixation. Most of these C cycling pathways were further strengthened in the microplastisphere of BMPs. Furthermore, MPs selectively enriched specific taxa on their surface and subsequently increased their abundance in soils; Bradyrhizobium, Ramlibacter and Variovorax (enriched by BMPs) positively regulated labile C degradation, and Amycolatopsis and Nocardia (enriched by PE–MPs) promoted recalcitrant C degradation. Moreover, Pseudonocardia and Variovorax that were enriched by BMPs improved C dissimilation and assimilation by regulating fermentation and C fixation processes, respectively. Taken together, these results improve the understanding of the influence of conventional and biodegradable MPs on soil C cycling pathways and related microbial communities, highlighting that the presence of BMPs rather than PE–MPs accelerates soil C turnover.

Scallop-bacteria symbiosis from the deep sea reveals strong genomic coupling in the absence of cellular integration.

Previous studies have revealed tight metabolic complementarity between bivalves and their endosymbiotic chemosynthetic bacteria, but little is known about their interactions with ectosymbionts. Our analysis of the ectosymbiosis between a deep-sea scallop (Catillopecten margaritatus) and a gammaproteobacterium showed that bivalves could be highly interdependent with their ectosymbionts as well. Our microscopic observation revealed abundant sulfur-oxidizing bacteria (SOB) on the surfaces of the gill epithelial cells. Microbial 16S rRNA gene amplicon sequencing of the gill tissues showed the dominance of the SOB. An analysis of the SOB genome showed that it is substantially smaller than its free-living relatives and has lost cellular components required for free-living. Genomic and transcriptomic analyses showed that this ectosymbiont relies on rhodanese-like proteins and SOX multienzyme complex for energy generation, mainly on the Calvin-Benson-Bassham (CBB) cycle and peripherally on a phosphoenolpyruvate carboxylase for carbon assimilation. Besides, the symbiont encodes an incomplete tricarboxylic acid (TCA) cycle. Observation of the scallop's digestive gland and its nitrogen metabolism pathways indicates it does not fully rely on the ectosymbiont for nutrition. Analysis of the host's gene expression provided evidence that it could offer intermediates for the ectosymbiont to complete its TCA cycle and some amino acid synthesis pathways using exosomes, and its phagosomes, endosomes, and lysosomes might be involved in harvesting nutrients from the symbionts. Overall, our study prompts us to rethink the intimacy between the hosts and ectosymbionts in Bivalvia and the evolution of chemosymbiosis in general.

Active dark carbon fixation evidenced by 14C isotope assimilation and metagenomic data across the estuarine-coastal continuum

Estuaries, as important land-ocean transitional zones across the Earth's surface, are hotspots of microbially driven dark carbon fixation (DCF), yet understanding of DCF process remains limited across the estuarine-coastal continuum. This study explored DCF activities and associated chemoautotrophs along the estuarine and coastal environmental gradients, using radiocarbon labelling and molecular techniques. Significantly higher DCF rates were observed at middle- and high-salinity regions (0.65–2.31 and 0.66–2.82 mmol C m−2 d−1, respectively), compared to low-salinity zone (0.07–0.19 mmol C m−2 d−1). Metagenomic analysis revealed relatively stable DCF pathways along the estuarine-coastal continuum, primarily dominated by Calvin-Benson-Bassham (CBB) cycle and Wood-Ljungdahl (WL) pathway. Nevertheless, chemoautotrophic communities driving DCF exhibited significant spatial variations. It is worth noting that although CBB cycle played an important role in DCF in estuarine sediments, WL pathway might play a more significant role, which has not been previously recognized. Overall, this study highlights that DCF activities coincide with the genetic potential of chemoautotrophy and the availability of reductive substrates across the estuarine-coastal continuum, and provides an important scientific basis for accurate quantitative assessment of global estuarine carbon sink.

Metagenomic Insights into Ecophysiology of Zetaproteobacteria and Gammaproteobacteria in Shallow Zones within Deep-sea Massive Sulfide Deposits.

Deep-sea massive sulfide deposits serve as energy sources for chemosynthetic ecosystems in dark, cold environments even after hydrothermal activity ceases. However, the vertical distribution of microbial communities within sulfide deposits along their depth from the seafloor as well as their ecological roles remain unclear. We herein conducted a culture-independent metagenomic ana-lysis of a core sample of massive sulfide deposits collected in a hydrothermally inactive field of the Southern Mariana Trough, Western Pacific, by drilling (sample depth: 0.52‍ ‍m below the seafloor). Based on the gene context of the metagenome-assembled genomes (MAGs) obtained, we showed the metabolic potential of as-yet-uncultivated microorganisms, particularly those unique to the shallow zone rich in iron hydroxides. Some members of Gammaproteobacteria have potential for the oxidation of reduced sulfur species (such as sulfide and thiosulfate) to sulfate coupled to nitrate reduction to ammonia and carbon fixation via the Calvin-Benson-Bassham (CBB) cycle, as the primary producers. The Zetaproteobacteria member has potential for iron oxidation coupled with microaerobic respiration. A comparative ana-lysis with previously reported metagenomes from deeper zones (~2‍ ‍m below the seafloor) of massive sulfide deposits revealed a difference in the relative abundance of each putative primary producer between the shallow and deep zones. Our results expand knowledge on the ecological potential of uncultivated microorganisms in deep-sea massive sulfide deposits and provide insights into the vertical distribution patterns of chemosynthetic ecosystems.

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeast Komagataella phaffii.

Synthetic autotrophs can serve as chassis strains for bioproduction from CO2 as a feedstock to take measures against the climate crisis. Integration of the Calvin-Benson-Bassham (CBB) cycle into the methylotrophic yeast Komagataella phaffii (Pichia pastoris) enabled it to use CO2 as the sole carbon source. The key enzyme in this cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzing the carboxylation step. However, this enzyme is error prone to perform an oxygenation reaction leading to the production of toxic 2-phosphoglycolate. Native autotrophs have evolved different recycling pathways for 2-phosphoglycolate. However, for synthetic autotrophs, no information is available for the existence of such pathways. Deletion of CYB2 in the autotrophic K. phaffii strain led to the accumulation of glycolate, an intermediate in phosphoglycolate salvage pathways, suggesting that such a pathway is enabled by native K. phaffii enzymes. 13C tracer analysis with labeled glycolate indicated that the yeast pathway recycling phosphoglycolate is similar to the plant salvage pathway. This orthogonal yeast pathway may serve as a sensor for RuBisCO oxygenation, and as an engineering target to boost autotrophic growth rates in K. phaffii.

The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals

For decades micoorganisms have been engineered for the utilization of lignocellulose-based second-generation (2G) feedstocks, but with the concerns of increased levels of atmospheric CO2 causing global warming there is an emergent need to transition from the utilization of 2G feedstocks to third-generation (3G) feedstocks such as CO2 and its derivatives. Here, we established a yeast platform that is capable of simultaneously converting 2G and 3G feedstocks into bulk and value-added chemicals. We demonstrated that by adopting 3G substrates such as CO2 and formate, the conversion of 2G feedstocks could be substantially improved. Specifically, formate could provide reducing power and energy for xylose conversion into valuable chemicals. Simultaneously, it can form a concentrated CO2 pool inside the cell, providing thermodynamically and kinetically favoured amounts of precursors for CO2 fixation pathways, e.g. the Calvin–Benson–Bassham (CBB) cycle. Furthermore, we demonstrated that formate could directly be utilized as a carbon source by yeast to synthesize endogenous amino acids. The engineered strain achieved a one-carbon (C1) assimilation efficiency of 9.2 %, which was the highest efficiency observed in the co-utilization of 2G and 3G feedstocks. We applied this strategy for productions of both bulk and value-added chemicals, including ethanol, free fatty acids (FFAs), and longifolene, resulting in yield enhancements of 18.4 %, 49.0 %, and ∼100 %, respectively. The strategy demonstrated here for co-utilization of 2G and 3G feedstocks sheds lights on both basic and applied research for the up-coming establishment of 3G biorefineries.

Genome sequencing and metabolic network reconstruction of a novel sulfur-oxidizing bacterium Acidithiobacillus Ameehan.

Sulfur-oxidizing bacteria play a crucial role in various processes, including mine bioleaching, biodesulfurization, and treatment of sulfur-containing wastewater. Nevertheless, the pathway involved in sulfur oxidation is highly intricate, making it complete comprehension a formidable and protracted undertaking. The mechanisms of sulfur oxidation within the Acidithiobacillus genus, along with the process of energy production, remain areas that necessitate further research and elucidation. In this study, a novel strain of sulfur-oxidizing bacterium, Acidithiobacillus Ameehan, was isolated. Several physiological characteristics of the strain Ameehan were verified and its complete genome sequence was presented in the study. Besides, the first genome-scale metabolic network model (AMEE_WP1377) was reconstructed for Acidithiobacillus Ameehan to gain a comprehensive understanding of the metabolic capacity of the strain.The characteristics of Acidithiobacillus Ameehan included morphological size and an optimal growth temperature range of 37-45°C, as well as an optimal growth pH range of pH 2.0-8.0. The microbe was found to be capable of growth when sulfur and K2O6S4 were supplied as the energy source and electron donor for CO2 fixation. Conversely, it could not utilize Na2S2O3, FeS2, and FeSO4·7H2O as the energy source or electron donor for CO2 fixation, nor could it grow using glucose or yeast extract as a carbon source. Genome annotation revealed that the strain Ameehan possessed a series of sulfur oxidizing genes that enabled it to oxidize elemental sulfur or various reduced inorganic sulfur compounds (RISCs). In addition, the bacterium also possessed carbon fixing genes involved in the incomplete Calvin-Benson-Bassham (CBB) cycle. However, the bacterium lacked the ability to oxidize iron and fix nitrogen. By implementing a constraint-based flux analysis to predict cellular growth in the presence of 71 carbon sources, 88.7% agreement with experimental Biolog data was observed. Five sulfur oxidation pathways were discovered through model simulations. The optimal sulfur oxidation pathway had the highest ATP production rate of 14.81 mmol/gDW/h, NADH/NADPH production rate of 5.76 mmol/gDW/h, consumed 1.575 mmol/gDW/h of CO2, and 1.5 mmol/gDW/h of sulfur. Our findings provide a comprehensive outlook on the most effective cellular metabolic pathways implicated in sulfur oxidation within Acidithiobacillus Ameehan. It suggests that the OMP (outer-membrane proteins) and SQR enzymes (sulfide: quinone oxidoreductase) have a significant impact on the energy production efficiency of sulfur oxidation, which could have potential biotechnological applications.

Phylogenetically and metabolically diverse autotrophs in the world’s deepest blue hole

The world’s deepest yongle blue hole (YBH) is characterized by sharp dissolved oxygen (DO) gradients, and considerably low-organic-carbon and high-inorganic-carbon concentrations that may support active autotrophic communities. To understand metabolic strategies of autotrophic communities for obtaining carbon and energy spanning redox gradients, we presented finer characterizations of microbial community, metagenome and metagenome-assembled genomes (MAGs) in the YBH possessing oxic, hypoxic, essentially anoxic and completely anoxic zones vertically. Firstly, the YBH microbial composition and function shifted across the four zones, linking to different biogeochemical processes. The recovery of high-quality MAGs belonging to various uncultivated lineages reflected high novelty of the YBH microbiome. Secondly, carbon fixation processes and associated energy metabolisms varied with the vertical zones. The Calvin–Benson–Bassham (CBB) cycle was ubiquitous but differed in affiliated taxa at different zones. Various carbon fixation pathways were found in the hypoxic and essentially anoxic zones, including the 3-hyroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle affiliated to Nitrososphaeria, and Wood-Ljungdahl (WL) pathway affiliated to Planctomycetes, with sulfur oxidation and dissimilatory nitrate reduction as primary energy-conserving pathways. The completely anoxic zone harbored diverse taxa (Dehalococcoidales, Desulfobacterales and Desulfatiglandales) utilizing the WL pathway coupled with versatile energy-conserving pathways via sulfate reduction, fermentation, CO oxidation and hydrogen metabolism. Finally, most of the WL-pathway containing taxa displayed a mixotrophic lifestyle corresponding to flexible carbon acquisition strategies. Our result showed a vertical transition of microbial lifestyle from photo-autotrophy, chemoautotrophy to mixotrophy in the YBH, enabling a better understanding of carbon fixation processes and associated biogeochemical impacts with different oxygen availability.

Proton Gradient Regulation 5 is required to avoid photosynthetic oscillations during light transitions.

The production of ATP and NADPH by the light reactions of photosynthesis and their consumption by the Calvin-Benson-Bassham (CBB) cycle and other downstream metabolic reactions requires careful regulation. Environmental shifts perturb this balance, leading to photo-oxidative stress and losses in CO2 assimilation. Imbalances in the production and consumption of ATP and NADPH manifest themselves as transient instability in the chlorophyll fluorescence, P700, electrochromic shift, and CO2 uptake signals recorded on leaves. These oscillations can be induced in wild-type plants by sudden shifts in CO2 concentration or light intensity; however, mutants exhibiting increased oscillatory behaviour have yet to be reported. This has precluded an understanding of the regulatory mechanisms employed by plants to suppress oscillations. Here we show that the Arabidopsis pgr5 mutant, which is deficient in Proton Gradient Regulation 5 (PGR5)-dependent cyclic electron transfer (CET), exhibits increased oscillatory behaviour. In contrast, mutants lacking the NADH-dehydrogenase-like-dependent CET are largely unaffected. The absence of oscillations in the hope2 mutant which, like pgr5, lacks photosynthetic control and exhibits high ATP synthase conductivity, ruled out loss of these photoprotective mechanisms as causes. Instead, we observed slower formation of the proton motive force and, by inference, ATP synthesis in pgr5 following environmental perturbation, leading to the transient reduction of the electron transfer chain and photosynthetic oscillations. PGR5-dependent CET therefore plays a major role in damping the effect of environmental perturbations on photosynthesis to avoid losses in CO2 fixation.