Improved production of RNA-inhibiting antimicrobial peptide by Bacillus licheniformis MCC 2514 facilitated by a genetic algorithm optimized medium.

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH4Cl and NaNO3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup.

Rapid detection of sunset yellow adulteration in tea powder with variable selection coupled to machine learning tools using spectral data.

In the present study sunset yellow (SY), a synthetic colour, which is a common adulterant in tea powders has been analysed using FT-IR spectral data coupled to machine learning tools for efficient classification and quantification of the SY adulteration. Earlier established real coded genetic algorithm (RCGA) was used as variable selection method to predict the key fingerprints of SY in the FT-IR spectra. Here, RCGA was used to select 20, 30, 40, 50 and 60 characteristic wavenumbers for SY. Classification was carried using support vector machine (SVM), random forest (RF) and extreme gradient boosting (XGB) classifiers. SVM classifier using 50 variables could give an accuracy of 0.90 amongst the three. Quantification of SY based on PLS (partial least squares), LS-SVM (least squares-SVM), RF and XGBoost were built on characteristic wavenumbers. Both RF and LS-SVM models were observed to be superior to PLS when coupled to RCGA obtained 20 fingerprint variables. Overall, RCGA-LS-SVM model resulted in lowest RMSECV (0.1956) with regression co-efficient values RC 2 = 0.9989 and RP 2 = 0.9979, when 50 fingerprint variables were used. These results demonstrated that FT-IR combined with RCGA-LS-SVM procedure could be a robust technique for rapid detection of SY in tea powder. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05694-3.

Recent advances in the microbial production of squalene.

Squalene is a triterpene hydrocarbon, a biochemical precursor for all steroids in plants and animals. It is a principal component of human surface lipids, in particular of sebum. Squalene has several applications in the food, pharmaceutical, and medical sectors. It is essentially used as a dietary supplement, vaccine adjuvant, moisturizer, cardio-protective agent, anti-tumor agent and natural antioxidant. With the increased demand for squalene along with regulations on shark-derived squalene, there is a need to find alternatives for squalene production which are low-cost as well as sustainable. Microbial platforms are being considered as a potential option to meet such challenges. Considerable progress has been made using both wild-type and engineered microbial strains for improved productivity and yields of squalene. Native strains for squalene production are usually limited by low growth rates and lesser titers. Metabolic engineering, which is a rational strain engineering tool, has enabled the development of microbial strains such as Saccharomyces cerevisiae and Yarrowia lipolytica, to overproduce the squalene in high titers. This review focuses on key strain engineering strategies involving both in-silico and in-vitro techniques. Emphasis is made on gene manipulations for improved precursor pool, enzyme modifications, cofactor regeneration, up-regulation of limiting reactions, and downregulation of competing reactions during squalene production. Process strategies and challenges related to both upstream and downstream during mass cultivation are detailed.

In silico target-based strain engineering of Saccharomyces cerevisiae for terpene precursor improvement.

Systems-based metabolic engineering enables cells to enhance product formation by predicting gene knockout and overexpression targets using modeling tools. FOCuS, a novel metaheuristic tool, was used to predict flux improvement targets in terpenoid pathway using the genome-scale model of Saccharomyces cerevisiae, iMM904. Some of the key knockout target predicted includes LYS1, GAP1, AAT1, AAT2, TH17, KGD-m, MET14, PDC1 and ACO1. It was also observed that the knockout reactions belonged either to fatty acid biosynthesis, amino acid synthesis pathways or nucleotide biosynthesis pathways. Similarly, overexpression targets such as PFK1, FBA1, ZWF1, TDH1, PYC1, ALD6, TPI1, PDX1 and ENO1 were established using three different existing gene amplification algorithms. Most of the overexpression targets belonged to glycolytic and pentose phosphate pathways. Each of these targets had plausible role for improving flux toward sterol pathway and were seemingly not artifacts. Moreover, an in vitro study as validation was carried with overexpression of ALD6 and TPI1. It was found that there was an increase in squalene synthesis by 2.23- and 4.24- folds, respectively, when compared with control. In general, the rationale for predicting these in silico targets was attributed to either increasing the acetyl-CoA precursor pool or regeneration of NADPH, which increase the sterol pathway flux.

Truncation of C-terminal amino acids of GH26 endo-mannanase (ManB-1601) affects biochemical properties and stability against anionic surfactants.

Hitherto, the contribution of C-terminal amino acids in structure, stability and function of GH26 endo-mannanases has not been demonstrated. Semi-logarithmic plot of endo-mannanase activity showed a progressive decline with increase in the number of truncated amino acids [ManB-CΔ5 (129 U/mL), ManB-CΔ10 (47 U/mL), ManB-CΔ15 (0.05 U/mL) and ManB-CΔ20 (0.02 U/mL)]. ManB-CΔ5 and ManB-CΔ10 exhibited similar temperature and pH optima and product profile but biochemical properties (kinetic constants, mannan hydrolysis, response to metal ions and enzyme inhibitors) and stability (in presence of commercial detergents, anionic surfactants and organic solvents and half-life) were markedly affected. Interaction of truncated proteins with anionic surfactants was probed using intrinsic, Nile red, acrylamide quenching, resonance light scattering and synchronous fluorescence spectroscopy studies. Truncation of ten amino acids increased vulnerability to anionic surfactants as conformational changes, exposure of the hydrophobic core and susceptibility to unfolding process were observed. The microenvironment around Trp residues was affected more with surfactants as compared to Tyr residues in truncated proteins. Zn2+ coordination might not play a role in providing stability against SDS. MD simulation studies corroborated that C-terminal amino acids (327-336) helps in structure stabilization, regulating flexibility of loops around the active site and preventing denaturation in the presence of SDS.

Alternative splicing regulates the α-glucosidase synthesis in Aspergillus neoniger NCIM 1400.

Aspergillus neoniger NCIM 1400 whose cell-free fraction was earlier established for transglycosylation activity conferred by α-glucosidase gene (agdA), was subjected to sequence analysis. Preliminary results revealed certain dynamics in the intron splicing mechanism, and to ascertain these molecular events, a detailed study was carried. The electrophoresis results from the cDNA portion (B-fragment) of agdA showed multiple bands, indicating the amplification of one or more fragments. The sequence results of cDNA cloned vector revealed the retention type of alternative splicing in the agdA. The splicing mechanism of agdA in NCIM 1400 was compared to different A. niger strains, which harbours agdA orthologues, using PCR. It was observed that effective intron splicing leads to higher α-glucosidase activity from these selected Aspergillus spp. To explore the dynamics of intron retention in A. neoniger NCIM 1400, time-course analysis of intron retention, enzyme activity, and sugar consumption were carried over a period of 168 h of fungal growth. RT-qPCR results revealed that introns retention was not detected during the initial growth phase when the maltose and its hydrolysed product, glucose were consumed. Here we demonstrate that exhaustion of maltose causes increase in retention of introns in the mRNA transcripts of agdA gene, and this could be the possible mode of regulating this gene.

Adaptive evolution of engineered yeast for squalene production improvement and its genome-wide analysis.

In the present study, the adaptive evolution of a metabolically engineered Saccharomyces cerevisiae strain in the presence of an enzyme inhibitor terbinafine for enhanced squalene accumulation via serial transfer leads to the development of robust strains. After adaptation for nearly 1500 h, a strain with higher squalene production efficiency was identified at a specific growth rate of 0.28 h-1 with a final squalene titer of 193 mg/L, which is 16.5-fold higher than the BY4741 and 3-fold higher over the metabolically engineered SK22 strain. Whole-genome sequencing comparison between the reference strain and the evolved variant SK23 has led to the identification of 462 single-nucleotide variants (SNVs) between both strains, with 102 SNVs affecting metabolism-related genes. It was also established that F420I mutation of ERG1 in S. cerevisiae improves squalene synthesis. Further, the effect of increased squalene on lipid droplet and neutral lipid pattern in the evolved mutant strains was investigated by fluorescent techniques proving that the neutral lipid content and clustering of lipid droplets increase with an increase in squalene.

Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides.

A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L bioreactor. Specific growth rate of 0.1 h-1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.

Modified chemical method for efficient transformation and diagnosis in Pichia pastoris.

In the present study, green fluorescence protein (GFP) was used as a candidate protein to test and optimize a robust chemical transformation procedure in P. pastoris. Towards this, it was adjudged that pretreatment of P. pastoris cells with lithium chloride (LiCl) and its optimal concentration is critical for efficient transformation. Using three different methods (M1: 100 mM LiCl, 10 min, M2: 1 M LiCl, 10 min and M3: 1 M LiCl, 1 h), it was found that concentration and incubation time for LiCl treatment significantly affects the transformation efficiency. The transformation efficiency (transformants/µg DNA) was observed to be 1.01 × 102, 5.07 × 103 and 6.52 × 103 using methods M1, M2 and M3, respectively, indicating the superiority of M3. Moreover, presence of the GFP gene in the positive transformants was confirmed using a novel colony PCR method where the colonies were treated with LiCl prior to GFP specific amplification. Also, it was established using fluorescence microscopy and western blot analysis that increasing zeocin concentration as a post transformational vector amplification (PTVA) strategy increased the fluorescence and gene expression, respectively. Further, RT-qPCR revealed that the gene copy number using methods M1, M2 and M3 were 2.97, 5.26 and 7.19, respectively, when 500 µg/ml zecocin was used for selection, thus corroborating western blot results. In conclusion, we demonstrate a cheap and robust chemical method for achieving higher transformation efficiency in P. pastoris and a simple procedure for colony-PCR based-diagnosis alleviating the need for enzymatic treatment.

Studies on Squalene Biosynthesis and the Standardization of Its Extraction Methodology from Saccharomyces cerevisiae.

In this study, a homogenization-based extraction method was developed and was compared to five conventional methods of squalene extraction. Squalene recovered from this novel procedure gave 3.5-fold, 10-fold, 16-fold, and 8.1-fold higher yield than standard procedures, viz., saponification with 60% KOH, acidic saponification, saponification with 18% KOH, and glass beads method, respectively. Furthermore, this procedure has been evaluated on laboratory Saccharomyces cerevisiae strains such as BY4742 and CEN.PK2-1C (native), deletion strains (ERG6 and ERG11), and tHMG1 overexpressed S. cerevisiae strains. When sonication method of cell lysis was replaced with homogenization, it was found that the yields were significantly higher and reached a value of 9 mg/g DCW in case of BY4742. In addition, squalene yield in ergosterol mutant strains has been analyzed and was found to be 1.8-fold and 3.4-fold higher in ERG6 and ERG11 deletion strains, respectively, than in BY4742. Squalene was also found to be higher at the optimized temperature of 30 °C and pH 6.0. Furthermore, tolerance of S. cerevisiae to external squalene at various concentrations has been carried and found that the organism was tolerant up to 25 g/L of squalene.

Dynamic optimization of fed-batch bioprocesses using flower pollination algorithm.

There exist several optimization strategies such as sequential quadratic programming (SQP), iterative dynamic programing (IDP), stochastic-based methods such as differential evolution (DE), genetic algorithm (GA), particle swarm optimization (PSA), and ant colony optimization (ACO) for finding optimal feeding profile(s) during fed-batch fermentations. Here in the present study, flower pollination algorithm (FPA) which is inspired by the pollination process in terrestrial flowering plants has been used for the first time to find the optimal feeding profile(s) during fed-batch fermentations. Single control variable, two control variables and state variable bounded problems were chosen to test the robustness of the FPA for optimal control problems. It was observed that FPA is computationally less intensive in comparison with other stochastic strategies. Thus, obtained results were compared to other studies and it has been found that the FPA converged either to newer optima or closer to the established global optimum for the cases studied.

Fed-Batch Strategies for Production of PHA Using a Native Isolate of Halomonas venusta KT832796 Strain.

In this study, polyhydroxyalkanoates (PHA) accumulation by Halomonas venusta KT832796, a moderate halophilic bacteria isolated from marine source was studied. Both nutritional requirements and process parameters for submerged cultivation of the organism in bioreactor have been standardized. From the shake flask studies, glucose and ammonium citrate as carbon and nitrogen source produced maximum PHA at a ratio 20 with 3.52 g/L of dry cell weight and 70.56% of PHA content. However, ammonium sulfate as the nitrogen source was found to be more suitable for fed-batch cultivation. Several feeding strategies including pH-based fed-batch and variants of pulse feeding were studied to improve the PHA levels. pH-based feeding, although improved PHA level to 26 g/L, most of the carbon flux was diverted towards biomass formation; hence, the percent PHA was only 39.15% of the dry cell weight. Maximum PHA of 33.4 g/L, which corresponded to 88.12% of the dry cell, was obtained from high concentration single pulse method. There was a net 8.65-fold increase in PHA using this feeding strategy when compared to batch studies. According to our knowledge, this is the highest amount of PHA reported for a Halomonas venusta strain.

Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in Saccharomyces cerevisiae.

Although overexpression of the tHMG1 gene is a well-known strategy for terpene synthesis in Saccharomyces cerevisiae, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the tHMG1 gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes', viz., ZWF1 and POS5 (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length POS5 has improved squalene synthesis in cytosol. Finally, when tHMG1 and full-length POS5 were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in S. cerevisiae.

FOCuS: a metaheuristic algorithm for computing knockouts from genome-scale models for strain optimization.

Although handful tools are available for constraint-based flux analysis to generate knockout strains, most of these are either based on bilevel-MIP or its modifications. However, metaheuristic approaches that are known for their flexibility and scalability have been less studied. Moreover, in the existing tools, sectioning of search space to find optimal knocks has not been considered. Herein, a novel computational procedure, termed as FOCuS (Flower-pOllination coupled Clonal Selection algorithm), was developed to find the optimal reaction knockouts from a metabolic network to maximize the production of specific metabolites. FOCuS derives its benefits from nature-inspired flower pollination algorithm and artificial immune system-inspired clonal selection algorithm to converge to an optimal solution. To evaluate the performance of FOCuS, reported results obtained from both MIP and other metaheuristic-based tools were compared in selected case studies. The results demonstrated the robustness of FOCuS irrespective of the size of metabolic network and number of knockouts. Moreover, sectioning of search space coupled with pooling of priority reactions based on their contribution to objective function for generating smaller search space significantly reduced the computational time.

Progress in terpene synthesis strategies through engineering of Saccharomyces cerevisiae.

Terpenes are natural products with a remarkable diversity in their chemical structures and they hold a significant market share commercially owing to their distinct applications. These potential molecules are usually derived from terrestrial plants, marine and microbial sources. In vitro production of terpenes using plant tissue culture and plant metabolic engineering, although receiving some success, the complexity in downstream processing because of the interference of phenolics and product commercialization due to regulations that are significant concerns. Industrial workhorses' viz., Escherichia coli and Saccharomyces cerevisiae have become microorganisms to produce non-native terpenes in order to address critical issues such as demand-supply imbalance, sustainability and commercial viability. S. cerevisiae enjoys several advantages for synthesizing non-native terpenes with the most significant being the compatibility for expressing cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40 g/l of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to envisage both molecular and process strategies for developing yeast cell factories to produce these compounds. This review contains a brief consideration of existing strategies to engineer S. cerevisiae toward the synthesis of terpene molecules. Some of the common targets for synthesis of terpenes in S. cerevisiae are as follows: overexpression of tHMG1, ERG20, upc2-1 in case of all classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and sesquiterpenes could be some generic targets. Efforts are made to consolidate various studies (including patents) on this subject to understand the similarities, to identify novel strategies and to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the S. cerevisiae genome are discussed. This review also focuses on process considerations and challenges during the mass production of these potential compounds from the engineered strain for commercial exploitation.

Model-based estimation of optimal temperature profile during simultaneous saccharification and fermentation of Arundo donax.

A kinetic model fitted to enzymatic hydrolysis of Arundo donax was coupled to a fermentation kinetic model derived from simultaneous saccharification and fermentation (SSF) experiments at different temperatures for the determination of optimal temperature profile (between 36 and 45°C) using iterative dynamic programming (IDP). A sensitivity analysis of enzyme kinetic model not only facilitated model reduction in terms of number of parameters, but also enabled artifacts from parameter estimations to be identified. In separate fermentation experiments conducted at 35, 40, 45, and 50°C using ∼40 g/L background glucose in fiber-free liquid fraction of Arundo it was found that growth was possible at 40°C, but the fermentation capacity was completely lost after 12 h at 50°C. The final ethanol concentration obtained after 120 h in isothermal SSF experiments at 36, 39, 42, and 45°C were 10.6, 13.7, 14.2, and 12.5 g/L, respectively. The predicted optimal temperature profile in SSF determined by iterative dynamic programming was (i) gradual decrease from 40 to 37.5°C until 16 h, (ii) a linear increase upto 45°C until 80 h, and (iii) gradual decrease by 1°C until 120 h. Experimental results were in good agreement with the model predictions. The ethanol concentration after 72 h obtained in the optimal case was 13.6 g/L in comparison to 9.1, 12.2, 12.6, and 11.6 g/L for ISO-SSF at 36, 39, 42, and 45°C, respectively. Moreover this value was 95.8% of the final value achieved at the end of 120 h, indicating that the process times could be significantly shortened by using non-isothermal SSF.

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.

BACKGROUND: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. RESULTS: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. CONCLUSIONS: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.