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Simultaneous coexpression of multiple proteins is essential for biotechnology and synthetic biology. Currently, the most popular polyprotein coexpression system utilizes the foot-and-mouth disease virus (FMDV) 2A peptide that mediates translational ribosome-skipping events. However, due to unfavorable consumer acceptance of transgenic products containing animal-virus sequences, novel non-viral 2A-like peptides from purple sea urchin (Strongylcentrotus purpuratus) and California sea slug (Aplysia californica) were investigated for polyprotein coexpression in this study. We demonstrated that these non-viral 2A sequences functioned similarly to their viral counterpart in polyprotein processing, in both plant and mammalian cells, and were successfully used to express a functional recombinant antibody. The new non-viral 2A-like sequences offer an alternative tool for engineering multigenic traits or production of protein complexes as biomedicine via coexpression of protein subunits.

Garlic, an economically important vegetable, spice, and medicinal crop, produces highly enlarged bulbs and unique organosulfur compounds. Here, we report a chromosome-level genome assembly for garlic, with a total size of approximately 16.24 Gb, as well as the annotation of 57 561 predicted protein-coding genes, making garlic the first Allium species with a sequenced genome. Analysis of this garlic genome assembly reveals a recent burst of transposable elements, explaining the substantial expansion of the garlic genome. We examined the evolution of certain genes associated with the biosynthesis of allicin and inulin neoseries-type fructans, and provided new insights into the biosynthesis of these two compounds. Furthermore, a large-scale transcriptome was produced to characterize the expression patterns of garlic genes in different tissues and at various growth stages of enlarged bulbs. The reference genome and large-scale transcriptome data generated in this study provide valuable new resources for research on garlic biology and breeding.

This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones.Key Points• Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production.• Astaxanthin is excreted and accumulated in the extracellular oil phase.• Astaxanthin is the predominant carotenoid in the extracellular oil phase.• Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction. Graphical abstract.

This review highlights main bioactive compounds and important biological functions especially anticancer effects of the garlic. In addition, we review current literature on the stability and bioavailability of garlic components. Finally, this review aims to provide a potential strategy for using nanotechnology to increase the stability and solubility of garlic components, providing guidelines for the qualities of garlic products to improve their absorption and prevent their early degradation, and extend their circulation time in the body. The application of nanotechnology to improve the bioavailability and targeting of garlic compounds are expected to provide a theoretical basis for the functional components of garlic to treat human health. We review the improvement of bioavailability and bioactivity of garlic bioactive compounds via nanotechnology, which could promisingly overcome the limitations of conventional garlic products, and would be used to prevent and treat cancer and other diseases in the near future.

This review highlights main bioactive compounds and important biological functions especially anticancer effects of the garlic. In addition, we review current literature on the stability and bioavailability of garlic components. Finally, this review aims to provide a potential strategy for using nanotechnology to increase the stability and solubility of garlic components, providing guidelines for the qualities of garlic products to improve their absorption and prevent their early degradation, and extend their circulation time in the body. The application of nanotechnology to improve the bioavailability and targeting of garlic compounds are expected to provide a theoretical basis for the functional components of garlic to treat human health. We review the improvement of bioavailability and bioactivity of garlic bioactive compounds via nanotechnology, which could promisingly overcome the limitations of conventional garlic products, and would be used to prevent and treat cancer and other diseases in the near future.

This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (, Highlights • 13C tracing was used to track Y. lipolytica metabolism of lipid-based feedstock. • Y. lipolytica has a segregated flux network for lipid and sugar co-utilizations. • Lipid feedstock and nitrogen sources affect cell morphology and optical density. • Lipid feedstock benefits both Y. lipolytica growth and carotenoid biosynthesis.

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coht), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Cohc) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (At) and C. cellulolyticum exoglucanase (Ec), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both Ec and At on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Cohc/Coht ratio (i.e., increased Ec/At ratio). Furthermore, among scaffoldins having the same Cohc/Coht ratio, highest rates of Avicel hydrolysis are noted when Coht, and hence an endoglucanase, is situated next to the CBM and not flanked by Cohc. These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

Summary A novel approach is developed for coordinated expression of multiple proteins from a single transgene in plants. An Ssp DnaE mini-intein variant engineered for hyper N-terminal autocleavage is covalently linked to the foot-and-mouth disease virus 2A (F2A) peptide with unique ribosome skipping property, via a peptide linker, to create an “IntF2A” self-excising fusion protein domain. This IntF2A domain acts, in cis, to direct highly effective release of its flanking proteins of interest (POI) from a “polyprotein” precursor in plants. This is successfully demonstrated in stably transformed cultured tobacco cells as well as in different organs of transgenic tobacco plants. Highly efficient polyprotein processing mediated by the IntF2A domain was also demonstrated in lettuce and Nicotiana benthamiana based on transient expression. Protein constituents released from the polyprotein precursor displayed proper function, and accumulated at similar levels inside the cells. Importantly, no C-terminal F2A extension remains on the released POIs. We demonstrated co-expression of as many as three proteins in plants without compromising expression levels when compared with those using single protein vectors. Accurate differential cellular targeting of released POIs is also achieved. In addition, we succeeded in expressing a fully assembled and functional chimeric anti-His Tag IgG antibody in N. benthamiana leaves. The IntF2A based polyprotein transgene system overcomes key impediments of existing strategies for multi-protein co-expression in plants, which is particularly important for gene/trait stacking. This article is protected by copyright. All rights reserved.

Efficient coproduction of multiple proteins or their subunits is important in many facets of life sciences and biotechnology. Here, we report a novel approach that exploits the synergy between an engineered mini-intein and an ubiquitin variant to achieve coordinated coexpression of multiple proteins in eukaryotic hosts, from a single open reading frame that encodes a polyprotein precursor consisting of proteins of interest (POIs) connected by an intervening intein-ubiquitin fusion domain. The intein variant mediates highly active autocatalytic cleavage at its N-terminus, whereas the endogenous deubiquitinases cleave at ubiquitin's C-terminus, leading to the release of the POIs. Using fluorescent reporter proteins for proof-of-concept, utility of the intein-ubiquitin domain was validated in higher plants and yeast systems. Essentially complete release of the POIs was achieved as demonstrated with western blots. Proteins expressed using the intein-ubiquitin system potentially preserve their intended sequences, which is important for preventing alteration of POI function.

A novel T-head Square-neck One-side Bolt (TSOB) was proposed and tensile tests on the T-head Square-neck One-side Bolts bolted Connection (TSOBC) for T-stub to Hollow Square Steel Tube (HSST) were conducted. In total, six TSOBCs and three Standard High-strength Bolts bolted Connections (SHBCs) were tested. Four failure modes were found in the test, namely, (1) column wall yielding, (2) column wall yielding with bolt hole shearing, (3) T-stub flange yielding, and (4) bolt failure. Compared to the SHBC, the tensile yield strength and tensile ultimate strength of TSOBCs with vertical and horizontal slotted bolt holes both decreased if the connections encountered the column wall yielding, the column wall yielding with bolt hole shearing, or the T-stub flange yielding failure. The Gomes and Yeomans models were modified to calculate the bending yield strength of the column wall of TSOBC under the failure mode of column wall yielding with bolt hole shearing. The modified Gomes model was recommended to design TSOBC failed by column wall yielding with bolt hole shearing. Theoretical models for calculating the bending yield strength of T-stub flange with slotted bolt holes of TSOBC was also presented, and calculated values were in good agreement with test results.

Papaya (Carica papaya) is widely cultivated in many tropical regions of the world. With an estimated 30–50% cull rate, there is a large amount of off-grade papaya produced. Here, we report very low-cost processing of culled papaya fruit waste, without needing any complex mechanized operations, to yield several products, including seed oil, sugar-rich puree, detoxified/defatted seed meal, and crude myrosinase and glucosinolates with antimicrobial and biofumigation applications. We then demonstrated that both puree and seed oil can serve as effective carbon substrates for cultivation of the oleaginous yeast Yarrowia lipolytica to produce single-cell proteins and high-value recombinant protein products. To use papaya seed oil for culturing Y. lipolytica, the concentration of the inhibitory substance benzyl isothiocyanate (BITC) in the oil needs to be minimized. If the culled fruits (and hence seeds) were stored frozen prior to drying, a very high level (>30 mM) of BITC was detected in the oil extracted from the dried seeds. However, if the seeds were not frozen prior to drying, oil from dried papaya seeds contained almost no BITC, and could support vigorous growth of Y. lipolytica, with efficient production of a functional nanobody fusion protein at a level similar to that achieved using olive oil. By using both juice and seed lipid, rather than juice alone, Y. lipolytica biomass produced per unit papaya more than doubled. As Y. lipolytica is amenable to genetic manipulation, and is known as a proficient cell factory with many industrial applications, the papaya waste valorization technology could potentially be extended to produce additional useful products such as biofuel and oleochemicals from Y. lipolytica.

In the present study, we fabricated magnetic oleosomes functionalized with recombinant proteins as a new carrier for oil-based lipophilic drugs for cancer treatment. The bioengineered oleosome is composed of neutral lipids surrounded by a phospholipid monolayer with embedded oleosin fusion proteins. The oleosin was genetically fused to a nanobody of a green fluorescent protein (GFP). A recombinant protein consisting of immunoglobulin-binding protein LG fused to GFP was used to couple the oleosome to an antibody for targeted delivery to breast cancer cells. The lipid core of the oleosome was loaded with magnetic nanoparticles and carmustine as the lipophilic drug. The magnetic oleosome was characterized using transmission electron microscopy and dynamic light scattering. Moreover, the specific delivery of oleosome into the target cancer cell was investigated via confocal microscopy. To examine the cell viability of the delivered oleosome, a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out. Furthermore, an animal study was conducted to confirm the effect resulting from the delivery of the anticancer drug-loaded oleosomes. Taken together, the fabricated lipophilic drug-loaded magnetic oleosome can be a powerful tool for oil-based drug delivery agent for cancer therapy.

Low cost electric power generation using raw juice from culled papaya or fruit waste as feedstock was achieved in a sugar-air alkaline battery (SAAB) with about 37% coulombic efficiency at 0.7–0.8 mW cm−2 near peak power density. Such fruit waste, juice-based electrochemical energy could be poised as sustainable distributed energy resource for remote or underdeveloped regions that need cheap electricity without complicated infrastructure.

Scaffold proteins play an important role in diverse cellular processes. Their unique molecular architecture helps to organize functionally interacting proteins in space and time. The resulting self-assembled protein complexes perform a wide variety of unique cellular functions. Inspired by these naturally occurring protein scaffolds, there has been an increasing interest in the development of novel artificial protein scaffolds for both in-vivo and in-vitro applications. Here two different molecular assembly strategies for effective biosynthesis of novel protein scaffolds are discussed. These scaffolds include amyloid-like nanofibril scaffolds and nano-scale oleosomal scaffolds derived from biological protein-bound lipid droplets synthesized in vivo. By recruiting protein components with distinctive functions (e.g. targeting, catalytic, and/or reporting) on the same scaffold, one can create intricate nanofactories to carry out novel tasks, including complex electrochemical biosensing.

The present study reports CO2 capture and conversion to bicarbonate using Escherichia coli expressing carbonic anhydrase (CA) on its cell surface in a novel foam bioreactor. The very large gas–liquid interfacial area in the foam bioreactor promoted rapid CO2 absorption while the CO2 in the aqueous phase was subsequently converted to bicarbonate ions by the CA. CO2 gas removal in air was investigated at various conditions such as gas velocity, cell density and CO2 inlet concentration. Regimes for kinetic and mass transfer limitations were defined. Very high removal rates of CO2 were observed: 9570 g CO2 m−3 bioreactor h−1 and a CO2 removal efficiency of 93% at 4% inlet CO2 when the gas retention time was 24 s, and cell concentration was 4 gdw L−1. These performances are superior to earlier reports of experimental bioreactors using CA for CO2 capture. Overall, this bioreactor system has significant potential as an alternative CO2 capture technology.

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH4)2SO4 (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH2PO4. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.

Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences and biomedicine.

Oleosomes are discrete organelles filled with neutral lipids surrounded by a protein-embedded phospholipid monolayer. Their simple yet robust structure, as well as their amenability to biological, chemical, and physical processing, can be exploited for various biotechnology applications. In this study, we report facile biosynthesis of functionalized oleosomes within oleaginous yeast Yarrowia lipolytica, through expression of oleosin fusion proteins. By fusing a cDNA clone of a sesame oleosin with either the coding sequence of a red fluorescent protein mCherry or a cellulosomal scaffolding protein cohesin from Clostridium cellulolyticum, these oleosin-fusion proteins were efficiently expressed and specifically targeted to and anchored on the surface of the oleosomes within the Y. lipolytica cells. The engineered oleosomes can be easily separated from the Y. lipolytica cell extract via floating centrifugation and both mCherry and cohesin domains are shown to be functional. Upon sonication, the engineered Yarrowia oleosomes exhibit a mean diameter of 200300nm and are found to be highly stable. The feasibility of co-displaying multiple proteins on the Yarrowia oleosomes was demonstrated by incubating cohesin-displaying oleosomes with different dockerin-fusion proteins. Based on this strategy, engineered oleosomes with both cell-targeting and reporting activities were created and shown to be functional. Taken together, the Yarrowia oleosome surface display system in which oleosin serves as an efficient membrane anchor motif shows great promise as a simple platform for creating tunable nanoparticles. Biotechnol. Bioeng. 2013; 110: 702710. (c) 2012 Wiley Periodicals, Inc.

To increase the thermostability of Rhizomucor miehei lipase, the software Disulfide by Design was used to engineer a novel disulfide bond between residues 96 and 106, and the corresponding double cysteine mutants were constructed. The R. miehei lipase mutant could be expressed by Pichia pastoris in a free secreted form or could be displayed on the cell surface. The new disulfide bond spontaneously formed in the mutant R. miehei lipase. Thermostability was examined by measuring of hydrolysis activity using 4-nitrophenyl caprylate as a substrate. The engineered disulfide bond contributed to thermostability in the free form of the R. miehei lipase variant. The variant displayed on the yeast cell surface had significantly increased residual hydrolytic activity in aqueous solution after incubation at 60 degrees C for 5 h and increased synthetic activity in organic solvent at 60 degrees C. These results indicated that yeast surface display might improve the stability of R. miehei lipase, as well as amplifying the thermostability through the engineered disulfide bond.

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