Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae.

Microbial proteins are promising substitutes for animal- and plant-based proteins. S. cerevisiae, a generally recognized as safe (GRAS) microorganism, has been frequently employed to generate heterologous proteins. However, constructing a universal yeast chassis for efficient protein production is still a challenge due to the varying properties of different proteins. With progress in synthetic biology, a multitude of molecular biology tools and metabolic engineering strategies have been employed to alleviate these issues. This review first analyses the advantages of protein production by S. cerevisiae. The most recent advances in improving heterologous protein yield are summarized and discussed in terms of protein hyperexpression systems, protein secretion engineering, glycosylation pathway engineering and systems metabolic engineering. Furthermore, the prospects for efficient and sustainable heterologous protein production by S. cerevisiae are also provided.

Development of Organogels for Live Yarrowia lipolytica Encapsulation.

Encapsulation of cells/microorganisms attracts great attention in many applications, but current studies mainly focus on hydrophilic encapsulation materials. Herein, we develop a new class of hydrophobic and lipophilic organogels for highly efficient encapsulation of Yarrowia lipolytica, an oleaginous yeast, by a mild and nonsolvent photopolymerization method. The organogels allow free diffusion of hydrophobic molecules that oleaginous yeasts require to survive and function. Moreover, they are mechanically robust and possess favorable biocompatibility, thus providing a free-standing platform and an ideal survival environment for oleaginous Y. lipolytica encapsulation. By tuning monomer structures and cross-linking densities, the optimized organogel, Gel12-1.5%, achieves the highest viability of ∼96%. Furthermore, organogels can inhibit the cryoinjuries to oleaginous yeasts in cryopreservation, exhibiting the potential for long-term storage. It is also found that with varying alkyl lengths, the organogels show different temperature-dependent phase transition properties, which enable the rapid selection of targeted yeasts for steganography. Findings in this work provide guidance for designing biocompatible, hydrophobic, and lipophilic encapsulation materials.

Engineering synthetic microbial consortium for cadaverine biosynthesis from glycerol.

OBJECTIVES: 1,5-pentanediamine (cadaverine) is a C5 platform chemical, also an important raw material for bio-polyamide PA5X. With increasing concerns about the depletion of fossil resources and global environmental protection, cadaverine bio-production has attracted more attentions. RESULTS: Here, a microbial consortium consisting of Corynebacterium glutamicum cgl-FDK and Escherichia coli BL-ABST-Spy was constructed to de novo synthesize cadaverine utilizing glycerol as the sole carbon resource. The glycerol utilization pathway was initially constructed in C. glutamicum cgl-FDK to produce lysine from glycerol. Then, the pyridoxal 5'-phosphate (PLP) biosynthesis pathway and SpyTag/SpyCatcher protein-ligation system for lysine decarboxylase (CadA) and cadaverine-lysine antiporter protein (CadB) were introduced into E. coli BL-ABST-Spy to synthesize cadaverine from lysine. Furthermore, the fermentation conditions of microbial consortium were optimized and the cadaverine production reached 9.3 g/L with glycerol as the sole carbon source. CONCLUSIONS: This work provides a promising strategy for efficiently producing cadaverine from glycerol with an artificial microbial consortium.

Metabolomics assisted metabolic network modeling and network wide analysis of metabolites in microbiology.

Metabolomics is the science of qualitatively and quantitatively analyzing low molecular weight metabolites occur in a given biological system. It provides valuable information to elucidate the functional roles and relations of different metabolites in a metabolic pathway. In recent years, a large amount of research on microbial metabolomics has been conducted. It has become a useful tool for achieving highly efficient synthesis of target metabolites. At the same time, many studies have been conducted over the years in order to integrate metabolomics data into metabolic network modeling, which has yielded many exciting results. Additionally, metabolomics also shows great advantages in analyzing the relationship of metabolites network wide. Integrating metabolomics data into metabolic network construction and applying it in network wide analysis of cell metabolism would further improve our ability to control cellular metabolism and optimize the design of cell factories for the overproduction of valuable biochemicals. This review will examine recent progress in the application of metabolomics approaches in metabolic network modeling and network wide analysis of microbial cell metabolism.

A facile modification to improve the biocompatibility and adsorbability of activated carbon with zwitterionic hydrogel.

In this work, poly(carboxybetaine methacrylate) hydrogel (pCBMA) was employed to modify the activated carbon (AC) for improving the biocompatibility and adsorption capacity of AC in biological environments. First, size-controlled hydrogel beads and hydrogel coated AC (pCBMA-AC) were fabricated with a homemade device, and the preparation conditions were optimized. Then the physical and biological properties of pCBMA-AC with different diameters were investigated. 2 mm pCBMA-AC dispalyed excellent stability with leakage rate only 0.16% after 72 h shaking incubation, as well as remarkable biocompatibility with merely 0.13% hemolysis rate and 3.41% cell death, while 14.72% and 70.11% for the bare AC, respectively, indicating the acceptable lower hemolysis and cytotoxicity according to ISO 10993. Furthermore, the adsorption capacities of pCBMA-AC were evaluated in biological environments with methylene blue as model molecules. The pCBMA-AC displayed 93.50% and 97.32% adsorption rates in BSA solution and FBS, respectively, but only 70.33% and 40.26% for the uncoated AC. These results indicated that pCBMA endows AC remarkable biocompatibility and adsorption capacity, which could extend the applications of AC in biological environments.

Integration of parallel 13 C-labeling experiments and in silico pathway analysis for enhanced production of ascomycin.

Herein, the hyper-producing strain for ascomycin was engineered based on 13 C-labeling experiments and elementary flux modes analysis (EFMA). First, the metabolism of non-model organism Streptomyces hygroscopicus var. ascomyceticus SA68 was investigated and an updated network model was reconstructed using 13 C- metabolic flux analysis. Based on the precise model, EFMA was further employed to predict genetic targets for higher ascomycin production. Chorismatase (FkbO) and pyruvate carboxylase (Pyc) were predicted as the promising overexpression and deletion targets, respectively. The corresponding mutant TD-FkbO and TD-ΔPyc exhibited the consistency effects between model prediction and experimental results. Finally, the combined genetic manipulations were performed, achieving a high-yield ascomycin engineering strain TD-ΔPyc-FkbO with production up to 610 mg/L, 84.8% improvement compared with the parent strain SA68. These results manifested that the integration of 13 C-labeling experiments and in silico pathway analysis could serve as a promising concept to enhance ascomycin production, as well as other valuable products. Biotechnol. Bioeng. 2017;114: 1036-1044. © 2016 Wiley Periodicals, Inc.

Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production.

Ascomycin (FK520), a macrocyclic polyketide natural antibiotic, displays high anti-fungal and immunosuppressive activity. In this study, the LysR family transcriptional regulator FkbR1 was characterized, and its role in ascomycin biosynthesis was explored by gene deletion, complementation, and overexpression. Inactivation of fkbR1 led to 67.5% reduction of ascomycin production, which was restored by complementation of fkbR1. Overexpression of fkbR1 resulted in a 33.5% increase in ascomycin production compared with the parent strain FS35. These findings indicated that FkbR1 was a positive regulator for ascomycin production. Quantitative RT-PCR analysis revealed that the expressions of fkbE, fkbF, fkbS, and fkbU were downregulated in the fkbR1 deletion strain and upregulated in the fkbR1 overexpression strain. Electrophoretic mobility shift assays (EMSAs) in vitro and chromatin immunoprecipitation (ChIP)-qPCR assays in vivo indicated that FkbR1 bound to the intergenic region of fkbR1-fkbE. To investigate the roles of the target genes fkbE and fkbF in ascomycin production, the deletion and overexpressions of fkbE and fkbF were implemented, respectively. Overexpression of fkbE resulted in a 45.6% increase in ascomycin production, but little change was observed in fkbF overexpression strain. To further enhance ascomycin production, the fkbR1 and fkbE combinatorial overexpression strain OfkbRE was constructed with the ascomycin yield increased by 69.9% to 536.7 mg/L compared with that of the parent strain. Our research provided a helpful strategy to increase ascomycin production via engineering FkbR1 and its target gene.

Enhancement of ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by combining resin HP20 addition and metabolic profiling analysis.

Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5 % m/v HP20 added at 24 h, ascomycin production was increased to 380 from 300 mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2 % v/v n-hexadecane was added at 24 h; 1.0 g/L valine was supplemented at 48 h; 1.0 g/L lysine was added at 72 h. The ascomycin production was ultimately improved to 460 mg/L, a 53.3 % enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds.

Mussel-inspired protein-based nanoparticles for curcumin encapsulation and promoting antitumor efficiency.

Curcumin demonstrated therapeutic potential for cancer. However, its medical application is limited due to low solubility, poor stability and low absorption rate. Here, we used the mussel-inspired functional protein (MPKE) to fabricate the curcumin-carrying nanoparticle (Cur-MPKE) for encapsulating and delivering curcumin. The protein MPKE is composed of the mussel module and zwitterionic peptide. The Dopa group bonding characteristic of the mussel module was leveraged for the self-assembly of nanoparticles, while the superhydrophilic property of the zwitterionic peptide was utilized to enhance the stability of nanoparticles. As expected, MPKE and Cur are tightly bound through hydrogen bonds and dynamic imide bonds to form nanoparticles. Cur-MPKE showed improved solubility and stability in aqueous solutions as well as excellent biocompatibility. Besides, Cur-MPKE also exhibited pH-triggered release and enhanced uptake of curcumin by tumor cells, promoting the antioxidant activity and antitumor effect of curcumin. Moreover, systemic experiments of Cur-MPKE to rats demonstrated that Cur-MPKE significantly inhibited tumor tissue growth and proliferation without causing obvious systemic toxicity. This work provides a new strategy for fabricating the delivery system of curcumin with improved stability, sustainability and bioavailability.

Comparative metabolic profiling-based improvement of rapamycin production by Streptomyces hygroscopicus.

Rapamycin is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces hygroscopicus. To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7 fermentation in medium M1 and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and 16 key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, and shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as follows: 1.0 g/L methyl oleate was added at 0 h; 1.0 g/L lysine was added at 12 h; 0.5 g/L shikimic acid was added at 24 h; 0.5 g/L sodium succinate, 0.1 g/L phenylalanine, 0.1 g/L tryptophan, and 0.1 g/L tyrosine were added at 36 h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3 was increased by 56.6 % compared with it in M2, reaching 307 mg/L at the end of fermentation (120 h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds.

Mussel-inspired proteins functionalize catheter with antifouling and antibacterial properties.

Bacterial adhesion and subsequent biofilm formation on catheter can cause inevitably infection. The development of multifunctional antibacterial coating is a promising strategy to resist the bacteria adhesion and biofilm formation. Herein, a mussel-inspired chimeric protein MZAgP is prepared and employed to modify a variety of polymeric catheters. The MZAgP is composed of mussel-adhesive peptide, zwitterionic peptide, and silver-binding peptide, which can endow catheters with antifouling, bactericidal and biocompatibility performances. Expectedly, negligible biofilm is observed on the MZAgP coated catheters after incubating with bacteria for 120 h. And ignorable hemolysis and cytotoxicity are obtained on coated catheters. In addition, the modified catheters also display persistent antifouling and bacteriostatic properties throughout 168 h under hydrodynamic conditions. Moreover, the coated catheters can still remain excellent antifouling and antibacterial properties even after 2 months of storage. This multifunctional coating may be promising as antibacterial and antibiofilm biomaterial, and the coated catheters are potential in clinical application.

Investigation into antifreeze performances of natural amino acids for novel CPA development.

Cryopreservation can prolong the viability of cells and help meet the demand for biosamples of high medical value. Cryoprotectants (CPAs) can mitigate unavoidable cell cryoinjury caused by the formation and growth of ice crystals during freeze-thaw cycles. Therefore, the development of efficient and biocompatible CPAs has attracted extensive attention. In this work, the antifreeze performances of 18 water-soluble natural amino acids were systematically investigated by wet experiments and molecular dynamics simulations to explore their potentials as CPAs. Phenylalanine has an amphipathic structure with excellent ice recrystallization inhibition (IRI) activity, and methionine exhibits optimal depressing freezing point ability. Combining phenylalanine or methionine with osmolyte (proline) as CPAs can maintain normal morphology of sheep red blood cells (RBCs), and the recovery of sheep RBCs reaching 85 and 71%, respectively, while only 32% in the 20% (w/v) glycerol solution. This study demonstrates the potential of amino acid-based CPAs and reveals the synergistic effect of IRI activity and osmotic pressure regulation in cryopreservation.

[Molecular engineering and immobilization of lysine decarboxylase for synthesis of 1, 5-diaminopentane: a review].

1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.

Beetle and mussel-inspired chimeric protein for fabricating anti-icing coating.

Ice accretion on surfaces can cause serious damages and economic losses in industries and civilian facilities. Antifreeze proteins (AFPs) as evolutionary adaptation products of organisms to cold climates, provide solutions for alleviating icing problems. In this work, a chimeric protein Mfp-AFP was rationally designed combining mussel-inspired adhesive domain with Tenebrio molitor-derived antifreeze protein domain. Expectedly, the multifunctional Mfp-AFP can lower the freezing point of water and inhibit ice recrystallization. The chimeric protein could also readily modify diverse solid surfaces due to the adhesive domain containing Dopa, and resist frosting and delay ice formation due to the beetle-derived antifreeze fragment. Moreover, Mfp-AFP coatings display excellent biocompatibility proved by cytocompatibility and hemolysis assays. Here, the designed multifunctional protein coatings provide an alternative strategy for fabricating anti-icing surfaces.

Zwitterionic peptide-functionalized highly dispersed carbon nanotubes for efficient wastewater treatment.

Multi-walled carbon nanotubes (MWCNTs) have displayed great potential as catalyst carriers due to their nanoscale structure and large specific surface area. However, their hydrophobicity and poor dispersibility in water restrict their applications in aqueous environments. Herein, the dispersibility of MWCNTs was significantly enhanced with a chimeric protein MPKE which consisted of a zwitterionic peptide unit and a mussel adhesive protein unit. The MPKE could be easily attached to MWCNTs (MPKE-MWCNTs) by a simple stirring process due to the versatile adhesion ability of mussel adhesive unit. As expected, the MPKE-MWCNTs displayed outstanding dispersibility in water (>7 months), as well as in alkaline solutions (pH = 12) and organic solvents (DMSO and ethanol) due to the hydrophilicity of the zwitterionic peptide unit. Moreover, the MPKE-MWCNTs were used as silver nanoparticle carriers for the reduction of 4-nitrophenol in wastewater, with the normalized rate constant knor up to 32.9 s-1 mmol-1. Meanwhile, they also exhibited excellent biocompatibility and antibacterial activity, which were favorable for wastewater treatment. This work provides a facile strategy for MWCNT modification, functionalization and applications in aqueous environments.

Facile fabrication of a high-efficient and biocompatibility biocatalyst for bisphenol A removal.

In this study, we developed a smart microfluidic device to prepare biocatalyst HRP-pCBMA. HRP-pCBMA is composed of horseradish peroxidase (HRP) and zwitterionic polymers poly(carboxybetaine methacrylate) (pCBMA), and could be applied to biodegrade bisphenol A (BPA) efficiently. Compared to free HRP, HRP-pCBMA exhibited an obviously enhanced degrading capability for 1 mM BPA with 99.42% degradation efficiency within 20 min, even being superior to 20-fold amount of free HPR. Besides, HRP-pCBMA displayed high stability against the abrupt changes of environmental factors (temperature, pH and organic solvents), and HRP-pCBMA exhibited a relatively high BPA degradation rate of more than 90% even after 10 cycles. The Kcat and Vmax values of HRP-pCBMA were both 7-fold higher than that of free HRP, indicating significant improvement of the catalytic activity. Furthermore, the cytotoxicity assay indicated HRP-pCBMA has excellent biocompatibility. These results demonstrated that HRP-pCBMA possessed great potential in the bioremediation of BPA.

Engineered a novel pH-sensitive short major ampullate spidroin.

The pH diversification has been proved as an important factor affecting the self-assembly of spidroin. Herein, we constructed a novel spider silk protein (NT-MaSp1s-CT) with the pH-dependent secondary structures, containing pH-sensitive N-terminal, C-terminal domains and a repeated core region with merely 191 amino acids. Then pH sensitivity of NT-MaSp1s-CT was detected at different pH conditions and NT-MaSp1s-CT displayed pH-dependent conformational transitions consistent with rational designed objective. Besides, the micelles theory was employed to inquiry the assembly mechanism of NT-MaSp1s-CT in high concentration spinning dope. As expected, NT-MaSp1s-CT protein can be spun into continuous and uniform fibers with the pH ranging from 2 to 11, which is the largest pH boundary for artificial spider silk formation, simplifying the assembly conditions and paving a broad path for spinning process. Moreover, the hemolysis and cytotoxicity of NT-MaSp1s-CT fibers were also determined and the novel fibers exhibit excellent biocompatibility, providing wider potential applications in the biomedical and pharmaceutical fields.

Mussel-inspired triblock functional protein coating with endothelial cell selectivity for endothelialization.

Surface modification of biomaterials for rapid endothelialization is a promising approach for improving long-term patency of artificial vascular grafts (e.g. polytetrafluoroethylene, PTFE) with small-caliber vascular (<6 mm). However, surfaces modified with traditional strategies using hydrophilic polymers may be excessively hydrophilic to limit endothelial cell adhesion and formation of confluent endothelial lining. In this study, a triblock functional protein cofp-MZY/R was fabricated with cell selectivity of endothelial cells (ECs) over smooth muscle cells (SMCs) for endothelialization on PTFE. This rational designed triblock protein consisted of mussel-inspired domain, zwitterionic polypeptide and bioactive peptides (YIGSR and REDV), in which Dopa was efficiently obtained with residue-specificity in vivo. The triblock protein could facilely form coating on PTFE surface and the resulting protein coating exhibited moderate nonspecific resistance of protein and platelets. Together with bioactive peptides tail, it was available for cell attachment on surfaces. As protein material, this coating displayed remarkable biocompatibility through cytotoxicity and hemolysis measurements. Moreover, cellular behavior assay demonstrated that triblock protein coating could selectively promote adhesion, proliferation and migration of ECs rather than SMCs. This mussel-inspired triblock functional protein coating indicated a promising strategy for endothelialization of artificial vascular grafts.

Novel Mussel-Inspired Universal Surface Functionalization Strategy: Protein-Based Coating with Residue-Specific Post-Translational Modification in Vivo.

Surface functionalization can effectively endow materials with desirable properties, promoting the performance between the material and environment, with extensive applications. However, a universal and straightforward surface functionalization method with biocompatibility is scarce. In this study, with synthetic biology strategy, recombinant mussel plaque protein with a zwitterionic peptide inspired by molecular chaperone was engineered through post-translational modification, in which 3,4-dihydroxyphenylalanine was residue-specifically obtained efficiently from tyrosine with tyrosinase coexpressed in vivo. The rational designed chimeric protein coating in this work could successfully anchor to various substrates and exhibit excellent antifouling performance in resisting protein adsorption, cell attachment, and bacterial adhesion with eminent biocompatibility.

Bioinspired Multifunctional Protein Coating for Antifogging, Self-Cleaning, and Antimicrobial Properties.

A multifunctional coating with antifogging, self-cleaning, and antimicrobial properties has been prepared based on a mussel-inspired chimeric protein MP-KE, which is the first example that these proteins were successfully applied to fabricate antifogging surfaces. The coating exhibits super hydrophilic properties, as indicated by contact angles less than 5° and high light transmittance similar to bare glass substrates about 90%. The zwitterionic peptides of MP-KE empower water molecules to expand into thin hydrated films rapidly, providing the protein coating with diverse surface functions. Moreover, the coatings have excellent stability and a convenient preparation process because of the mussel adhesive motif of MP-KE which makes the coating anchor onto the surface strongly. As a protein material, this multifunctional coating possesses remarkable biocompatibility and has a potential application prospects in the biomedical and pharmaceutical fields.