Bioprocess For Production Research Articles

Overcoming Bacteriophage Contamination in Bioprocessing: Strategies and Applications.

Bacteriophage contamination has a devastating impact on the viability of bacterial hosts and can significantly reduce the productivity of bioprocesses in biotechnological industries. The consequences range from widespread fermentation failure to substantial economic losses, highlighting the urgent need for effective countermeasures. Conventional prevention methods, which focus primarily on the physical removal of bacteriophages from equipment, bioprocess units, and the environment, have proven ineffective in preventing phage entry and contamination. The coevolutionary dynamics between phages and their bacterial hosts have spurred the development of a diverse repertoire of antiviral defense mechanisms within microbial communities. These naturally occurring defense strategies can be harnessed through genetic engineering to convert phage-sensitive hosts into robust, phage-resistant cell factories, providing a strategic approach to mitigate the threats posed by bacteriophages to industrial bacterial processes. In this review, an overview of the various defense strategies and immune systems that curb the propagation of bacteriophages and highlight their applications in fermentation bioprocesses to combat phage contamination is provided. Additionally, the tactics employed by phages to circumvent these defense strategies are also discussed, as preventing the emergence of phage escape mutants is a key component of effective contamination management.

Chemical composition and bio-efficacy of agro-waste plant extracts and their potential as bioinsecticides against Culex pipiens mosquitoes

Mosquitoes are considered one of the most lethal creatures on the planet and are responsible for millions of fatalities annually through the transmission of several diseases to humans. Green trash is commonly employed in agricultural fertilizer manufacturing and microbial bioprocesses for energy production. However, there is limited information available on the conversion of green waste into biocides. This study investigates the viability of utilizing green waste as a new biopesticide against Culex pipiens mosquito larvae. The current study found that plant extracts from Punica granatum (98.4 % mortality), Citrus sinensis (92 % mortality), Brassica oleracea (88 % mortality), Oryza sativa (81.6 % mortality), and Colocasia esculenta (53.6 % mortality) were very good at killing Cx. pipiens larvae 24 h post-treatment. The LC50 values were 314.43, 370.72, 465.59, 666.67, and 1798.03 ppm for P. granatum, C. sinensis, B. oleracea, O. sativa, and C. esculenta, respectively. All plant extracts, particularly P. granatum extract (14.93 and 41.87 U/g), showed a significant reduction in acid and alkaline phosphate activity. Additionally, pomegranate extract showed a significant decrease (90 %) in field larval density, with a stability of up to five days post-treatment. GC–MS results showed more chemical classes, such as terpenes, esters, fatty acids, alkanes, and phenolic compounds. HPLC analysis revealed that the analyzed extracts had a high concentration of phenolic and flavonoid components. Moreover, there are many variations among these plants in the amount of each compound. The docking interaction showed a simulation of the atomic-level interaction between a protein and a small molecule through the binding site of target proteins, explaining the most critical elements influencing the enzyme's activity or inhibitions. The study's findings showed that the various phytochemicals found in agro-waste plants had high larvicidal activity and provide a safe and efficient substitute to conventional pesticides for pest management, as well as a potential future in biotechnology.

BayesianSSA: a Bayesian statistical model based on structural sensitivity analysis for predicting responses to enzyme perturbations in metabolic networks

BackgroundChemical bioproduction has attracted attention as a key technology in a decarbonized society. In computational design for chemical bioproduction, it is necessary to predict changes in metabolic fluxes when up-/down-regulating enzymatic reactions, that is, responses of the system to enzyme perturbations. Structural sensitivity analysis (SSA) was previously developed as a method to predict qualitative responses to enzyme perturbations on the basis of the structural information of the reaction network. However, the network structural information can sometimes be insufficient to predict qualitative responses unambiguously, which is a practical issue in bioproduction applications. To address this, in this study, we propose BayesianSSA, a Bayesian statistical model based on SSA. BayesianSSA extracts environmental information from perturbation datasets collected in environments of interest and integrates it into SSA predictions.ResultsWe applied BayesianSSA to synthetic and real datasets of the central metabolic pathway of Escherichia coli. Our result demonstrates that BayesianSSA can successfully integrate environmental information extracted from perturbation data into SSA predictions. In addition, the posterior distribution estimated by BayesianSSA can be associated with the known pathway reported to enhance succinate export flux in previous studies.ConclusionsWe believe that BayesianSSA will accelerate the chemical bioproduction process and contribute to advancements in the field.

Combined bioprocess for fermentative hydrogen production from food waste: A review

Bio hydrogen is a cheaper, sustainable and safer source to produce fuel comparable to energy obtained from fossil fuels. There are many experimental methods to produce bio hydrogen using food wastes as substrates that are acted upon by specific bacterial and fungal strains. Some of the methods include batch-dark fermentation, solid-state dark fermentation, dark-anaerobic hydrogen fermentation and integrated light-dark fermentation. Different food wastes are used in these fermentation processes such as kitchen food waste, potatoes peels, sugary waste water, fish, meats, grains, cassava residues, corn pulp and starchy solution etc. These food wastes are rich source of main raw materials that are required for bio hydrogen production such as cellulose, carbohydrates, fats, proteins, lipids, starch, phosphorus, volatile solids, Published experimental and research approaches revealed that the use of mixed dark-photo fermentative bacterial consortium in flat photo bioreactors and fermenters resulted in higher yield. Combined dark-photo fermentation is an advanced and promising strategy for increasing overall yield of bio hydrogen.

Design of Thermoresponsive Genetic Controls with Minimal Heat-Shock Response.

As temperature serves as a versatile input signal, thermoresponsive genetic controls have gained significant interest for recombinant protein production and metabolic engineering applications. The conventional thermoresponsive systems normally require the continuous exposure of heat stimuli to trigger the prolonged expression of targeted genes, and the accompanied heat-shock response is detrimental to the bioproduction process. In this study, we present the design of thermoresponsive quorum-sensing (ThermoQS) circuits to make Escherichia coli record transient heat stimuli. By conversion of the heat input into the accumulation of quorum-sensing molecules such as acyl-homoserine lactone derived from Pseudomonas aeruginosa, sustained gene expressions were achieved by a minimal heat stimulus. Moreover, we also demonstrated that we reprogrammed the E. coli Lac operon to make it respond to heat stimuli with an impressive signal-to-noise ratio (S/N) of 15.3. Taken together, we envision that the ThermoQS systems reported in this study are expected to remarkably diminish both design and experimental expenditures for future metabolic engineering applications.

Scalable bioprocess for high-yield production of SARS-CoV-2 trimeric spike protein-based immunogen (IMT-CVAX) using suspension CHO cells

The COVID-19 pandemic has brought global attention towards the readiness of biotechnology platforms for rapid development of immunogens to be used as vaccine antigens, in diagnostics and in fundamental research. In the present study we provide a bioprocess that is both robust and industry-compatible for high level expression, efficient purification, and scale-up of IMT-CVAX, a prefusion-stabilized trimeric spike protein of SARS-CoV-2. The recombinant IMT-CVAX protein was produced using stable cell pool of Chinese Hamster Ovary (CHO-S) cells that were banked in compliance with cGMP (Current Good Manufacturing Practices) regulations. High quantity expression of IMT-CVAX was achieved through optimization of the shake flask-based process with regards to media, nutrient feeding regimen, incubation temperature, viable cell density and cell viability. We employed refined expression procedures to scale up IMT-CVAX using bioreactor, resulting in a significantly higher yield of around 500 mg/L. Subsequently, a straightforward and simple purification process was developed to produce high-quality IMT-CVAX protein. This procedure consisted of centrifugation, tangential flow filtration (TFF), and either one- or two-step liquid chromatography. A robust anti-spike IgG response was observed in mice after immunization with purified adjuvanted IMT-CVAX. In pseudoviral neutralization assay, mice-generated anti-IMT-CVAX sera could neutralize various SARS-CoV-2 variants. The human convalescent sera from COVID-19-recovered patients recognized IMT-CVAX effectively, which confirms the conformational integrity of IMT-CVAX epitopes. The bioprocess demonstrated here is able to produce sufficient quantities of biopharmaceutical-quality spike protein, which can be used as immunogen against emerging SARS-CoV-2 variants, and can aid in the rapid response to any other pandemic-potential pathogens.

Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO–K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO–K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

Inline monitoring of lactobionic acid production from cheese whey by Pseudomonas taetrolens in a stirred bioreactor using electrical conductivity

AbstractIn this study, we introduce a novel experimental approach and present a simplified mathematical model for a quick monitoring of a biotec process producing lactobionic acid (LBA). It relies on monitoring the electrical conductivity of the fermentation broth and it is designed to predict the concentration of LBA throughout the microbial cheese whey valorization via LBA production. Following a systematic series of experiments conducted to refine the mathematical model, we performed conductivity monitoring during LBA production from “caciotta” and “squacquerone” wheys by Pseudomonas taetrolens in a 3 L stirred tank bioreactor. Throughout the bioproduction process, the conductivity values exhibited an upward trend corresponding to the increase in LBA concentration. Our findings underscore the feasibility and advantages of employing inline conductivity monitoring during LBA production from various cheese wheys. The results emphasize that conductivity measurements can effectively estimate product concentration in a fermentation process, particularly when there is a shift in ionic concentration. Furthermore, these conductivity measurements offer valuable insights for monitoring and optimizing the working conditions in a fermentation process.

Integrated bioprocess for carbon dioxide sequestration and methanol production

Carbon dioxide (CO2) poses a significant threat, contributing to global warming and climate change. This study focused on isolating efficient CO2-reducing methanogens and methanotrophs for converting methane into methanol. Samples from diverse regions in India were collected and processed, yielding 82 methanogenic and 48 methylotrophic isolates. Methanogenic isolate M11 produced a higher amount of methane, reaching 2.9 mol L−1 on the sixth day of incubation at 35 °C, pH 7.0, and CO2:H2 (80:20) as feeding rates. Under optimized conditions, isolate M11 effectively converted 8.3 mol CO2 to 7.9 mol methane in 24 h. Methylotrophic isolate M31 demonstrated significant soluble methane monooxygenase activity (450 nmol/ml) and produced 0.4 mol methanol in 24 h. 16S rRNA analysis identified Methanobacterium sp. and Methyloceanibacter sp. among the isolates, elucidating their taxonomic diversity. This study offers valuable insights into methanogens’ potential in CO2 sequestration and methane conversion to methanol through methanotrophism, a promising sustainable biofuel production.

A novel bio-based water reducer for concrete application: Facile process and techno-economic analysis of xylonate bioproduction from lignocellulosic residues

The xylonate was bioproduced directly from lignocellulosic residues, and the application properties of crude xylonate powder were investigated with a promising potential as a bio-based concrete water reducer. The overall integrated process was evaluated on the view of mass balance and techno-economic analysis using Aspen Plus modeling. 56 g/L of calcium xylonate (XA-Ca) can be obtained within 8 h from acidic corncob hydrolysate (ACH) at a 100 % yield with the whole-cell catalysis of Gluconobacter oxydans. At the addition of 0.2 wt% in the dry concrete content, XA-Ca powder and sodium xylonate powder prepared from pure xylose and ACH could reduce water used in concrete by 15 %, 14 %, 13 %, and 13 %, respectively. XA-Ca from ACH led to a 33% increase in concrete compressive strength and a 24% increase in concrete flexural strength at day 7 compared to the blank group. The techno-economic analysis showed the minimum XA-Ca powder price to be $ 0.806/kg which can be economically feasible. This study provides a practical and economical process prototype for the industrial concrete water reducer application of novel bio-based xylonate produced from lignocellulosic residues.

Customized 16S-23S rDNA ITS Amplicon Metagenomics for Acetic Acid Bacteria Species Identification in Vinegars and Kombuchas.

Acetic acid bacteria (AAB) are involved in food and beverage production bioprocesses, like those in vinegar and kombucha. They oxidize sugars and alcohols into various metabolites, resulting in the final products' pleasant taste and aroma. The 16S rDNA amplicon metagenomics using Illumina technology is usually used to follow the microbiological development of these processes. However, the 16S rRNA gene sequences among different species of AAB are very similar, thus not enabling a reliable identification down to the species level but only to the genus. In this study, we have constructed primers for amplifying half of the 16S-23S rRNA gene internal transcribed spacer (ITS) for library construction and further sequencing using Illumina technology. This approach was successfully used to estimate the relative abundance of AAB species in defined consortia. Further application of this method for the analysis of different vinegar and kombucha samples proves it suitable for assessing the relative abundance of AAB species when these bacteria represent a predominant part of a microbial community.

Exploring predictive kinetic modelling of thermal degradation from laboratory to production scale – A case study on three vitamins in milk

Kinetic thermal degradation models are vital components for optimization of food and bioproducts processing. Typically, models are fitted to laboratory-scale experiments where vials are heated and held. However, these conditions are highly dissimilar from the thermal processes experienced in industrial production. Whereas fitting kinetic data to industrial-scale production is often impossible due to cost and flexibility issues, this discrepancy between what is typically measured and what the models are intended for could be of concern. This study presents a case study where traditional laboratory experiments are fitted to vitamin degradation kinetic models in milk (vitamins B1, B2, and E). The best-fitted kinetic models are then validated using five, carefully controlled industrial-scale dairy processes. Results show that predictions are surprisingly close to the validation data (mean relative percentual deviation is −1.1 %–+3 % depending on vitamin), given the substantial difference between the laboratory and production scale setup. This offers empirical support for the conventional method of fitting kinetic parameters through simplified laboratory experiments to predict vitamin degradation during industrial scale processing of foods and bioproducts.

Non-sterile cultivation of oleaginous organisms

Cultivating oleaginous organisms in non-sterile conditions can reduce the energy and cost of microbial oil production. Recent studies use strategies that enable non-sterile cultivation without affecting bioprocess productivity. This forum article discusses the trends, strategies, and prospects of non-sterile cultivation, as successful non-sterile cultivation could make microbial oil production economically viable.

Bioprocess in-line monitoring and control using Raman spectroscopy and Indirect Hard Modeling (IHM).

Process in-line monitoring and control are crucial to optimize the productivity of bioprocesses. A frequently applied Process Analytical Technology (PAT) tool for bioprocess in-line monitoring is Raman spectroscopy. However, evaluating bioprocess Raman spectra is complex and calibrating state-of-the-art statistical evaluation models is effortful. To overcome this challenge, we developed an Indirect Hard Modeling (IHM) prediction model in a previous study. The combination of Raman spectroscopy and the IHM prediction model enables non-invasive in-line monitoring of glucose and ethanol mass fractions during yeast fermentations with significantly less calibration effort than comparable approaches based on statistical models. In this study, we advance this IHM-based approach and successfully demonstrate that the combination of Raman spectroscopy and IHM is capable of not only bioprocess monitoring but also bioprocess control. For this purpose, we used this combination's in-line information as input of a simple on-off glucose controller to control the glucose mass fraction in Saccharomyces cerevisiae fermentations. When we performed two of these fermentations with different predefined glucose set points, we achieved similar process control quality as approaches using statistical models, despite considerably smaller calibration effort. Therefore, this study reaffirms that the combination of Raman spectroscopy and IHM is a powerful PAT tool for bioprocesses.

Multivariate data analysis on multisensor measurement for inline process monitoring of adenovirus production in HEK293 cells.

In the era of Biopharma 4.0, process digitalization fundamentally requires accurate and timely monitoring of critical process parameters (CPPs) and quality attributes. Bioreactor systems are equipped with a variety of sensors to ensure process robustness and product quality. However, during the biphasic production of viral vectors or replication-competent viruses for gene and cell therapies and vaccination, current monitoring techniques relying on a single working sensor can be affected by the physiological state change of the cells due to infection/transduction/transfection step required to initiate production. To address this limitation, a multisensor (MS) monitoring system, which includes dual-wavelength fluorescence spectroscopy, dielectric signals, and a set of CPPs, such as oxygen uptake rate and pH control outputs, was employed to monitor the upstream process of adenovirus production in HEK293 cells in bioreactor. This system successfully identified characteristic responses to infection by comparing variations in these signals, and the correlation between signals and target critical variables was analyzed mechanistically and statistically. The predictive performance of several target CPPs using different multivariate data analysis (MVDA) methods on data from a single sensor/source or fused from multiple sensors were compared. An MS regression model can accurately predict viable cell density with a relative root mean squared error (rRMSE) as low as 8.3% regardless of the changes occurring over the infection phase. This is a significant improvement over the 12% rRMSE achieved with models based on a single source. The MS models also provide the best predictions for glucose, glutamine, lactate, and ammonium. These results demonstrate the potential of using MVDA on MS systems as a real-time monitoring approach for biphasic bioproduction processes. Yet, models based solely on the multiplicity and timing of infection outperformed both single-sensor and MS models, emphasizing the need for a deeper mechanistic understanding in virus production prediction.

A non-aseptic bioprocess for production and recovery of 2,3-butanediol via conversion of crude glycerol and corn steep liquor at pilot-scale

The production and recovery of 2,3-butanediol (BDO) through biodiesel derived glycerol valorization by Klebsiella oxytoca ACA-DC 1581 was holistically optimized with regard to the efficiency and cost of the bioprocess. The absence of thermal treatment of the substrate had no negative effect upon the growth of microorganism and the bioconversion of crude glycerol into BDO, enabling the development of a non-aseptic and lower-cost bioprocess. Both digestate and corn steep liquor (CSL), the main by-products of the biogas and corn industries respectively, successfully served as the sole source of nitrogen, contributing to the complete replacement of more expensive sources (e.g., yeast extract). The biochemical pathway of glycerol catabolism was examined under varying concentrations of dissolved oxygen and BDO production was optimized in a fully aerobic environment (volumetric mass transfer coefficient; kLa = 70.5 1/h.) The glycerol consumption rate was 2.80 g/L/h, the BDO productivity reached 1.12 g/L/h and the yield of BDO produced per unit of glycerol consumed was 0.46 g/g, with these values being among the highest ones reported in the literature for wild-type strains cultivated on crude glycerol. In all fed-batch fermentations, final BDO and acetoin concentration reached ∼80 g/L, while a plateau was observed at ∼68 g/L of BDO. Finally, the culture was carried out efficiently in the pilot-scale reactor (250 L). The salting-out extraction (SOE), consisting of ethanol (24 %) and K2HPO4 (25 %), recovered 91.7 % of BDO from the fermentation medium and was studied for the first time in a glycerol-based medium. The study suggests the potential industrialization of the bioprocess through sustainable, pilot-scale and low-cost bioconversion of biodiesel-derived crude glycerol and CSL or digestate into BDO.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.

Development of an Integrated Bioprocess System for Bioethanol and Arabitol Production from Sugar Beet Cossettes.

An innovative integrated bioprocess system for bioethanol production from raw sugar beet cossettes (SBC) and arabitol from remaining exhausted sugar beet cossettes (ESBC) was studied. This integrated three-stage bioprocess system is an example of the biorefinery concept to maximise the use of raw SBC for the production of high value-added products such as sugar alcohols and bioethanol. The first stage of the integrated bioprocess system was simultaneous sugar extraction from SBC and its alcoholic fermentation to produce bioethanol in an integrated bioreactor system (vertical column bioreactor and stirred tank bioreactor) containing a high-density suspension of yeast Saccharomyces cerevisiae (30 g/L). The second stage was the pretreatment of ESBC with dilute sulfuric acid to release fermentable sugars. The resulting liquid hydrolysate of ESBC was used in the third stage as a nutrient medium for arabitol production by non-Saccharomyces yeasts (Spathaspora passalidarum CBS 10155 and Spathaspora arborariae CBS 11463). The obtained results show that the efficiency of bioethanol production increased with increasing temperature and prolonged residence time in the integrated bioreactor system. The maximum bioethanol production efficiency (87.22 %) was observed at a time of 60 min and a temperature of 36 °C. Further increase in residence time (above 60 min) did not result in the significant increase of bioethanol production efficiency. Weak acid hydrolysis was used for ESBC pretreatment and the highest sugar yield was reached at 200 °C and residence time of 1 min. The inhibitors of the weak acid pretreatment were produced below bioprocess inhibition threshold. The use of the obtained liqiud phase of ESBC hydrolysate for the production of arabitol in the stirred tank bioreactor under constant aeration clearly showed that S. passalidarum CBS 10155 with 8.48 g/L of arabitol (YP/S=0.603 g/g and bioprocess productivity of 0.176 g/(L.h)) is a better arabitol producer than Spathaspora arborariae CBS 10155. An innovative integrated bioprocess system for the production of bioethanol and arabitol was developed based on the biorefinery concept. This three-stage bioprocess system shows great potential for maximum use of SBC as a feedstock for bioethanol and arabitol production and it could be an example of a sustainable 'zero waste' production system.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing

BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Just around the Corner: Advances in the Optimization of Yeasts and Filamentous Fungi for Lactic Acid Production.

Lactic acid (LA) production has seen significant progress over the past ten years. LA has seen increased economic importance due to its broadening use in different sectors such as the food, medicine, polymer, cosmetic, and pharmaceutical industries. LA production bioprocesses using microorganisms are economically viable compared to chemical synthesis and can benefit from metabolic engineering for improved productivity, purity, and yield. Strategies to optimize LA productivity in microorganisms on the strain improvement end include modifying metabolic routes, adding gene coding for lactate transporters, inducing tolerance to organic acids, and choosing cheaper carbon sources as fuel. Many of the recent advances in this regard have involved the metabolic engineering of yeasts and filamentous fungi to produce LA due to their versatility in fuel choice and tolerance of industrial-scale culture conditions such as pH and temperature. This review aims to compile and discuss metabolic engineering innovations in LA production in yeasts and filamentous fungi over the 2013-2023 period, and present future directions of research in this area, thus bringing researchers in the field up to date with recent advances.