Shake Flask Conditions Research Articles

Scale-up of CHO cell cultures: from 96-well-microtiter plates to stirred tank reactors across three orders of magnitude

Abstract Background For process development in mammalian cell cultivations, scale-up approaches are essential. A lot of studies concern the scale transfer between different-sized stirred tank reactors. However, process development usually starts in even smaller cultivation vessels like microtiter plates or shake flasks. A scale-up from those small shaken devices to a stirred tank reactor is barely stated in literature for mammalian cells. Thus, this study aims to address data-driven scale-up for CHO DP12 cells. The oxygen transfer rate is used as a database. Results The cultivation conditions in microtiter plates and shake flasks are comparable when choosing the maximum oxygen transfer capacity as a scale-up parameter. The minimum cultivation volume was reduced to 400 µL in round and square 96-deep-well microtiter plates. Using a scale-up based on the maximum oxygen transfer capacity to a stirred tank reactor led to conditions with excessive hydromechanical stress. However, cultivation conditions could be reproduced in a stirred tank reactor by utilizing the volumetric power input as a scale-up parameter. Key metabolites behaved the same in all three scales and the final antibody titer was equal. Conclusion This study presents a successful replication of cultivation results for mammalian cells in microtiter plates, shake flasks and stirred tank reactors. The working volumes ranged from 0.4 to 50 and 600 mL. It offers the opportunity to adapt the method to other, more sensitive mammalian cells and to perform cost- and time-effective experiments in high-throughput.

Yeast Synthesis and Herbicidal Activity Evaluation of Aspterric Acid.

Aspterric acid (AA) is a novel natural product herbicide that targets dihydroxyacid dehydratase in plants. In this study, we introduced two distinct AA biosynthesis-related gene clusters into Saccharomyces cerevisiae and screened the core biosynthetic enzyme, sesquiterpene cyclase, from various fungi. The combination of sesquiterpene cyclase from Aspergillus taichungensis IBT 19404 and two cytochrome P450s from Penicillium brasilianum resulted in the optimal AA synthesis efficiency in yeast, with the highest titer of 33.21 mg/L achieved by optimizing fermentation conditions in shake flasks. Moreover, the herbicidal effects of AA on weed germination and growth were evaluated. Notably, AA strongly inhibited the germination of Amaranthus tricolor, Portulaca oleracea, Bidens pilosa, Lolium perenne, and Leptochloa chinensis. Furthermore, AA could also inhibit the shoot and root growth of weeds with a superior inhibitory effect on roots relative to shoots. Our work not only provides a sustainable method for the biosynthesis of AA in yeast but also paves the way for the application of AA as a preemergence herbicide.

Solubilization and enhanced degradation of benzene phenolic derivatives-Bisphenol A/Triclosan using a biosurfactant producing white rot fungus Hypocrea lixii S5 with plant growth promoting traits.

Endocrine disrupting chemicals (EDCs) as benzene phenolic derivatives being hydrophobic partition to organic matter in sludge/soil sediments and show slow degradation rate owing to poor bioavailability to microbes. In the present study, the potential of a versatile white rot fungal isolate S5 identified as Hypocrea lixii was monitored to degrade bisphenol A (BPA)/triclosan (TCS) under shake flask conditions with concomitant production of lipopeptide biosurfactant (BS) and plant growth promotion. Sufficient growth of WRF for 5 days before supplementation of 50 ppm EDC (BPA/TCS) in set B showed an increase in degradation rates by 23% and 29% with corresponding increase in secretion of lignin-modifying enzymes compared to set A wherein almost 84% and 97% inhibition in fungal growth was observed when BPA/TCS were added at time of fungal inoculation. Further in set B, EDC concentration stimulated expression of laccase and lignin peroxidase (Lip) with 24.44 U/L of laccase and 281.69 U/L of Lip in 100 ppm BPA and 344 U/L Lip in 50 ppm TCS supplemented medium compared to their respective controls (without EDC). Biodegradation was also found to be correlated with lowering of surface tension from 57.02 mN/m (uninoculated control) to 44.16 mN/m in case of BPA and 38.49 mN/m in TCS, indicative of biosurfactant (BS) production. FTIR, GC-MS, and LC-ESI/MSMS confirmed the presence of surfactin lipopeptide isoforms. The WRF also displayed positive plant growth promoting traits as production of ammonia, indole acetic acid, siderophores, Zn solubilization, and 1-1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, reflecting its soil restoration ability. The combined traits of biosurfactant production, EDC degradation and plant growth promotion displayed by WRF will help in emulsifying the hydrophobic pollutants favoring their fast degradation along with restoration of contaminated soil in natural conditions.

Engineering Rhodosporidium toruloides for sustainable production of value-added punicic acid from glucose and wood residues

Rhodosporidium toruloides has emerged as a prominent candidate for producing single-cell oil from cost-effective feedstocks. In this study, the capability of R. toruloides to produce punicic acid (PuA), a representative plant unusual fatty acid, was investigated. The introduction of acyl lipid desaturase and conjugase (PgFADX) allowed R. toruloides to accumulate 3.7 % of total fatty acids as PuA. Delta-12 acyl lipid desaturase (PgFAD2) and diacylglycerol acyltransferase 2 were shown to benefit PuA production. The strain with PgFADX and PgFAD2 coexpression accumulated 12 % of its lipids as PuA from glucose, which translated into a PuA titer of 451.6 mg/L in shake flask condition. Utilizing wood hydrolysate as the feedstock, this strain produced 6.4 % PuA with a titer of 310 mg/L. Taken together, the results demonstrated that R. toruloides could serve as an ideal platform for the production of plant-derived high-value conjugated fatty acid using agricultural and forestry waste as feedstock.

Description and Assessment of an Antimicrobial Substance from Marine Actinobacteria

Streptomyces is thought to be the primary source of bacteria for the synthesis of bioactive substances, such as naturally occurring antibiotics. Finding and identifying the actinomycetes exhibiting antagonistic activity was the primary goal of the current investigation. A strain of actinomycetes that was isolated from marine sand samples that were collected off the Indian coast of Vedharanyam exhibited antimicrobial activity against a number of microbial pathogens. The nutritional requirements and cultural conditions for maximal growth and yield of bioactive compounds have been optimized under shake flask condition. Analyze the compound characterization of the sample such as UV, FT-IR, GC-MS and sequencing 16S rRNA for species identification. The actinomycetes isolates which are isolated from sediment sample can be further used as antimicrobial activity agents against bacterial and fungal pathogens and their compound characterization.

Isolation and Molecular Characterization of Polyester-Degrading Bacterial Strains from Municipal Wastewater and Their Application in Microplastic Degradation in Contaminated Wastewater

Diverse synthetic polymers find their regular application in routine life and are disposed of directly into the environment through various sources. The released microplastic and microfibres from the sources creates contamination in both aquatic and terrestrial environments. When these micropollutants enter the food chain, they have adverse effects on the environment and human health. This study focuses on the application of native bacterial strains isolated for the effective degradation of polyester microfibers. The isolated bacterial strains, namely MFB 6, MFB 8, and MFB 11, tolerant to polyethylene glycol (PEG), were identified as Bacillus sp., Paenibacillus sp., and Aeromonas veronii, respectively, with NCBI gene bank accession numbers ON318306, ON318390, and PP237260. Polyester biodegradation efficiency of 10.5%, 12.6% and 8.4% was achieved by MFB 6, MFB 8 and MFB 11, respectively, after 42 days of investigation in minimal salt medium under shake flask condition. In future the application of these microorganisms will help in sustainable management of synthetic microfiber pollutants and find their suitable application in micro plastic degradation in contaminated wastewater.

Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Pilot-Scale Fermentation of Pseudoalteromonas sp. Strain FDHY-MZ2: An Effective Strategy for Increasing Algicidal Activity.

The role of microorganisms in effectively terminating harmful algal blooms (HABs) is crucial for maintaining environmental stability. Recent studies have placed increased emphasis on bio-agents capable of inhibiting HABs. The bacterium Pseudoalteromonas sp. strain FDHY-MZ2 has exhibited impressive algicidal abilities against Karenia mikimotoi, a notorious global HAB-forming species. To augment this capability, cultures were progressively scaled from shake flask conditions to small-scale (5 L) and pilot-scale (50 L) fermentation. By employing a specifically tailored culture medium (2216E basal medium with 1.5% soluble starch and 0.5% peptone), under precise conditions (66 h, 20 °C, 450 rpm, 30 L/min ventilation, 3% seeding, and constant starch flow), a notable increase in algicidal bacterial biomass was observed; the bacterial dosage required to entirely wipe out K. mikimotoi within a day decreased from 1% to 0.025%. Compared to an unoptimized shake flask group, the optimized fermentation culture caused significant reductions in algal chlorophyll and protein levels (21.85% and 78.3%, respectively). Co-culturing induced increases in algal malondialdehyde and H2O2 by 5.98 and 5.38 times, respectively, leading to further disruption of algal photosynthesis. This study underscores the unexplored potential of systematically utilized microbial agents in mitigating HABs, providing a pathway for their wider application.

Utilization of lignocellulosic hydrolysates for photomixotrophic chemical production in Synechococcus elongatus PCC 7942

To meet the need for environmentally friendly commodity chemicals, feedstocks for biological chemical production must be diversified. Lignocellulosic biomass are an carbon source with the potential for effective use in a large scale and cost-effective production systems. Although the use of lignocellulosic biomass lysates for heterotrophic chemical production has been advancing, there are challenges to overcome. Here we aim to investigate the obligate photoautotroph cyanobacterium Synechococcus elongatus PCC 7942 as a chassis organism for lignocellulosic chemical production. When modified to import monosaccharides, this cyanobacterium is an excellent candidate for lysates-based chemical production as it grows well at high lysate concentrations and can fix CO2 to enhance carbon efficiency. This study is an important step forward in enabling the simultaneous use of two sugars as well as lignocellulosic lysate. Incremental genetic modifications enable catabolism of both sugars concurrently without experiencing carbon catabolite repression. Production of 2,3-butanediol is demonstrated to characterize chemical production from the sugars in lignocellulosic hydrolysates. The engineered strain achieves a titer of 13.5 g L−1 of 2,3-butanediol over 12 days under shake-flask conditions. This study can be used as a foundation for industrial scale production of commodity chemicals from a combination of sunlight, CO2, and lignocellulosic sugars.

OPTIMIZATION OF PROCESS PARAMETERS FOR IMPROVED PRODUCTION OF ANTIMICROBIAL METABOLITES FROM NOCARDIOPSIS FLAVESCENS VJMS-18 ISOLATED FROM SOUTH-COASTAL REGIONS OF ANDHRA PRADESH, INDIA

Objective: The objective of the present study is to optimize the cultural parameters for Nocardiopsis flavescens VJMS-18 strain isolated from marine sediment samples of the south-coast of Andhra Pradesh, India. Methods: The strain N. flavescensVJMS-18 was identified based on morphological, physiological, biochemical and molecular approaches. The effect of environmental parameters such as incubation period, pH, temperature and salt concentration and the effect of various nutrients such as carbon and nitrogen sources and minerals on the bioactive metabolite production by N. flavescens VJMS-18 was evaluated by employing agar well diffusion assay. Results: The nutritional requirements and cultural conditions to enhance the yield of secondary metabolites are optimized under shake flask conditions. ISP-2 medium supplemented with sodium chloride at 3% maintained at pH 7.0 supported the maximum yield of secondary metabolites by the strain when incubated at 35°C for 8 days. The strain exhibited a broad spectrum of antagonistic activity against Gram-positive (Staphylococcus aureus and Bacillus megaterium) and Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa) as well as fungus (Candida albicans). Conclusion: It was found that the antimicrobial metabolite production by the strain was positively influenced by carbohydrates, nitrogen sources and minerals.

Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry.

Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a paraBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development.

Fluctuating pH for efficient photomixotrophic succinate production

Cyanobacteria are attracting increasing attention as a photosynthetic chassis organism for diverse biochemical production, however, photoautotrophic production remains inefficient. Photomixotrophy, a method where sugar is used to supplement baseline autotrophic metabolism in photosynthetic hosts, is becoming increasingly popular for enhancing sustainable bioproduction with multiple input energy streams. In this study, the commercially relevant diacid, succinate, was produced photomixotrophically. Succinate is an important industrial chemical that can be used for the production of a wide array of products, from pharmaceuticals to biopolymers. In this system, the substrate, glucose, is transported by a proton symporter and the product, succinate, is hypothesized to be transported by another proton symporter, but in the opposite direction. Thus, low pH is required for the import of glucose and high pH is required for the export of succinate. Succinate production was initiated in a pH 7 medium containing bicarbonate. Glucose was efficiently imported at around neutral pH. Utilization of bicarbonate by CO2 fixation raised the pH of the medium. As succinate, a diacid, was produced, the pH of the medium dropped. By repeating this cycle with additional pH adjustment, those contradictory requirements for transport were overcome. pH affects a variety of biological factors and by cycling from high pH to neutral pH processes such as CO2 fixation rates and CO2 solubility can vary. In this study the engineered strains produced succinate during fluctuating pH conditions, achieving a titer of 5.0 g L−1 after 10 days under shake flask conditions. These results demonstrate the potential for photomixotrophic production as a viable option for the large-scale production of succinate.

Efficient secretion of mussel adhesion proteins using a chaperone protein Spy as fusion tag in Bacillus subtilis.

Mussel foot proteins (Mfps) are considered as remarkable materials due to their extraordinary adhesive capability. Recombinant expression is an ideal way to synthesis these proteins at large scale. However, secretory expression of Mfps into culture medium has not been achieved in a heterologous host. Here, to realize the secretion of Mfp3 and Mfp5 in Bacillus subtilis, signal peptide screening was first performed. Minimal Mfp3-6×His was targeted into the growth medium with AmyE signal peptide. We found that a small chaperone protein Spy was secreted efficiently in B. subtilis, and the fusion proteins Spy-Mfp3-6×His and Spy-Mfp5-6×His could also be delivered into growth medium well. The yield of Spy-Mfp3-6×His and Spy-Mfp5-6×His reached 255 and 119mgL-1 at shake flask conditions, respectively. Mfp3-6×His and Mfp5-6×His were finally purified via TEV protease cleavage and NTA affinity chromatography. Mfp3-6×His and Mfp5-6×His could be efficiently secreted using a chaperone protein Spy as fusion tag in B. subtilis.

Single batch co-production of Pseudomonad polyhydroxybutyrate and rhamnolipids on wasted cooking oil under salinity stress

Abstract Polyhydroxyalkanoates (PHAs) are emerging biodegradable bioplastics, and rhamnolipids are sustainable biosurfactants; both are considered as alternatives to synthetic petroleum-based plastics and surfactants, respectively. Herein, the effects of three distinct nitrogen sources on PHAs and rhamnolipid yields during the fermentation process have been undertaken. The study focuses on the optimum co-production of PHAs and rhamnolipid using a Pseudomonas aeruginosa strain grown on wasted soybean oil (WSO) with different nitrogen sources in the minimal salts’ media under salinity stress with shake flask conditions. The yields of PHAs and rhamnolipids were observed to be 2.20 g L−1 and 1.02 g L−1, respectively, with carbamide as the sole nitrogen and WSO (2 %, w/v) as the sole carbon source. The fermentative yields of both PHAs (2.37 g L−1) and rhamnolipids (1.24 g L−1) roughly increased up to 8 % under salinity stress. The products were characterized using advanced analytical techniques. The chemical structure of rhamnolipid with this strain had previously been elucidated, whereas the PHA produced was identified as a poly(hydroxybutyrate).

Citric Acid Production by Yarrowia lipolytica NRRL Y-1094: Optimization of pH, Fermentation Time and Glucose Concentration Using Response Surface Methodology

In this study, three Yarrowia lipolytica strains (Y. lipolytica NRRL Y-1094, Y. lipolytica NRRL YB-423 and Y. lipolytica IFP29) were screened for acid-production capacity and the maximum zone-area was formed by Y. lipolytica NRRL Y-1094. The strain was then selected as a potential citric-acid (CA) producer for further studies. The CA production by Y. lipolytica NRRL Y-1094 was optimized using response surface methodology (RSM) and considering three factors, comprising initial pH-value, fermentation time, and initial glucose-concentration. The highest CA-concentration was 30.31 g/L under optimum conditions (pH 5.5, 6 days, and 125 g/L glucose) in shake flasks. It has been reported that this result gives better results than many productions with shake flasks. According to estimated regression-coefficients for CA concentration, the fermentation time had the greatest impact on CA production, followed by the substrate concentration and initial pH-level, respectively. On the other hand, this study is a fundamental step in solving and optimizing the production mechanism of Y. lipolytica NRRL Y-1094, a microorganism that has not yet been used in CA production with a glucose-based medium. The results suggest that future studies can perform higher yields by optimizing other medium constituents and environmental factors.

Modulating DHA-Producing Schizochytrium sp. toward Astaxanthin Biosynthesis via a Seamless Genome Editing System.

Schizochytrium sp. is commercially used for the production of docosahexaenoic acid (DHA). Some strains of Schizochytrium sp. are also known to produce low amounts of carotenoids, including astaxanthin and β-carotene. In order to enhance the production of astaxanthin in Schizochytrium sp., we established a seamless genome editing system with a dual selection marker for rapid screening of positive transformants. By using this system, we strengthened the endogenous mevalonate pathway, enhanced the supply of geranylgeranyl diphosphate and β-carotene, upregulated endogenous β-carotene hydroxylase, and introduced the algal astaxanthin pathway. The highest astaxanthin production in the engineered Schizochytrium sp. was achieved at 8.1 mg/L (307.1 μg/g dry cell weight) under shake-flask conditions, which was 2.6-fold higher than that in the start strain. Meanwhile, the percentage of DHA to total fatty acids was not obviously affected. We then eliminated the dual selection marker by using the Cre-loxP recombination system, and the engineered strain was ready for iterative editing. The developed system could be applied to seamlessly engineer DHA-producing Schizochytrium sp. toward astaxanthin and other value-added terpenoids, which broadens the application of this strain.

Role of RpoS in Regulating Stationary Phase Salmonella Typhimurium Pathogenesis-Related Stress Responses under Physiological Low Fluid Shear Force Conditions.

The discovery that biomechanical forces regulate microbial virulence was established with the finding that physiological low fluid shear (LFS) forces altered gene expression, stress responses, and virulence of the enteric pathogen Salmonella enterica serovar Typhimurium during the log phase. These log phase LFS-induced phenotypes were independent of the master stress response regulator, RpoS (σS). Given the central importance of RpoS in regulating stationary-phase stress responses of S. Typhimurium cultured under conventional shake flask and static conditions, we examined its role in stationary-phase cultures grown under physiological LFS. We constructed an isogenic rpoS mutant derivative of wild-type S. Typhimurium and compared the ability of these strains to survive in vitro pathogenesis-related stresses that mimic those encountered in the infected host and environment. We also compared the ability of these strains to colonize (adhere, invade, and survive within) human intestinal epithelial cell cultures. Unexpectedly, LFS-induced resistance of stationary-phase S. Typhimurium cultures to acid and bile salts stresses did not rely on RpoS. Likewise, RpoS was dispensable for stationary-phase LFS cultures to adhere to and survive within intestinal epithelial cells. In contrast, the resistance of these cultures to challenges of oxidative and thermal stresses, and their invasion into intestinal epithelial cells was influenced by RpoS. These findings expand our mechanistic understanding of how physiological fluid shear forces modulate stationary-phase S. Typhimurium physiology in unexpected ways and provide clues into microbial mechanobiology and nuances of Salmonella responses to microenvironmental niches in the infected host. IMPORTANCE Bacterial pathogens respond dynamically to a variety of stresses in the infected host, including physical forces of fluid flow (fluid shear) across their surfaces. While pathogens experience wide fluctuations in fluid shear during infection, little is known about how these forces regulate microbial pathogenesis. This is especially important for stationary-phase bacterial growth, which is a critical period to understand microbial resistance, survival, and infection potential, and is regulated in many bacteria by the general stationary-phase stress response protein RpoS. Here, we showed that, unlike conventional culture conditions, several stationary-phase Salmonella pathogenic stress responses were not impacted by RpoS when bacteria were cultured under fluid shear conditions relevant to those encountered in the intestine of the infected host. These findings offer new insight into how physiological fluid shear forces encountered by Salmonella during infection might impact pathogenic responses in unexpected ways that are relevant to their disease-causing ability.

Production of fatty acids by engineered Ogataea polymorpha

Fatty acids (FA) are widely used as feed stocks for the production of cosmetics, personal hygiene products, lubricants and biofuels. Ogataea polymorpha is considered as an ideal chassis for bio-manufacturing, due to its outstanding characteristics such as methylotroph, thermal-tolerance and wide substrate spectrum. In this study, we harnessed O. polymorpha for overproduction of fatty acids by engineering its fatty acid metabolism and optimizing the fermentation process. The engineered strain produced 1.86 g/L FAs under the optimized shake-flask conditions (37℃, pH 6.4, a C/N ratio of 120 and an OD600 of seed culture of 6-8). The fed-batch fermentation process was further optimized by using a dissolved oxygen (DO) control strategy. The C/N ratio of initial medium was 17.5, and the glucose medium with a C/N ratio of 120 was fed when the DO was higher than 30%. This operation resulted in a titer of 18.0 g/L FA, indicating the potential of using O. polymorpha as an efficient cell factory for the production of FA.

Life cycle assessment of bioethanol production fromPennisetumsp. using fed‐batch simultaneous saccharification and cofermentation at high solid loadings

Considerable progress has been achieved for the production of bioethanol from lignocellulosic biomass. However, increasing the substrate concentration has been shown to decrease ethanol productivity. Therefore, Saccharomyces cerevisiae (S. cerevisiae) and Pichia sp. were used for ethanol production from glucose and xylose sugars in the present investigation. Furthermore, coculture fermentations were conducted for 13 g/L of sugar (10 g of glucose and 3 g/L of xylose). It was further used for ethanol production from ultrasonication-assisted NaOH (UA-NaOH) pretreated and enzymatically saccharified biomass in batch and fed-batch fermentation conditions. Furthermore, fed-batch fermentation was used for separate hydrolysis and cofermentation (SHCF) and simultaneous saccharification and cofermentation (SSCF) in shake flask conditions. The highest ethanol production of 12.2 and 7.9 g/L was observed for fed-batch SSCF deenanath grass (DG) and hybrid Napier grass (HNG) (Palkonal MBW as the enzyme) biomass (80 g/L), respectively, in shake flask conditions. However, increasing the biomass concentration to 270 g/L produced an ethanol concentration of 77.6 and 51.3 g/L for DG and HNG, respectively, in fed-batch SSCF conditions in a bioreactor. Furthermore, nuclear magnetic resonance studies of the residual biomass of DG revealed lower carbohydrate content, demonstrating the efficiency of the fermentation strategy. Life cycle analysis of DG and HNG revealed DG had a higher yield as well as lower environmental impact than HNG. Thus, the fed-batch SSCF process can be considered a promising technique for biorefinery-based bioethanol production from Pennisetum sp. in the future. Novelty Statement For making lignocellulosic fuel ethanol economically competitive as compared to fossil fuels, it is important to achieve higher titers of ethanol from increased substrate concentration. But an increase in the substrate concentration decreases the ethanol productivity. In this study, the fed-batch simultaneous saccharification and fermentation produced higher titers of about 77.6 g/L ethanol even at higher substrate loadings. Besides, The LCA results demonstrated lower environmental toxicity, indicating the efficiency of the pretreatment and simultaneous saccharification and cofermentation process.

Functional characterization and substrate promiscuity of sesquiterpene synthases from Tripterygium wilfordii

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii, we observed that Ses-TwTPS1-1 and Ses-TwTPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses-TwTPS1-2, Ses-TwTPS3, and Ses-TwTPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E, E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses-TwTPS1-1:V199I and Ses-TwTPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio.