Smart proteins as a new paradigm for meeting dietary protein sufficiency of India: a critical review on the safety and sustainability of different protein sources.

India, a global leader in agriculture, faces sustainability challenges in feeding its population. Although primarily a vegetarian population, the consumption of animal derived proteins has tremendously increased in recent years. Excessive dependency on animal proteins is not environmentally sustainable, necessitating the identification of alternative smart proteins. Smart proteins are environmentally benign and mimic the properties of animal proteins (dairy, egg and meat) and are derived from plant proteins, microbial fermentation, insects and cell culture meat (CCM) processes. This review critically evaluates the technological, safety, and sustainability challenges involved in production of smart proteins and their consumer acceptance from Indian context. Under current circumstances, plant-based proteins are most favorable; however, limited land availability and impending climate change makes them unsustainable in the long run. CCM is unaffordable with high input costs limiting its commercialization in near future. Microbial-derived proteins could be the most sustainable option for future owing to higher productivity and ability to grow on low-cost substrates. A circular economy approach integrating agri-horti waste valorization and C1 substrate synthesis with microbial biomass production offer economic viability. Considering the use of novel additives and processing techniques, evaluation of safety, allergenicity, and bioavailability of smart protein products is necessary before large-scale adoption.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Production of single cell oil by Lipomyces starkeyi from waste plant oil generated by the palm oil mill industry.

Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by Lipomyces starkeyi. In this study, the ability of L. starkeyi to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. L. starkeyi was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when L. starkeyi was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for L. starkeyi.

Genomic insights into Dactylosporangium sp. AC04546, a rubber degrader with three latex clearing proteins.

With more than 100 rubber-degrading strains being reported, only 9 Lcp proteins isolated from Nocardia, Gordonia, Streptomyces, Rhodococcus, Actinoplanes, and Solimonas have been purified and biochemically characterized. A new strain, Dactylosporangium sp. AC04546 (strain JCM34239), isolated from soil samples collected in Sarawak Forest, was able to grow and utilize natural or synthetic rubber as the sole carbon source. Complete genome of Strain AC04546 was obtained from the hybrid assembly of PacBio Sequel II and Illumina MiSeq. Strain AC04546 has a large circular genome of 13.08 Mb with a G+C content of 72.1%. The genome contains 11,865 protein-coding sequences with 3 latex clearing protein (lcp) genes located on its chromosome. The genetic organization of the lcp gene cluster is similar to two other reported rubber-degrading strains-Actinoplanes sp. OR16 and Streptomyces sp. CFMR 7. All 3 Lcp from strain AC04546 were expressed in Escherichia coli and exhibited degrading activity against natural rubber. The distinctiveness of strain AC04546, along with other characterized rubber-degrading strains, is reported here.

Recent advances in 3D bioprinting for cancer research: From precision models to personalized therapies.

Cancer remains one of the most devastating diseases, necessitating innovative and precise therapeutic solutions. The emergence of 3D bioprinting has revolutionized the platform of cancer therapy by offering bespoke solutions for drug screening, tumor modeling, and personalized medicine. The utilization of 3D bioprinting enables the fabrication of complex tumor models that closely mimic the in vivo microenvironment, facilitating more accurate drug testing and personalized treatment strategies. Moreover, 3D bioprinting also provides a platform for the development of implantable scaffolds as a therapeutic solution to cancer. In this review, we highlight the application of 3D bioprinting for cancer therapy along with current advancements in cancer 3D model development with recent case studies.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Development and Characterization of a Tunable Metal-Organic Framework (MOF) for the Synthesis of a Rare Sugar D-Tagatose.

D-tagatose is a valuable rare sugar with potential health benefits such as antiobesity, low-calorie, prebiotic, and anticancer. However, its production is mainly depending on chemical or enzymatic catalysis. Herein, a cobalt-based metal-organic framework (MOF) was developed at room temperature in an aqueous system using a self-assembly method. The L-arabinose isomerase (L-AI) was immobilized into this unique MOF by an in situ encapsulation process. The morphology and structural aspects of the MOF preparations were characterized by different analytical techniques such as scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FT-IR), and X-Ray diffraction (XRD). Moreover, thermogravimetric analysis (TGA) suggested the high thermal stability of the L-AI@MOF. Significantly, the immobilized catalyst exhibited enhanced catalytic efficiency (kcat/Km) of 3.22 mM-1 s-1 and improved turnover number (kcat) of 57.32 s-1. The L-AI@MOF efficiently catalyzes the synthesis of D-tagatose from D-galactose up to the equilibrium level (~ 50%) of isomerization in heterogeneous catalysis. Interestingly, L-AI@MOF was found stable and reusable for more than five cycles without the requirement of additional metal ions during catalysis. Thus, L-AI stabilized in the MOF system demonstrated a higher catalytic activity and potential guidance for the sustainable synthesis of rare sugar D-tagatose.

Effects of poly(3-hydroxybutyrate) [P(3HB)] coating on the bacterial communities of artificial structures.

The expanding urbanization of coastal areas has led to increased ocean sprawl, which has had both physical and chemical adverse effects on marine and coastal ecosystems. To maintain the health and functionality of these ecosystems, it is imperative to develop effective solutions. One such solution involves the use of biodegradable polymers as bioactive coatings to enhance the bioreceptivity of marine and coastal infrastructures. Our study aimed to explore two main objectives: (1) investigate PHA-degrading bacteria on polymer-coated surfaces and in surrounding seawater, and (2) comparing biofilm colonization between surfaces with and without the polymer coating. We applied poly(3-hydroxybutyrate) [P(3HB)) coatings on concrete surfaces at concentrations of 1% and 6% w/v, with varying numbers of coating cycles (1, 3, and 6). Our findings revealed that the addition of P(3HB) indeed promoted accelerated biofilm growth on the coated surfaces, resulting in an occupied area approximately 50% to 100% larger than that observed in the negative control. This indicates a remarkable enhancement, with the biofilm expanding at a rate roughly 1.5 to 2 times faster than the untreated surfaces. We observed noteworthy distinctions in biofilm growth patterns based on varying concentration and number of coating cycles. Interestingly, treatments with low concentration and high coating cycles exhibited comparable biofilm enhancements to those with high concentrations and low coating cycles. Further investigation into the bacterial communities responsible for the degradation of P(3HB) coatings identified mostly common and widespread strains but found no relation between the concentration and coating cycles. Nevertheless, this microbial degradation process was found to be highly efficient, manifesting noticeable effects within a single month. While these initial findings are promising, it's essential to conduct tests under natural conditions to validate the applicability of this approach. Nonetheless, our study represents a novel and bio-based ecological engineering strategy for enhancing the bioreceptivity of marine and coastal structures.

Efficacy of the collapsed cone algorithm calculated radiotherapy plans in intensity-modulated radiation therapy (IMRT) and volumetric-modulated arc therapy (VMAT): A comparative dosimetric study in tumors of thorax.

AIM: In this study, efficacy of collapsed cone algorithm-generated intensity-modulated radiation therapy (IMRT) and volumetric-modulated arc therapy (VMAT) were evaluated for treatment of thoracic esophageal cancer. MATERIALS AND METHODS: Ten previously treated patients with VMAT were considered for evaluation. The planning parameters were evaluated in terms of max dose, mean dose, Homogeneity Index, Conformity Index for planning target volume, and organ at risk doses. Total monitor unit, treatment time, and gamma passing index were also reported. RESULTS: The target dose coverage of the VMAT and IMRT plans achieved the clinical dosimetric criteria for all ten patients in the evaluation. Under the condition of equivalent target dose distribution, the VMAT plan's Conformity Index, monitor unit, treatment time, and gamma passing index rate were superior than in the IMRT plan, and the result was statistically significant. CONCLUSION: Collapsed cone algorithm-based VMAT can have a more effective and better approach for esophageal cancer than IMRT.

Evaluation of Radiation Treatment Planning Algorithms in IMRT and VMAT: A Comparative Dosimetric Study in Lung Equivalent Heterogeneous Medium.

Background: In Radiotherapy, computation of dose is important since in a small field with heterogeneity, dose is usually computed with discrepancies. Objective: The present study was aimed to evaluate the dosimetry of treatment planning algorithms in lung equivalent heterogeneous medium for Volumetric Modulated Arc Therapy (VMAT) with step and shoot Intensity-Modulated Radiation Therapy (ss-IMRT), and dynamic Intensity-Modulated Radiation Therapy (d-IMRT). Material and Methods: In this experimental study, Computerized Imaging Reference System (CIRS) phantom was used with an inhomogeneous Racemosa wood cylinder for two types of tumors, namely, Left Lung Central Tumor (LCT) and Left Lung Peripheral Tumor (LPT) in the CIRS left lung cavity. The computed tomography (CT) datasets were employed with the generation of VMAT, d-IMRT and ss-IMRT plans for the LCT and LPT irradiated with 6 MV photon beams. In this study, the accuracy and efficacy of two algorithms: Monte Carlo (MC) and the Pencil Beam (PB), from the Monaco treatment planning system (TPS), were tested by using Gafchromic EBT3 films and CIRS thorax phantom. Results: Regardless of treatment techniques, both algorithms exhibited higher divergence in LPT than LCT. In both LCT and LPT, the highest deviation was near the tumor-lung junction. However, the deviation was higher in the PB algorithm than MC algorithm, with a minimally acceptable variation of -0.8%. Conclusion: The MC algorithm shows more consistency for EBT3 measured dose in lung equivalent heterogeneous medium. However, accurate dose predictions are complicated due to electronic disequilibrium within and at the interface of inhomogeneity. These constraints may cause variations from the anticipated outcomes of the treatments.