Biosynthetic Proteins Research Articles

Effects of Individual Amino Acids on the Blood Circulation of Biosynthetic Protein Nanocages: Toward Guidance on Surface Engineering.

Protein nanocages (PNCs) hold great promise for developing multifunctional nanomedicines. Long blood circulation is a key requirement of PNCs for most in vivo application scenarios. In addition to the classical PEGylation strategy, short peptides with a specific sequence screened via phage display are also very effective in prolonging the blood half-life (t1/2 ) of PNCs. However, there is a lack of knowledge on how individual amino acids affect the circulation of PNCs. Here the effects of the 20 proteinogenic amino acids in the form of an X3 or X5 tag (X represents an amino acid) are explored on the pharmacokinetics of PNCs, which lead to the formation of a heatmap illustrating the extent of t1/2 prolongation by each proteinogenic amino acid. Significantly, oligo-lysine and oligo-arginine can effectively prolong the t1/2 of strongly negatively charged PNCs through charge neutralization, while oligo-cysteine can also do so, but via a different mechanism, mediating the covalent binding of PNCs with plasma albumin as a stealth material. These findings are extendible and offer guidance for surface-engineering biosynthetic PNCs and other nanoparticles.

MoaB1 Homologs Contribute to Biofilm Formation and Motility by Pseudomonas aeruginosa and Escherichia coli.

moaB homologs, encoding the molybdopterin biosynthetic protein B1, have been reported to be expressed under anoxic conditions and during biofilm growth in various microorganisms; however, little is known about MoaB's function. Here, we demonstrate that in Pseudomonas aeruginosa, MoaB1 (PA3915) contributes to biofilm-related phenotypes. Specifically, moaB1 expression is induced in biofilms, and insertional inactivation of moaB1 reduced biofilm biomass accumulation and pyocyanin production while enhancing swarming motility, and pyoverdine abundance without affecting attachment, swimming motility, or c-di-GMP levels. Inactivation of the highly conserved E. coli homolog of moaB1, moaBEc, likewise coincided with reduced biofilm biomass accumulation. In turn, heterologous expression of moaBEc restored biofilm formation and swarming motility by the P. aeruginosa moaB1 mutant to wild-type levels. Moreover, MoaB1 was found to interact with other conserved biofilm-associated proteins, PA2184 and PA2146, as well as the sensor-kinase SagS. However, despite the interaction, MoaB1 failed to restore SagS-dependent expression of brlR encoding the transcriptional regulator BrlR, and inactivation of moaB1 or moaBEc had no effect on the antibiotic susceptibility phenotype of biofilms formed by P. aeruginosa and E. coli, respectively. While our findings did not establish a link between MoaB1 and molybdenum cofactor biosynthesis, they suggest that MoaB1 homologs contribute to biofilm-associated phenotypes across species boundaries, possibly hinting at the existence of a previously undescribed conserved biofilm pathway. IMPORTANCE Proteins contributing to the biogenesis of molybdenum cofactors have been characterized; however, the role of the molybdopterin biosynthetic protein B1 (MoaB1) has remained elusive, and solid evidence to support its role in biosynthesis of molybdenum cofactor is lacking. Here, we demonstrate that, in Pseudomonas aeruginosa, MoaB1 (PA3915) contributes to biofilm-related phenotypes in a manner that does not support a role of MoaB1 in the biosynthesis of molybdenum cofactors.

Vagococcus luciliae sp. nov., isolated from the common green bottle fly Lucilia sericata.

The genus Vagococcus belongs to the family Enterococcaceae (order Lactobacillales) and is closely related to the genus Enterococcus. Currently, 19 species of the genus have been validly named. In this study, we isolated strain G314FT from the common green bottle fly Lucilia sericata collected in Germany. Sequencing of its almost-complete 16S rRNA gene revealed that the isolate belongs to the genus Vagococcus, being closely related to Vagococcus bubulae SS1994T with high sequence identity (99.50 %), followed by Vagococcus martis D7T301T (98.86 %), Vagococcus vulneris SS1995T (98.71 %), Vagococcus teuberi DSM 21459T (98.64 %), Vagococcus silagei 2B-2T (98.64 %) and Vagococcus penaei CD276T (98.64 %). Genome sequencing of strain G314FT was performed by a combination of Illumina and Oxford Nanopore technology, yielding a circular genome with a size of 2 139 468 bp and an 11 kb plasmid. Average nucleotide identity and digital DNA-DNA hybridization values were calculated between G314FT and its closest-related taxa, and found to be <91 % and <40 %, respectively, thus strongly supporting that strain G314FT represents a novel species of the genus Vagococcus. Phylogenetic and core protein-based phylogenomic trees revealed that G314FT was closely related to a group of three species, V. bubulae SS1994T, V. martis D7T301T and V. teuberi DSM 21459T. Comparatively, the genome of G314FT is the smallest in the group of the four related species, and the biochemical pathway comparison using BlastKOALA revealed that G314FT has lost some amino acid biosynthetic proteins; however, it has gained enzymes for carbohydrate metabolism. Phenotypically, G314FT was consistent with other species of the genus Vagococcus including a negative catalase reaction and non-motility. Using the polyphasic approach, our data supports that the isolate represents a new species, for which we propose the name Vagococcus luciliae G314FT (=DSM 112651T= CCM 9164T).

Quantitative functionalization of biosynthetic caged protein materials

BackgroundAs one of the representative protein materials, protein nanocages (PNCs) are self‐assembled supramolecular structures with multiple advantages, such as good monodispersity, biocompatibility, structural addressability, and facile production. Precise quantitative functionalization is essential to the construction of PNCs with designed purposes.ResultsWith three modifiable interfaces, the interior surface, outer surface, and interfaces between building blocks, PNCs can serve as an ideal platform for precise multi‐functionalization studies and applications. This review summarizes the currently available methods for precise quantitative functionalization of PNCs and highlights the significance of precise quantitative control in fabricating PNC‐based materials or devices. These methods can be categorized into three groups, genetic, chemical, and combined modification.ConclusionThis review would be constructive for those who work with biosynthetic PNCs in diverse fields.

The Hypolipidemic Effect of Hawthorn Leaf Flavonoids through Modulating Lipid Metabolism and Gut Microbiota in Hyperlipidemic Rats.

The purpose of this study was to explore the potential mechanisms of the lipid-regulating effects and the effect on modulating the gut microbiota of hawthorn leaf flavonoids (HLF) in the high-fat diet-induced hyperlipidemic rats. The hypolipidemic effect of HLF was investigated in the high-fat diet-induced hyperlipidemic rats. The action targets of HLF in the treatment of hyperlipidemia were predicted by network pharmacology and KEGG enrichment bubble diagram, which were verified by the test of western blotting. Meanwhile, we used 16S rRNA sequencing to evaluate the effects of HLF on the microbes. The results of animal experiments showed that HLF could reduce the body weight and regulate the levels of serum lipid in high-fat diet (HFD) rats. Meanwhile, for the related targets of cholesterol metabolism, HLF could significantly upregulate the expression of LDLR, NR1H3, and ABCG5/ABCG8; reduce the expression of PCSK9; and increase the level of CYP7A1 in the intestinal tissue, whereas cholesterol biosynthetic protein expressions including HMGCR and SCAP were lowered by HLF. In addition, HLF increased the activities of plasma SOD, CAT, and GSH-Px and decreased the levels of Casp 1, NLRP3, IL-1β, IL-18, and TNF-α, improving the degree of hepatocyte steatosis and inflammatory infiltration of rats. Notably, HLF significantly regulated the relative abundance of major bacteria such as g_Lactobacillus, g_Anaerostipes, g_[Eubacterium]_hallii_group, g_Fusicatenibacter, g_Akkermansia, and g_Collinsella. Synchronously, we found that HLF could regulate the disorder of plasma HEPC and TFR levels caused by HFD. This study demonstrates that HLF can regulate metabolic hyperlipidemia syndromes and modulate the relative abundance of major bacteria, which illustrated that it might be associated with the modulation of gut microbiota composition and metabolites.

Identification of Host Molecules Involved in the Proliferation of Nucleopolyhedrovirus in Bombyx mori.

The Bombyx mori nucleopolyhedrovirus (BmNPV), a foodborne infectious virus, is the pathogen causing nuclear polyhedrosis and high lethality in the silkworm. In this study, we characterized the molecules involved in BmNPV-silkworm interaction by RNA sequencing of the fat body isolated from the virus-susceptible strain P50. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation showed that the upregulated differentially expressed genes (DEGs) were mainly involved in translation, signal transduction, folding, sorting, and degradation, as well as transport and catabolism, while the downregulated DEGs were predominantly enriched in the metabolism of carbohydrates, amino acids, and lipids at 72 h post BmNPV infection. Knockout of the upregulated somatomedin-B and thrombospondin type-1 domain-containing protein, probable allantoicase, trifunctional purine biosynthetic protein adenosine-3, and Psl and pyoverdine operon regulator inhibited the proliferation of BmNPV, while knockout of the downregulated clip domain serine protease 3 and carboxylesterase clade H, member 1 promoted it. The molecules herein identified provide a foundation for developing strategies and designing drugs against BmNPV.

Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways

ABSTRACT The molecular repertoire of Trypanosoma cruzi effects its virulence and impacts the clinical course of the resulting Chagas disease. This study aimed to determine the mechanism underlying the pathogenicity of T. cruzi. Two T. cruzi cell lines (C8C3 hvir and C8C3 lvir ), obtained from the clone H510 C8C3 and exhibiting different virulence phenotypes, were used to evaluate the parasite’s infectivity in mice. The organ parasite load was analysed by qPCR. The proteomes of both T. cruzi cell lines were compared using nLC-MS/MS. Cruzipain (Czp), complement regulatory protein (CRP), trans-sialidase (TS), Tc-85, and sialylated epitope expression levels were evaluated by immunoblotting. High-virulence C8C3 hvir was highly infectious in mice and demonstrated three to five times higher infectivity in mouse myocardial cells than low-virulence C8C3 lvir . qPCR revealed higher parasite loads in organs of acute as well as chronically C8C3 hvir -infected mice than in those of C8C3 lvir -infected mice. Comparative quantitative proteomics revealed that 390 of 1547 identified proteins were differentially regulated in C8C3 hvir with respect to C8C3 lvir . Amongst these, 174 proteins were upregulated in C8C3 hvir and 216 were downregulated in C8C3 lvir . The upregulated proteins in C8C3 hvir were associated with the tricarboxylic acid cycle, ribosomal proteins, and redoxins. Higher levels of Czp, CRP, TS, Tc-85, and sialylated epitopes were expressed in C8C3 hvir than in C8C3 lvir . Thus, T. cruzi virulence may be related to virulence factor expression as well as upregulation of bioenergetic and biosynthetic pathways proteins.

Characterization of Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase Genes of Tomato (Solanum lycopersicum L.) and Analysis of Their Differential Expression in Response to Temperature.

In plants, the trehalose biosynthetic pathway plays key roles in the regulation of carbon allocation and stress adaptation. Engineering of the pathway holds great promise to increase the stress resilience of crop plants. The synthesis of trehalose proceeds by a two-step pathway in which a trehalose-phosphate synthase (TPS) uses UDP-glucose and glucose-6-phosphate to produce trehalose-6 phosphate (T6P) that is subsequently dephosphorylated by trehalose-6 phosphate phosphatase (TPP). While plants usually do not accumulate high amounts of trehalose, their genome encodes large families of putative trehalose biosynthesis genes, with many members lacking obvious enzymatic activity. Thus, the function of putative trehalose biosynthetic proteins in plants is only vaguely understood. To gain a deeper insight into the role of trehalose biosynthetic proteins in crops, we assessed the enzymatic activity of the TPS/TPP family from tomato (Solanum lycopersicum L.) and investigated their expression pattern in different tissues as well as in response to temperature shifts. From the 10 TPS isoforms tested, only the 2 proteins belonging to class I showed enzymatic activity, while all 5 TPP isoforms investigated were catalytically active. Most of the TPS/TPP family members showed the highest expression in mature leaves, and promoter–reporter gene studies suggest that the two class I TPS genes have largely overlapping expression patterns within the vasculature, with only subtle differences in expression in fruits and flowers. The majority of tomato TPS/TPP genes were induced by heat stress, and individual family members also responded to cold. This suggests that trehalose biosynthetic pathway genes could play an important role during temperature stress adaptation. In summary, our study represents a further step toward the exploitation of the TPS and TPP gene families for the improvement of tomato stress resistance.

TFG regulates secretory and endosomal sorting pathways in neurons to promote their activity and maintenance

Molecular pathways that intrinsically regulate neuronal maintenance are poorly understood, but rare pathogenic mutations that underlie neurodegenerative disease can offer important insights into the mechanisms that facilitate lifelong neuronal function. Here, we leverage a rat model to demonstrate directly that the TFG p.R106C variant implicated previously in complicated forms of hereditary spastic paraplegia (HSP) underlies progressive spastic paraparesis with accompanying ventriculomegaly and thinning of the corpus callosum, consistent with disease phenotypes identified in adolescent patients. Analyses of primary cortical neurons obtained from CRISPR-Cas9-edited animals reveal a kinetic delay in biosynthetic secretory protein transport from the endoplasmic reticulum (ER), in agreement with prior induced pluripotent stem cell-based studies. Moreover, we identify an unexpected role for TFG in the trafficking of Rab4A-positive recycling endosomes specifically within axons and dendrites. Impaired TFG function compromises the transport of at least a subset of endosomal cargoes, which we show results in down-regulated inhibitory receptor signaling that may contribute to excitation-inhibition imbalances. In contrast, the morphology and trafficking of other organelles, including mitochondria and lysosomes, are unaffected by the TFG p.R106C mutation. Our findings demonstrate a multifaceted role for TFG in secretory and endosomal protein sorting that is unique to cells of the central nervous system and highlight the importance of these pathways to maintenance of corticospinal tract motor neurons.

Modulation of sensitivity to gaseous signaling by sterol-regulatory hypoxic transcription factors in Aspergillus nidulans biofilm cells

Fungal biofilm founder cells experience self-generated hypoxia leading to dramatic changes in their cell biology. For example, during Aspergillus nidulans biofilm formation microtubule (MT) disassembly is triggered causing dispersal of EB1 from MT tips. This process is dependent on SrbA, a sterol regulatory element-binding transcription factor required for adaptation to hypoxia. We show that SrbA, an ER resident protein prior to activation, is proteolytically activated during early stages of biofilm formation and that, like SrbA itself, its activating proteases are also required for normal biofilm MT disassembly. In addition to SrbA, the AtrR transcription factor is also found to be required to modulate cellular responses to gaseous signaling during biofilm development. Using co-cultures, we further show that cells lacking srbA or atrR are capable of responding to biofilm generated gaseous microenvironments but are actually more sensitive to this signal than wild type cells. SrbA is a regulator of ergosterol biosynthetic genes and we find that the levels of seven GFP-tagged Erg proteins differentially accumulate during biofilm formation with various dependencies on SrbA for their accumulation. This uncovers a complex pattern of regulation with biofilm accumulation of only some Erg proteins being dependent on SrbA with others accumulating to higher levels in its absence. Because different membrane sterols are known to influence cell permeability to gaseous molecules, including oxygen, we propose that differential regulation of ergosterol biosynthetic proteins by SrbA potentially calibrates the cell’s responsiveness to gaseous signaling which in turn modifies the cell biology of developing biofilm cells.

Increased rates of brain protein synthesis during [N1,N2] sleep: L-[1-11C]leucine PET studies in human subjects

During sleep, reduced brain energy demands provide an opportunity for biosynthetic processes like protein synthesis. Sleep is required for some forms of memory consolidation which requires de novo protein synthesis. We measured regional cerebral protein synthesis rates (rCPS) in human subjects to ascertain how rCPS is affected during sleep. Subjects underwent three consecutive L-[1-11C]leucine PET scans with simultaneous polysomnography: 1. rested awake, 2. sleep-deprived awake, 3. sleep. Measured rCPS were similar across the three conditions. Variations in sleep stage times during sleep scans were used to estimate rCPS in sleep stages under the assumption that measured rCPS is the weighted sum of rCPS in each stage, with weights reflecting time and availability of [11C]leucine in that stage. During sleep scans, subjects spent most of the time in N2, N3, and awake and very little time in N1 and REM; rCPS in N1 and REM could not be reliably estimated. When stages N1 and N2 were combined [N1,N2], estimates of rCPS were more robust. In selective regions, estimated rCPS were statistically significantly higher (30–39%) in [N1,N2] compared with N3; estimated rCPS in N3 were similar to values measured in sleep-deprived awake scans. Results indicate increased rates of protein synthesis linked to [N1,N2] sleep.

Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals.

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8–18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.

Biosynthetic and Unfolded Protein Nanocarriers for Chemotherapeutic Drugs in Oral Cancers: Improved Bioavailability and Safety of Chemotherapeutics

AbstractConventional nanocarriers for oral squamous cell carcinoma (OSCC) chemotherapy are greatly limited due to undesired adverse effects and low bioavailability. As an FDA‐approved protein nanocarrier, i.e., human serum albumin (HSA), the folded conformation limits its versatility for loading OSCC chemotherapeutics with different molecular structures. Here, a new type of biosynthetic and unfolded protein nanocarrier is developed, which shows outstanding efficiency for loading and delivering a series of chemotherapeutic agents, including chlormethine, docetaxel, and pingyangmycin. Moreover, all the nanoformulated drugs exhibit rather prolonged half‐lives as evidenced by sustained release of the payloads for up to 5 days. In stark contrast to polymeric micelles and liposomes, in this system the loading efficiency is increased 3–4 times and the corresponding half‐life is even extended one order of magnitude. Enhanced antitumor effects in rat OSCC models are realized. Additionally, off‐target side effects such as liver and kidney toxicity and immunogenicity are significantly mitigated with the nanomedications. Thus, this versatile formulation strategy boosts the efficacy, bioavailability, and safety of chemotherapeutics in OSCC therapy, and promisingly enables combined chemotherapy in comprehensive treatment for OSCC.

Bioinformatics and Genomic Analyses of the Suitability of Eight Riboswitches for Antibacterial Drug Targets.

Antibiotic resistance (AR) is an acute problem that results in prolonged and debilitating illnesses. AR mortality worldwide is growing and causes a pressing need to research novel mechanisms of action and untested target molecules. This article presents in silico analyses of eight bacterial riboswitches for their suitability for antibacterial drug targets. Most bacterial riboswitches are located in the 5′-untranslated region of messenger RNAs, act as allosteric cis-acting gene control elements, and have not been found in humans before. Sensing metabolites, the riboswitches regulate the synthesis of vital cellular metabolites in various pathogenic bacteria. The analyses performed in this article represent a complete and informative genome-wide bioinformatics analysis of the adequacy of eight riboswitches as antibacterial drug targets in different pathogenic bacteria based on four criteria. Due to the ability of the riboswitch to control biosynthetic pathways and transport proteins of essential metabolites and the presence/absence of alternative biosynthetic pathways, we classified them into four groups based on their suitability for use as antibacterial drug targets guided by our in silico analyses. We concluded that some of them are promising targets for antibacterial drug discovery, such as the PreQ1, MoCo RNA, cyclic-di-GMP I, and cyclic-di-GMP II riboswitches.

Pseudocitrobacter corydidari sp. nov., isolated from the Asian emerald cockroach Corydidarum magnifica.

A Gram-negative bacterial strain, G163CMT, was isolated from the gut of the Asian emerald cockroach Corydidarum magnifica. The 16S rRNA gene sequence (1416 bp) of strain G163CMT showed 99.22% similarity to Pseudocitrobacter faecalis CCM 8479T and Pseudocitrobacter vendiensis CPO20170097T. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values of strain G163CMT were 92.4, 48.8 and 95.7% to P. faecalis CCM 8479T, and 93.3, 52.4 and 95.7% to P. vendiensis CPO20170097T. This strongly supports the designation of G163CMT as representing a new species in the genus Pseudocitrobacter. Phylogenetic trees based on the alignment of 16S rRNA, multilocus sequence analysis of six single-copy genes (fusA, pyrG, leuS, rpoB, recN and mnmE) and 107 core protein sequences consistently showed G163CMT to be a member of the genus Pseudocitrobacter, closely related to P. vendiensis CPO20170097T. In contrast to P. faecalis CCM 8479T and P. vendiensis CPO20170097T, the genome of G163CMT did not encode for proteins conferring resistance to antibiotics. However, all three genomes encoded a similar number of virulence factors and specialized metabolite biosynthetic proteins. The major fatty acids of strain G163CMT were C16:0 (31.5 %), C18:1 ω7c (22.6 %), C17:0 cyclo (15.3 %) and C14:0 (6.5 %). Based on the polyphasic results, we conclude that strain G163CMT represents a novel species of the genus Pseudocitrobacter and we propose the name Pseudocitrobacter corydidari sp. nov. with the type strain G163CMT (=DSM 112648T=CCM 9160T).

Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway.

Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.

Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon.

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses. Integrated metabolomic and proteomic data support a link between lignin biosynthesis and primary metabolism mediated by the ammonia released from ALs that is recycled for the synthesis of amino acids via glutamine. RNA interference knockdown of lignin genes confirmed that the route of the canonical pathway using shikimate ester intermediates is not essential for lignin formation in Brachypodium, and there is an alternative pathway from Tyr via sinapic acid for the synthesis of syringyl lignin involving yet uncharacterized enzymatic steps. Our findings support a model in which plant ALs play a central role in coordinating the allocation of carbon for lignin synthesis and the nitrogen available for plant growth. Collectively, these data also emphasize the value of integrative multiomic analyses to advance our understanding of plant metabolism.

Starch Biosynthetic Protein Complex Formation in Rice ss2a be2b (+) Double Mutant Differs from Their Parental Single Mutants.

Amylopectin, which consists of highly branched glucose polymers, is a major component of starch. Biochemical processes that regulate the elongation of glucose polymers and the generation and removal of glucose branches are essential for determining the properties of starch. Starch synthases (SSs) and branching enzyme (BE) mainly form complexes consisting of SSI, SSIIa, and BEIIb during endosperm development. Loss of BEIIb in rice is complemented by BEIIa, but the compensatory effects differ depending on the presence or absence of inactive BEIIb. To better understand these compensatory mechanisms, ss2a be2b (+) double mutant, which possessed truncated inactive SSIIa and inactive BEIIb, were analyzed. Soluble proteins separated by gel filtration chromatography showed that SSIIa and BEIIb proteins in the wild-type exhibited a broad range of elution patterns and only small amounts were detected in high molecular mass fractions. In contrast, most of truncated inactive SSIIa and inactive BEIIb from ss2a be2b (+) were found in high molecular mass fractions, and the SSI-SSIIa-BEIIb trimeric protein complex found in the wild-type was likely absent in ss2a be2b (+). Those SSIIa and BEIIb proteins in high molecular mass fractions in ss2a be2b (+) were also identified by mass spectrometry. Parental ss2a single mutant had negligible amounts of SSIIa suggesting that the truncated inactive SSIIa was recruited to high-molecular mass complexes in the presence of inactive BEIIb in ss2a be2b (+) double mutant. In addition, SSIVb might be involved in the formation of alternative protein complexes with < 300 kDa in ss2a be2b (+).

Biosynthetic protein and nanocellulose composite fibers with extraordinary mechanical performance

Typical cellulose fibers exhibit a high mechanical strength but very low toughness, which severely limits their high-tech applications. It is thus becoming urgently important to develop new strategies to simultaneously improve their strength and toughness. In this regard, a new type of biomacromolecular composite fibers is fabricated by wet-spinning a solution of biosynthetic protein and nanocellulose. In stark contrast to pristine cellulose fibers, significantly improved mechanical performance, e.g., 551.2 MPa maximum breaking strength, 40.6 MJ m−3 toughness, and 12.5% extensibility were realized in a versatile way. Notably, robust fiber meshes with good impact resistance ability were developed. They can withstand a gravity potential energy of 0.020 J, which is 1.4-fold higher than their pristine nanocellulose counterparts. Moreover, their excellent biocompatibility and superior mechanical properties allow the composite biological fibers for efficient surgical suturing. This work offers a new strategy to fabricate high-performance biological fibers for high-tech applications.